2017-4

Volume 34 Issue 4 December 2017 80 TL

ISSN 1300-7777

Review

The Scope of Kidney Affection in Monoclonal Gammopathies at All Levels of Clinical Significance

Şadiye Mehtat Ünlü, et al.; İzmir, Turkey

Research Articles

Retrospective Evaluation of Hairy Cell Leukemia Patients Treated with Three Different First-Line Treatment

Modalities in the Last Two Decades: A Single-Center Experience

Şeniz Öngören, et al.; İstanbul, Turkey

FMS-Like Tyrosine Kinase 3 (FLT3) and Nucleophosmin 1 (NPM1) in Iranian Adult Acute Myeloid Leukemia Patients

with Normal Karyotypes: Mutation Status and Clinical and Laboratory Characteristics

Narges Rezaei, et al.; Shiraz, Iran

Autoantibodies Against Carbonic Anhydrase I and II in Patients with Acute Myeloid Leukemia

Ahmet Menteşe, et al.; Trabzon, Rize, Turkey

Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast and Accurate Human Umbilical Cord Blood

Hematopoietic Stem Cell Assessment

Emine Begüm Gençer, et al.; Ankara, Turkey

Effectiveness of Visual Methods in Information Procedures for Stem Cell Recipients and Donors

Çağla Sarıtürk, et al.; Adana, Turkey

Influence of L-Carnitine on Stored Rat Blood: A Study on Plasma

Carl Hsieh and Vani Rajashekharaiah, et al.; Bangalore, India

Antioxidants Attenuate Oxidative Stress-Induced Hidden Blood Loss in Rats

Hong Qian, et al.; Nanjing, China

Cover Picture:

Yasushi Kubota et al.

Peculiar Cold-Induced

Leukoagglutination in Mycoplasma

pneumoniae Pneumonia

4

Editor-in-Chief

Reyhan Küçükkaya

İstanbul, Turkey

rkucukkaya@hotmail.com

Associate Editors

Ayşegül Ünüvar

İstanbul, Turkey

aysegulu@hotmail.com

Cengiz Beyan

TOBB University of Economics and Technology,

Ankara, Turkey

cengizbeyan@hotmail.com

Hale Ören

Dokuz Eylül University, İzmir, Turkey

hale.oren@deu.edu.tr

İbrahim C. Haznedaroğlu

Hacettepe University, Ankara, Turkey

haznedar@yahoo.com

M. Cem Ar

İstanbul University Cerrahpaşa Faculty of

Medicine, İstanbul, Turkey

mcemar68@yahoo.com

Selami Koçak Toprak

Ankara University, Ankara, Turkey

sktoprak@yahoo.com

Semra Paydaş

Çukurova University, Adana, Turkey

sepay@cu.edu.tr

Assistant Editors

A. Emre Eşkazan

İstanbul University Cerrahpaşa Faculty of

Medicine, İstanbul, Turkey

Ali İrfan Emre Tekgündüz

Dr. A. Yurtaslan Ankara Oncology Training and

Research Hospital, Ankara, Turkey

Claudio Cerchione

University of Naples Federico II Napoli,

Campania, Italy

Elif Ünal İnce

Ankara University, Ankara, Turkey

İnci Alacacıoğlu

Dokuz Eylül University, İzmir, Turkey

Müge Sayitoğlu

İstanbul University, İstanbul, Turkey

Nil Güler

Ondokuz Mayıs University, Samsun, Turkey

Olga Meltem Akay

Koç University, İstanbul, Turkey

Şule Ünal


Veysel Sabri Hançer

İstinye University, İstanbul, Turkey

Zühre Kaya

Gazi University, Ankara, Turkey

International Review Board

Nejat Akar

Görgün Akpek

Serhan Alkan

Çiğdem Altay

Koen van Besien

Ayhan Çavdar

M. Sıraç Dilber

Ahmet Doğan

Peter Dreger

Thierry Facon

Jawed Fareed

Gösta Gahrton

Dieter Hoelzer

Marilyn Manco-Johnson

Andreas Josting

Emin Kansu

Winfried Kern

Nigel Key

Korgün Koral

Abdullah Kutlar

Luca Malcovati

Robert Marcus

Jean Pierre Marie

Ghulam Mufti

Gerassimos A. Pangalis

Antonio Piga

Ananda Prasad

Jacob M. Rowe

Jens-Ulrich Rüffer

Norbert Schmitz

Orhan Sezer

Anna Sureda

Ayalew Tefferi

Nükhet Tüzüner

Catherine Verfaillie

Srdan Verstovsek

Claudio Viscoli

Past Editors

Erich Frank

Orhan Ulutin

Hamdi Akan

Aytemiz Gürgey

Senior Advisory Board

Yücel Tangün

Osman İlhan

Muhit Özcan

Teoman Soysal

Ahmet Muzaffer Demir

TOBB Economy Technical University Hospital, Ankara, Turkey

Maryland School of Medicine, Baltimore, USA

Cedars-Sinai Medical Center, USA

Ankara, Turkey

Chicago Medical Center University, Chicago, USA

Ankara, Turkey

Karolinska University, Stockholm, Sweden

Mayo Clinic Saint Marys Hospital, USA

Heidelberg University, Heidelberg, Germany

Lille University, Lille, France

Loyola University, Maywood, USA

Karolinska University Hospital, Stockholm, Sweden

Frankfurt University, Frankfurt, Germany

Colorado Health Sciences University, USA

University Hospital Cologne, Cologne, Germany


Albert Ludwigs University, Germany

University of North Carolina School of Medicine, NC, USA

Southwestern Medical Center, Texas, USA

Georgia Health Sciences University, Augusta, USA

Pavia Medical School University, Pavia, Italy

Kings College Hospital, London, UK

Pierre et Marie Curie University, Paris, France

King’s Hospital, London, UK

Athens University, Athens, Greece

Torino University, Torino, Italy

Wayne State University School of Medicine, Detroit, USA

Rambam Medical Center, Haifa, Israel

University of Köln, Germany

AK St Georg, Hamburg, Germany

Memorial Şişli Hospital, İstanbul, Turkey

Santa Creu i Sant Pau Hospital, Barcelona, Spain

Mayo Clinic, Rochester, Minnesota, USA

İstanbul Cerrahpaşa University, İstanbul, Turkey

University of Minnesota, Minnesota, USA

The University of Texas MD Anderson Cancer Center, Houston, USA

San Martino University, Genoa, Italy

Language Editor

Leslie Demir

Statistic Editor

Hülya Ellidokuz

Editorial Office

İpek Durusu

Bengü Timoçin

A-I

Publishing

Services

GALENOS PUBLISHER

Molla Gürani Mah. Kaçamak Sk. No: 21/1, Fındıkzade, İstanbul, Turkey

Phone: +90 212 621 99 25 • Fax: +90 212 621 99 27 • www. galenos.com.tr

Contact Information

Editorial Correspondence should be addressed to Dr. Reyhan Küçükkaya

E-mail : rkucukkaya@hotmail.com

All Inquiries Should be Addressed to

TURKISH JOURNAL OF HEMATOLOGY

Address : İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No: 8 06550 Çankaya, Ankara / Turkey

Phone : +90 312 490 98 97

Fax : +90 312 490 98 68

E-mail : info@tjh.com.tr

ISSN: 1300-7777

Publishing Manager

Sorumlu Yazı İşleri Müdürü

Muhlis Cem Ar

Management Address

Yayın İdare Adresi

Türk Hematoloji Derneği

İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk.

No: 8 06550 Çankaya, Ankara / Turkey

Online Manuscript Submission

http://mc.manuscriptcentral.com/tjh

Web page

www.tjh.com.tr

Owner on behalf of the Turkish Society of Hematology

Türk Hematoloji Derneği adına yayın sahibi

Güner Hayri Özsan

Üç ayda bir yayımlanan İngilizce süreli yayındır.

International scientific journal published quarterly.

Publishing House / Yayınevi

Molla Gürani Mah. Kaçamak Sk. No: 21, 34093

Fındıkzade, İstanbul, Turkey

Tel: +90 212 621 99 25 Faks: +90 212 621 99 27

E-posta: info@galenos.com.tr

Baskı: Özgün Ofset Ticaret Ltd. Şti.

Yeşilce Mah. Aytekin Sk. No: 21 34418 4. Levent / İstanbul

Printing Date / Basım Tarihi

20.11.2017

Cover Picture

Yasushi Kubota et al.,

Peculiar Cold-Induced Leukoagglutination in Mycoplasma pneumoniae

Pneumonia

Peripheral blood smear showed not only RBC agglutination but also

neutrophil aggregates, eosinophil aggregates, and monocyte aggregates.

Türk Hematoloji Derneği, 07.10.2008 tarihli ve 6 no’lu kararı ile Turkish

Journal of Hematology’nin Türk Hematoloji Derneği İktisadi İşletmesi

tarafından yayınlanmasına karar vermiştir.

A-II

AIMS AND SCOPE

The Turkish Journal of Hematology is published quarterly (March, June,

September, and December) by the Turkish Society of Hematology. It is an

independent, non-profit peer-reviewed international English-language

periodical encompassing subjects relevant to hematology.

The Editorial Board of The Turkish Journal of Hematology adheres to

the principles of the World Association of Medical Editors (WAME),

International Council of Medical Journal Editors (ICMJE), Committee on

Publication Ethics (COPE), Consolidated Standards of Reporting Trials

(CONSORT) and Strengthening the Reporting of Observational Studies in

Epidemiology (STROBE).

The aim of The Turkish Journal of Hematology is to publish original

hematological research of the highest scientific quality and clinical

relevance. Additionally, educational material, reviews on basic

developments, editorial short notes, images in hematology, and letters

from hematology specialists and clinicians covering their experience and

comments on hematology and related medical fields as well as social

subjects are published. As of December 2015, The Turkish Journal of

Hematology does not accept case reports. Important new findings or data

about interesting hematological cases may be submitted as a brief report.

General practitioners interested in hematology and internal medicine

specialists are among our target audience, and The Turkish Journal of

Hematology aims to publish according to their needs. The Turkish Journal

of Hematology is indexed, as follows:

- PubMed Medline

- PubMed Central

- Science Citation Index Expanded

- EMBASE

- Scopus

- CINAHL

- Gale/Cengage Learning

- EBSCO

- DOAJ

- ProQuest

- Index Copernicus

- Tübitak/Ulakbim Turkish Medical Database

- Turk Medline

Impact Factor: 0.686

Open Access Policy

Turkish Journal of Hematology is an Open Access journal. This journal

provides immediate open access to its content on the principle that

making research freely available to the public supports a greater global

exchange of knowledge.

Open Access Policy is based on the rules of the Budapest Open Access

Initiative (BOAI) http://www.budapestopenaccessinitiative.org/.

Subscription Information

The Turkish Journal of Hematology is sent free-of-charge to members

of Turkish Society of Hematology and libraries in Turkey and abroad.

Hematologists, other medical specialists that are interested in hematology,

and academicians could subscribe for only 40 $ per printed issue. All

published volumes are available in full text free-of-charge online at www.

tjh.com.tr.

Address: İlkbahar Mah., Turan Güneş Bulvarı, 613 Sok., No: 8, Çankaya,

Ankara, Turkey

Telephone: +90 312 490 98 97

Fax: +90 312 490 98 68

Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh

Web page: www.tjh.com.tr

E-mail: info@tjh.com.tr

Permissions

Requests for permission to reproduce published material should be sent to

the editorial office.

Editor: Professor Dr. Reyhan Küçükkaya

Adress: İlkbahar Mah, Turan Günes Bulvarı, 613 Sok., No: 8, Çankaya,

Ankara, Turkey

Telephone: +90 312 490 98 97

Fax: +90 312 490 98 68

Online Manuscript Submission: http://mc.manuscriptcentral.com/tjh

Web page: www.tjh.com.tr

E-mail: info@tjh.com.tr

Publisher

Galenos Yayınevi

Molla Gürani Mah. Kaçamak Sk. No:21 34093 Fındıkzade-İstanbul, Turkey

Telephone : +90 212 621 99 25

Fax : +90 212 621 99 27

info@galenos.com.tr

Instructions for Authors

Instructions for authors are published in the journal and at www.tjh.com.tr

Material Disclaimer

Authors are responsible for the manuscripts they publish in The Turkish

Journal of Hematology. The editor, editorial board, and publisher do not

accept any responsibility for published manuscripts.

If you use a table or figure (or some data in a table or figure) from another

source, cite the source directly in the figure or table legend.

The journal is printed on acid-free paper.

Editorial Policy

Following receipt of each manuscript, a checklist is completed by the

Editorial Assistant. The Editorial Assistant checks that each manuscript

contains all required components and adheres to the author guidelines,

after which time it will be forwarded to the Editor in Chief. Following the

Editor in Chief’s evaluation, each manuscript is forwarded to the Associate

Editor, who in turn assigns reviewers. Generally, all manuscripts will be

reviewed by at least three reviewers selected by the Associate Editor, based

on their relevant expertise. Associate editor could be assigned as a reviewer

along with the reviewers. After the reviewing process, all manuscripts are

evaluated in the Editorial Board Meeting.

Turkish Journal of Hematology’s editor and Editorial Board members are

active researchers. It is possible that they would desire to submit their

manuscript to the Turkish Journal of Hematology. This may be creating

a conflict of interest. These manuscripts will not be evaluated by the

submitting editor(s). The review process will be managed and decisions

made by editor-in-chief who will act independently. In some situation, this

process will be overseen by an outside independent expert in reviewing

submissions from editors.

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TURKISH JOURNAL OF HEMATOLOGY

INSTRUCTIONS FOR AUTHORS

The Turkish Journal of Hematology accepts invited review articles, research

articles, brief reports, letters to the editor, and hematological images that

are relevant to the scope of hematology, on the condition that they have

not been previously published elsewhere. Basic science manuscripts,

such as randomized, cohort, cross-sectional, and case-control studies,

are given preference. All manuscripts are subject to editorial revision

to ensure they conform to the style adopted by the journal. There is a

double-blind reviewing system. Review articles are solicited by the Editorin-Chief.

Authors wishing to submit an unsolicited review article should

contact the Editor-in-Chief prior to submission in order to screen the

proposed topic for relevance and priority.

The Turkish Journal of Hematology does not charge any article submission

or processing charges.

Manuscripts should be prepared according to ICMJE guidelines (http://

www.icmje.org/). Original manuscripts require a structured abstract. Label

each section of the structured abstract with the appropriate subheading

(Objective, Materials and Methods, Results, and Conclusion). Letters to

the editor do not require an abstract. Research or project support should

be acknowledged as a footnote on the title page. Technical and other

assistance should be provided on the title page.

Original Manuscripts

Title Page

Title: The title should provide important information regarding the

manuscript’s content. The title must specify that the study is a cohort

study, cross-sectional study, case-control study, or randomized study (i.e.

Cao GY, Li KX, Jin PF, Yue XY, Yang C, Hu X. Comparative bioavailability

of ferrous succinate tablet formulations without correction for baseline

circadian changes in iron concentration in healthy Chinese male

subjects: A single-dose, randomized, 2-period crossover study. Clin Ther

2011;33:2054-2059).

The title page should include the authors’ names, degrees, and institutional/

professional affiliations and a short title, abbreviations, keywords, financial

disclosure statement, and conflict of interest statement. If a manuscript

includes authors from more than one institution, each author’s name

should be followed by a superscript number that corresponds to their

institution, which is listed separately. Please provide contact information

for the corresponding author, including name, e-mail address, and

telephone and fax numbers.

Running Head: The running head should not be more than 40 characters,

including spaces, and should be located at the bottom of the title page.

Word Count: A word count for the manuscript, excluding abstract,

acknowledgments, figure and table legends, and references, should be

provided and should not exceed 2500 words. The word count for the

abstract should not exceed 300 words.

Conflict of Interest Statement: To prevent potential conflicts of

interest from being overlooked, this statement must be included in each

manuscript. In case there are conflicts of interest, every author should

complete the ICMJE general declaration form, which can be obtained at

http://www.icmje.org/downloads/coi_disclosure.zip

Abstract and Keywords: The second page should include an abstract

that does not exceed 300 words. For manuscripts sent by authors in

Turkey, a title and abstract in Turkish are also required. As most readers

read the abstract first, it is critically important. Moreover, as various

electronic databases integrate only abstracts into their index, important

findings should be presented in the abstract.

Objective: The abstract should state the objective (the purpose of the

study and hypothesis) and summarize the rationale for the study.

Materials and Methods: Important methods should be written

respectively.

Results: Important findings and results should be provided here.

Conclusion: The study’s new and important findings should be

highlighted and interpreted.

Other types of manuscripts, such as reviews, brief reports, and

editorials, will be published according to uniform requirements.

Provide 3-10 keywords below the abstract to assist indexers. Use

terms from the Index Medicus Medical Subject Headings List

(for randomized studies a CONSORT abstract should be provided: http://

www.consort-statement.org).

Introduction: The introduction should include an overview of the

relevant literature presented in summary form (one page), and whatever

remains interesting, unique, problematic, relevant, or unknown about

the topic must be specified. The introduction should conclude with the

rationale for the study, its design, and its objective(s).

Materials and Methods: Clearly describe the selection of observational

or experimental participants, such as patients, laboratory animals, and

controls, including inclusion and exclusion criteria and a description of the

source population. Identify the methods and procedures in sufficient detail

to allow other researchers to reproduce your results. Provide references

to established methods (including statistical methods), provide references

to brief modified methods, and provide the rationale for using them and

an evaluation of their limitations. Identify all drugs and chemicals used,

including generic names, doses, and routes of administration. The section

should include only information that was available at the time the plan

or protocol for the study was devised (https://www.strobe-statement.org/

fileadmin/Strobe/uploads/checklists/STROBE_checklist_v4_combined.

pdf).

Statistics: Describe the statistical methods used in enough detail to

enable a knowledgeable reader with access to the original data to verify

A-IV

the reported results. Statistically important data should be given in the

text, tables, and figures. Provide details about randomization, describe

treatment complications, provide the number of observations, and specify

all computer programs used.

Results: Present your results in logical sequence in the text, tables, and

figures. Do not present all the data provided in the tables and/or figures

in the text; emphasize and/or summarize only important findings, results,

and observations in the text. For clinical studies provide the number of

samples, cases, and controls included in the study. Discrepancies between

the planned number and obtained number of participants should be

explained. Comparisons and statistically important values (i.e. p-value

and confidence interval) should be provided.

Discussion: This section should include a discussion of the data. New and

important findings/results and the conclusions they lead to should be

emphasized. Link the conclusions with the goals of the study, but avoid

unqualified statements and conclusions not completely supported by

the data. Do not repeat the findings/results in detail; important findings/

results should be compared with those of similar studies in the literature,

along with a summarization. In other words, similarities or differences in

the obtained findings/results with those previously reported should be

discussed.

Study Limitations: Limitations of the study should be detailed. In

addition, an evaluation of the implications of the obtained findings/

results for future research should be outlined.

Conclusion: The conclusion of the study should be highlighted.

References

Cite references in the text, tables, and figures with numbers in square

brackets. Number references consecutively according to the order in

which they first appear in the text. Journal titles should be abbreviated

according to the style used in Index Medicus (consult List of Journals

Indexed in Index Medicus). Include among the references any paper

accepted, but not yet published, designating the journal followed by “in

press”.

Examples of References:

1. List all authors

Deeg HJ, O’Donnel M, Tolar J. Optimization of conditioning for marrow

transplantation from unrelated donors for patients with aplastic anemia

after failure of immunosuppressive therapy. Blood 2006;108:1485-1491.

2. Organization as author

Royal Marsden Hospital Bone Marrow Transplantation Team. Failure of

syngeneic bone marrow graft without preconditioning in post-hepatitis

marrow aplasia. Lancet 1977;2:742-744.

3. Book

Wintrobe MM. Clinical Hematology, 5th ed. Philadelphia, Lea & Febiger,

1961.

4. Book Chapter

Perutz MF. Molecular anatomy and physiology of hemoglobin. In:

Steinberg MH, Forget BG, Higs DR, Nagel RI, (eds). Disorders of Hemoglobin:

Genetics, Pathophysiology, Clinical Management. New York, Cambridge

University Press, 2000.

5. Abstract

Drachman JG, Griffin JH, Kaushansky K. The c-Mpl ligand (thrombopoietin)

stimulates tyrosine phosphorylation. Blood 1994;84:390a (abstract).

6. Letter to the Editor

Rao PN, Hayworth HR, Carroll AJ, Bowden DW, Pettenati MJ. Further

definition of 20q deletion in myeloid leukemia using fluorescence in situ

hybridization. Blood 1994;84:2821-2823.

7. Supplement

Alter BP. Fanconi’s anemia, transplantation, and cancer. Pediatr Transplant

2005;9(Suppl 7):81-86.

Brief Reports

Abstract length: Not to exceed 150 words.

Article length: Not to exceed 1200 words.

Introduction: State the purpose and summarize the rationale for the study.

Materials and Methods: Clearly describe the selection of the observational

or experimental participants. Identify the methods and procedures in

sufficient detail. Provide references to established methods (including

statistical methods), provide references to brief modified methods, and

provide the rationale for their use and an evaluation of their limitations.

Identify all drugs and chemicals used, including generic names, doses, and

routes of administration.

Statistics: Describe the statistical methods used in enough detail to

enable a knowledgeable reader with access to the original data to verify

the reported findings/results. Provide details about randomization,

describe treatment complications, provide the number of observations,

and specify all computer programs used.

Results: Present the findings/results in a logical sequence in the text,

tables, and figures. Do not repeat all the findings/results in the tables and

figures in the text; emphasize and/or summarize only those that are most

important.

Discussion: Highlight the new and important findings/results of the

study and the conclusions they lead to. Link the conclusions with the

goals of the study, but avoid unqualified statements and conclusions not

completely supported by your data.

Invited Review Articles

Abstract length: Not to exceed 300 words.


Review articles should not include more than 100 references. Reviews

should include a conclusion, in which a new hypothesis or study about the

A-V

subject may be posited. Do not publish methods for literature search or

level of evidence. Authors who will prepare review articles should already

have published research articles on the relevant subject. The study’s new

and important findings should be highlighted and interpreted in the

Conclusion section. There should be a maximum of two authors for review

articles.

Images in Hematology


Authors can submit for consideration illustrations or photos that are

interesting, instructive, and visually attractive, along with a few lines of

explanatory text and references. Images in Hematology can include no

more than 200 words of text, 5 references, and 3 figures or tables. No

abstract, discussion, or conclusion is required, but please include a brief

title.

Letters to the Editor


Letters can include no more than 500 words of text, 5-10 references, and

1 figure or table. No abstract is required, but please include a brief title.

Tables

Supply each table in a separate file. Number tables according to the order

in which they appear in the text, and supply a brief caption for each.

Give each column a short or abbreviated heading. Write explanatory

statistical measures of variation, such as standard deviation or standard

error of mean. Be sure that each table is cited in the text.

Figures

Figures should be professionally drawn and/or photographed. Authors

should number figures according to the order in which they appear in the

text. Figures include graphs, charts, photographs, and illustrations. Each

figure should be accompanied by a legend that does not exceed 50 words.

Use abbreviations only if they have been introduced in the text. Authors

are also required to provide the level of magnification for histological

slides. Explain the internal scale and identify the staining method used.

Figures should be submitted as separate files, not in the text file. Highresolution

image files are not preferred for initial submission as the file

sizes may be too large. The total file size of the PDF for peer review should

not exceed 5 MB.

Authorship

Each author should have participated sufficiently in the work to assume

public responsibility for the content. Any portion of a manuscript that

is critical to its main conclusions must be the responsibility of at least

one author.

Contributor’s Statement

All submissions should contain a contributor’s statement page. Each

statement should contain substantial contributions to idea and design,

acquisition of data, and analysis and interpretation of findings. All

persons designated as an author should qualify for authorship, and all

those that qualify should be listed. Each author should have participated

sufficiently in the work to take responsibility for appropriate portions of

the text.

Acknowledgments

Acknowledge support received from individuals, organizations, grants,

corporations, and any other source. For work involving a biomedical

product or potential product partially or wholly supported by corporate

funding, a note stating, “This study was financially supported (in part)

with funds provided by (company name) to (authors’ initials)”, must

be included. Grant support, if received, needs to be stated and the

specific granting institutions’ names and grant numbers provided when

applicable.

Authors are expected to disclose on the title page any commercial or

other associations that might pose a conflict of interest in connection

with the submitted manuscript. All funding sources that supported the

work and the institutional and/or corporate affiliations of the authors

should be acknowledged on the title page.

Ethics

When reporting experiments conducted with humans indicate that

the procedures were in accordance with ethical standards set forth

by the committee that oversees human subject research. Approval of

research protocols by the relevant ethics committee, in accordance

with international agreements (Helsinki Declaration of 1975, revised

2013 available at https://www.wma.net/policies-post/wma-declarationof-helsinki-ethical-principles-for-medical-research-involving-humansubjects/),

is required for all experimental, clinical, and drug studies.

Patient names, initials, and hospital identification numbers should not

be used. Manuscripts reporting the results of experimental investigations

conducted with humans must state that the study protocol received

institutional review board approval and that the participants provided

informed consent.

Non-compliance with scientific accuracy is not in accord with scientific

ethics. Plagiarism: To re-publish, in whole or in part, the contents of

another author’s publication as one’s own without providing a reference.

Fabrication: To publish data and findings/results that do not exist.

Duplication: Use of data from another publication, which includes republishing

a manuscript in different languages. Salami slicing: To create

more than one publication by dividing the results of a study unnecessarily.

We disapprove of such unethical practices as plagiarism, fabrication,

duplication, and salami slicing, as well as efforts to influence the

review process with such practices as gifting authorship, inappropriate

acknowledgments, and references. Additionally, authors must respect

participants‘ right to privacy.

On the other hand, short abstracts published in congress books that do

not exceed 400 words and present data of preliminary research, and

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those that are presented in an electronic environment, are not considered

as previously published work. Authors in such a situation must declare

this status on the first page of the manuscript and in the cover letter.

(The COPE flowchart is available at http://publicationethics.org.)

We use iThenticate to screen all submissions for plagiarism before

publication.

Conditions of Publication

All authors are required to affirm the following statements before their

manuscript is considered: 1. The manuscript is being submitted only

to The Turkish Journal of Hematology; 2. The manuscript will not be

submitted elsewhere while under consideration by The Turkish Journal

of Hematology; 3. The manuscript has not been published elsewhere,

and should it be published in The Turkish Journal of Hematology it will

not be published elsewhere without the permission of the editors (these

restrictions do not apply to abstracts or to press reports for presentations

at scientific meetings); 4. All authors are responsible for the manuscript’s

content; 5. All authors participated in the study concept and design,

analysis and interpretation of the data, and drafting or revising of the

manuscript and have approved the manuscript as submitted. In addition,

all authors are required to disclose any professional affiliation, financial

agreement, or other involvement with any company whose product

figures prominently in the submitted manuscript.

Authors of accepted manuscripts will receive electronic page proofs and

are responsible for proofreading and checking the entire article within

two days. Failure to return the proof in two days will delay publication. If

the authors cannot be reached by email or telephone within two weeks,

the manuscript will be rejected and will not be published in the journal.

Copyright

At the time of submission all authors will receive instructions for

submitting an online copyright form. No manuscript will be considered

for review until all authors have completed their copyright form. Please

note, it is our practice not to accept copyright forms via fax, e-mail, or

postal service unless there is a problem with the online author accounts

that cannot be resolved. Every effort should be made to use the online

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A-VIII

CONTENTS

Review

282 The Scope of Kidney Affection in Monoclonal Gammopathies at All Levels of Clinical Significance

Şadiye Mehtat Ünlü, Hayri Özsan, Sülen Sarıoğlu

Commentary

289 Time to Cure Hairy Cell Leukemia

Ilana Levy, Tamar Tadmor

Research Articles

291 Retrospective Evaluation of Hairy Cell Leukemia Patients Treated with Three Different First-Line Treatment Modalities in the Last Two

Decades: A Single-Center Experience

Şeniz Öngören, Ahmet Emre Eşkazan, Selin Berk, Tuğrul Elverdi, Ayşe Salihoğlu, Muhlis Cem Ar, Zafer Başlar, Yıldız Aydın,

Nükhet Tüzüner, Teoman Soysal

300 FMS-Like Tyrosine Kinase 3 (FLT3) and Nucleophosmin 1 (NPM1) in Iranian Adult Acute Myeloid Leukemia Patients with Normal

Karyotypes: Mutation Status and Clinical and Laboratory Characteristics

Narges Rezaei, Nargess Arandi, Behnaz Valibeigi, Sezaneh Haghpanah, Mehdi Khansalar, Mani Ramzi

307 Autoantibodies Against Carbonic Anhydrase I and II in Patients with Acute Myeloid Leukemia

Ahmet Menteşe, Nergiz Erkut, Selim Demir, Serap Özer Yaman, Ayşegül Sümer, Şeniz Doğramacı, Ahmet Alver, Mehmet Sönmez

314 Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast and Accurate Human Umbilical Cord Blood

Hematopoietic Stem Cell Assessment

Emine Begüm Gençer, Pınar Yurdakul, Klara Dalva, Meral Beksaç

321 Effectiveness of Visual Methods in Information Procedures for Stem Cell Recipients and Donors

Çağla Sarıtürk, Çiğdem Gereklioğlu, Aslı Korur, Süheyl Asma, Mahmut Yeral, Soner Solmaz, Nurhilal Büyükkurt, Songül Tepebaşı,

İlknur Kozanoğlu, Can Boğa, Hakan Özdoğu

328 Influence of L-Carnitine on Stored Rat Blood: A Study on Plasma

Carl Hsieh, Vani Rajashekharaiah

334 Antioxidants Attenuate Oxidative Stress-Induced Hidden Blood Loss in Rats

Hong Qian, Tao Yuan, Jian Tong, Wen-shuang Sun, Jiajia Jin, Wen-xiang Chen, Jia Meng, Nirong Bao, Jianning Zhao

Brief Reports

340 High Infection-Related Mortality in Pediatric Acute Myeloid Leukemia without Preventive Antibiotics and Antifungals: Retrospective

Cohort Study of a Single Center from a Middle-Income Country

Emine Zengin, Nazan Sarper, Sema Aylan Gelen, Uğur Demirsoy, Meriban Karadoğan, Suar Çakı Kılıç, Selim Öncel,

Emin Sami Arısoy, Devrim Dündar

345 Hematopoietic Stem Cell Transplantation in Primary Immunodeficiency Patients in the Black Sea Region of Turkey

Alişan Yıldıran, Mehmet Halil Çeliksoy, Stephan Borte, Şükrü Nail Güner, Murat Elli, Tunç Fışgın, Emel Özyürek, Recep Sancak, Gönül Oğur

Images in Hematology

350 Bullous Pyoderma Gangrenosum in a Patient with Acute Myelogenous Leukemia as a Pathergic Reaction after Bone Marrow Biopsy

Nur Efe İris, Reyhan Diz-Küçükkaya, Mutlu Arat, Zahide Eriş

A-IX

352 Giant Intracranial Solitary Plasmacytoma

Osman Kara, Tayfur Toptaş, Işık Atagündüz, Süheyla Bozkurt, Önder Şirikçi, Tülin Fıratlı Tuğlular

354 Peculiar Cold-Induced Leukoagglutination in Mycoplasma pneumoniae Pneumonia

Yasushi Kubota, Yuka Hirakawa, Kazuo Wakayama, Shinya Kimura

Letters to the Editor

356 Liver Transplantation in a Patient with Acquired Dysfibrinogenemia Who Presented with Subdural Hematoma: A Case Report

Şencan Acar, Gökhan Güngör, Murat Dayangaç, Reyhan Diz-Küçükkaya, Yaman Tokat, Murat Akyıldız

358 Chronic Active Parietal Osteomyelitis Due to Salmonella typhi in a Patient with Sickle Cell Anemia

Ahmad Antar, George Karam, Maurice Kfoury, Nadim El-Majzoub

359 Acquired Leukocyte Inclusion Bodies Resembling Döhle Bodies During Acute Cholangitis

Gökhan Özgür, Musa Barış Aykan, Murat Yıldırım, Selim Sayın, Ahmet Uygun, Cengiz Beyan

360 Three Novel Calreticulin Mutations in Two Turkish Patients

Veysel Sabri Hançer, Hüseyin Tokgöz, Serkan Güvenç, Ümran Çalışkan, Murat Büyükdoğan

362 Imatinib-Induced Interstitial Pneumonitis Successfully Switched to Nilotinib in a Patient with Prior History of

Mycobacterium tuberculosis Infection

Zhuan-Bo Luo, Ning Xu, Xiao-Ping Huang, Guifang Ouyang

364 Prostate Involvement in a Patient with Follicular Lymphoma

Seda Yılmaz, Sinan Demircioğlu, Özlen Bektaş, Özcan Çeneli, Sıdıka Fındık

366 Coexistence of EZH2, NOTCH1, IL7R, and PHF6 Mutations in Adult T-cell Acute Lymphoblastic Leukemia

Xilian Zhou, Yan Gu, Qi Han, Mario Soliman, Chunhua Song, Zheng Ge

369 Circulating Tumor Cells in Neuroblastoma

Mili Jain, Ashutosh Kumar, Sanjay Mishra, Nishant Verma, Madhu Mati Goel

370 Megakaryocytic Emperipolesis Associated with Thrombocytopenia: Causative or Coincidence?

Manu Goyal, Sreeja Thandilath Thekkelakayil, Anurag Gupta

372 First Observation of Hemoglobin San Diego, a High Oxygen Affinity Hemoglobin Variant, in Turkey

Ebru Yılmaz Keskin, Ali Fettah, Ana Catarina Oliveira, Şule Toprak, Andreia Lopes, Celeste Bento

374 A Case of Para-Bombay Phenotype Caused by Homozygous Mutation of the FUT1 Gene

Jung-Kuang Yu, Yi-Hong Liu, Tze-Kiong Er

376 A Myopathy, Lactic Acidosis, Sideroblastic Anemia (MLASA) Case Due to a Novel PUS1 Mutation

Çiğdem Seher Kasapkara, Leyla Tümer, Nadia Zanetti, Fatih Ezgü, Eleonora Lamantea, Massimo Zeviani

378 Frequency and Risk Factors for Secondary Malignancies in Patients with Mycosis Fungoides

Fatma Pelin Cengiz, Nazan Emiroğlu, Nahide Onsun

380 Leishmaniasis: Bone Marrow Aspirate Smear and Rapid Antibody Test

Beuy Joob, Viroj Wiwanitkit

381 Receiver Operating Characteristic Curve Analysis May be Helpful to Study the Prognostic Value of miR-155 in B-Cell Non-Hodgkin Lymphoma

Long Su

34 th Volume Index

Author Index 2017

Subject Index 2017

A-X

Advisory Board of This Issue (December 2017)

Abdulahad Doğan, Turkey

Ahmet Emre Eşkazan, Turkey

Ahmet Muzaffer Demir, Turkey

Akif Selim Yavuz, Turkey

Anıl Tombak, Turkey

Bülent Eser, Turkey

Burhan Engin, Turkey

Cem Ar, Turkey

Chloe Anthlas, UK

Dilber Talia İleri, Turkey

Dushyant Kumar, India

Düzgün Özatlı, Turkey

Ebru Koca, Turkey

Emre Tekgündüz, Turkey

Engin Özçivici, Turkey

Erdal Karaöz, Turkey

Erdal Kurtoğlu, Turkey

Eren Altun, Turkey

Graeme Quest, Canada

Hale Ören, Turkey

Handan Dinçaslan, Turkey

Hüseyin Onay, Turkey

James Yu, USA

Jamile Shammo, USA

Jansen Seheult, USA

John M. Bennett, USA

Kanjaksha Ghosh, India

Mahmut Bayık, Turkey

Majid Shahabi, Iran

Maria Papaioannou, Greece

Mehmet Sönmez, Turkey

Melih Aktan, Turkey

Meral Beksaç, Turkey

Michael Grever, USA

Müge Sayitoğlu, Turkey

Mutlu Arat, Turkey

Nader Cohan, Iran

Nejat Akar, Turkey

Oliver Karanfılski, Macedonia

Poojan Agarwal, India

Rajat Kumar Agarwal, India

Rejin Kebudi, Turkey

Rosemary Sparrow, Australia

Ruchi Gupta, India

Rümeyza Kazancıoğlu, Turkey

Sema Anak, Turkey

Sevil Çatal, Turkey

Sigbjørn Berentsen, Norway

Sujata Mallick, India

Tamar Tadmor, Israel

Tiraje Celkan, Turkey

Türkan Patıroğlu, Turkey

Ujjwayini Ray, India

Ümit Yavuz Malkan, Turkey

Vildan Güngörer, Turkey

Vildan Özkocaman, Turkey

Volkan Hazar, Turkey

Yahya Büyükaşık, Turkey

Yüksel Aliyazıcıoğlu, Turkey

REVIEW

DOI: 10.4274/tjh.2017.0197

Turk J Hematol 2017;34:282-288

The Scope of Kidney Affection in Monoclonal Gammopathies at All

Levels of Clinical Significance

Monoclonal Gammopatilerin Her Klinik Evresinde Böbrek Etkilenim Paternleri

Şadiye Mehtat Ünlü 1 , Hayri Özsan 2 , Sülen Sarıoğlu 1

1

Dokuz Eylül University Faculty of Medicine, Department of Pathology, İzmir, Turkey

2

Dokuz Eylül University Faculty of Medicine, Department of Hematology, İzmir, Turkey

Abstract

Multiple myeloma (MM) is one of the most important clonal malignant

plasma cell disorders and renal involvement is associated with poor

prognosis. Although there are several reasons for renal impairment

in MM, the main cause is the toxic effects of monoclonal proteins.

Although cast nephropathy is the best known and unchallenged

diagnosis for hematologists and pathologists, the renal effects of

monoclonal gammopathy can be various. Monoclonal gammopathy

of renal significance was proposed by the International Kidney

and Monoclonal Gammopathy Research Group for renal lesions in

monoclonal gammopathy in recent years. Renal lesions in monoclonal

gammopathy can be grouped as follows: light chain (cast) nephropathy,

acute tubular injury/necrosis, tubulointerstitial nephritis, amyloidosis,

monoclonal Ig deposition diseases, immunotactoid glomerulopathy,

type I cryoglobulinemia, proliferative glomerulonephritis with

monoclonal IgG deposits, C3 glomerulopathy with monoclonal

gammopathy, and crystal-storing histiocytosis, considering the

previous and new terminology. In this study, renal involvement of

monoclonal gammopathies, in terms of previous and new terminology,

was reviewed.

Keywords: Monoclonal gammopathy of renal significance, Plasma

cell disorders, Multiple myeloma, Renal involvement, Kidney, Cast

nephropathy

Öz

Multipl miyelom (MM) malign plazma hücre hastalıklarının en

önemlilerinden biridir ve böbrek tutulumu kötü prognozla ilişkilidir.

Multipl miyelomda böbrek fonksiyonunu etkileyecek çeşitli faktörler

bulunmakla birlikte ana etken monoklonal proteinlerin toksik etkisidir.

Monoklonal gammopatilerde böbrek tutulumları içinde hematolog

ve patologlar tarafından en iyi bilinen ve tanısı sorunsuz olan “kast

nefropatisi” olmakla birlikte çok farklı tutulum tipleri olabilir. Yakın

zamanda “International Kidney and Monoclonal Gammopathy

Research Group” tarafından, monoklonal gammopatilerdeki böbrek

tutulumlarını bir çatı altında toplayan “renal öneme sahip monoklonal

gammopati” terimi önerildi. Monoklonal gammopatilerdeki böbrek

lezyonları, önceki ve yeni terminoloji uyarınca; hafif zincir kast

nefropati, akut tübüler zedelenme/nekroz, tubulointerstisyel

nefrit, amiloidoz, monoklonal Ig depo hastalıkları, immünotaktoid

glomerülopati, tip I kriyoglobulinemi, monoklonal IgG depoziti

ilişkili proliferatif glomerülonefrit, monoklonal gammopati ilişkili C3

glomerülopati ve kristal/histiyosit depo hastalığı olarak gruplanabilir.

Bu makalede, monoklonal gammopatilerdeki böbrek tutulumu yeni ve

önceki terminolojiler gözden geçirildi.

Anahtar Sözcükler: Renal öneme sahip monoklonal gammopati,

Plazma hücre hastalıkları, Multipl miyelom, Böbrek tutulumu, Böbrek,

Kast nefropati

Introduction

Each specific immunoglobulin (Ig) molecule, which is synthesized

by plasma cells, includes two identical heavy chains and two

light chains (LCs). There are 5 types of heavy chains (ϒ/α/μ/δ/Є),

and immunoglobulins get their names (IgG, IgA, IgM, IgD, IgE)

according to these, while there are 2 types of LC (К/λ). Each

heavy chain and LC has constant domains and variable domains.

Variable domains include the antigen-binding region. Heavy

chains and LCs are synthesized independently of each other and

finally unite in the endoplasmic reticulum, and the structure

called immunoglobulin is unified here [1,2].

Normally, LCs are freely filtered through the glomerular basal

membrane because they are low-molecular-weight proteins

and are reabsorbed by the proximal tubules, endocytosed and

catabolized in the lysosome. After the catabolization process,

their amino acids return to the circulation. There are cubilinmegalin

receptors on the brush-border of proximal tubular cells,

which are very important for the control of LC endocytosis.

©Copyright 2017 by Turkish Society of Hematology

Turkish Journal of Hematology, Published by Galenos Publishing House

Address for Correspondence/Yazışma Adresi: Şadiye Mehtat ÜNLÜ, M.D.,

Dokuz Eylül University Faculty of Medicine, Department of Pathology, İzmir, Turkey

Phone : +90 232 412 34 15

E-mail : mehtat.unlu@deu.edu.tr ORCID-ID: orcid.org/0000-0002-7170-7594

Received/Geliş tarihi: May 18, 2017

Accepted/Kabul tarihi: August 22, 2017

282


Ünlü ŞM, et al: The Scope of Kidney Affection in Monoclonal Gammopathies at All Levels of Clinical Significance

The heavy chains do not cross the glomerular filtration barrier

[3,4,5].

In plasma cell dyscrasia, there is monoclonal plasma cell

proliferation and a single type of whole Ig or a subunit or just

a LC, which is synthesized by the clone, and the type of these

protein fragments causes damage to the kidneys at a varying

degree or the amount of the monoclonal LC in the filtrate exceeds

the capacity of proximal tubular cells. Approximately 85% of all

LCs with plasma cell dyscrasia are nephrotoxic. Most of them

are tubulopathic (70%) and so they affect the tubulointerstitial

compartment, and the rest of them are glomerulopathic and

affect the glomerular compartment. Of course, some host factors

are deterministic for this pattern and the grade of the damage,

while the molecular and physicochemical characteristics of the

LC affecting different compartments are unknown [6].

Monoclonal protein secretion is a typical feature of plasma cell

disorders and may affect the kidneys in several ways. Multiple

myeloma (MM) is one of the most important clonal malignant

plasma cell disorders and renal involvement is associated with

poor prognosis [7]. Clonal evaluation steps for MM include

monoclonal gammopathy of undetermined significance (MGUS)

and smoldering MM [8,9]. While MGUS and smoldering MM

patients do not require therapy except for clinical trial settings,

if the patients have myeloma defining events such as CRAB

(hypercalcemia, renal impairment, anemia, and bone lesions),

treatment becomes obligatory. One of the CRAB finding is

renal impairment and its incidence ranges from 20% to 50%

according to how it is defined [7,9]. The International Myeloma

Working Group consensus defines kidney impairment as an

acute deterioration of kidney function that results in a serum

creatinine level of more than 2.0 mg/dL, but kidney biopsy is

currently not proposed. That means that the real incidence of

nephropathy is not clear. Although the main cause for renal

impairment is the toxic effects of monoclonal proteins, there

are several other factors like hypercalcemia, dehydration,

nephrotoxic drugs, and contrast agents that can aggravate the

underlying disease [9]. Although MGUS and smoldering MM

seem to be more benign disorders and do not require therapy,

they may cause some renal conditions and those should be

treated.

Until recent time, in the writings on this subject, “MM, MGUS,

or smoldering myeloma” has probably been the most important

classification for hematologists while “glomerulopathic/

tubulopathic pattern” has been the most important for

pathologists. Although these classifications still maintain their

importance, they have become inadequate for the diagnosis,

monitoring, and treatment of kidney lesions.

Although MM and cast nephropathy are the most straightforward

diagnoses for hematologists and pathologists, there are uncertain

areas in this wide clinical and morphologic spectrum for both

hematologists and pathologists. According to the treatment

guidelines, chemotherapy is indicated when the patient has

symptoms related to the underlying plasmacytic or lymphocytic

proliferation [10,11]. At this stage, due to the lack of end-stage

organ involvement according to the definitions of smoldering

MM (>3 g/dL of monoclonal protein and/or >10% bone marrow

plasma cells) and MGUS (<3 g/dL of monoclonal protein and/

or <10% bone marrow plasma), they do not receive treatment.

Although this group looks like benign disease to hematologists,

the same situation is not true for the kidneys [11,12,13].

Some patients with proteinuria or acute renal failure consult

with nephrologists before hematologists and they get a

diagnosis from renal biopsy if the pathologist is experienced

in nephropathology, because renal injury patterns other than

cast nephropathy and amyloidosis can be very silent or can be

confused with other renal diseases.

Until recently, in cases of plasma cell dyscrasia, renal

involvements have been grouped into the following

categories: LC (cast) nephropathy, acute tubular injury/necrosis

(ATD), tubulointerstitial nephritis (TIN), amyloidosis, and

monoclonal Ig deposition diseases (MIDDs).

Light chain (cast) nephropathy is characterized by acute renal

deterioration or clear renal failure and an uncomplicated

histopathological diagnosis. Because the formation of the

myeloma cast begins in the collecting ducts, the medulla has

special importance in the biopsy. The casts generally contain the

LCs and Tamm-Horsfall protein and sometimes cellular debris

and crystals and giant cells. While monoclonal LC staining is

important in immunofluorescence, equally strong staining must

also not be ignored (Figure 1) [6,14,15,16].

Figure 1. Light chain cast nephropathy: A) classical large myeloma

casts show irregular shape and fracture planes (hematoxylin and

eosin; 100 x ); B) giant cell surrounding the casts (hematoxylin and

eosin; 200 x ); C) metachromatic staining of the myeloma casts

(Masson’s trichrome; 200 x ).

283



ATD is especially characterized by proximal tubular damage

because the LCs are metabolized in the lysosomes of these cells.

ATD can be the only histological lesion in a biopsy or it may

be combined with other findings with associated monoclonal

gammopathy. The clinical manifestation is a varying degree

of renal failure according to the severity of the tubular

injuries. Light chain proximal tubulopathy is characterized by

periodic acid-Schiff-negative crystals or non-crystallized LCs

in the cytoplasm of proximal tubular cells. Pathologic LCs are

almost always kappa in proximal tubulopathy with crystal

formation and appear to be hypereosinophilic under a light

microscope. In addition, round/rectangular free structures can

be detected in the cytoplasm of proximal tubules by electron

microscope. Due to the loss of crystals during standard frozen

sectioning, direct immunofluorescence (DIF) is not a proper

method to demonstrate these structures [17,18].

In the non-crystallized cases, pathologic LCs can be kappa or

lambda and can be detected in classical DIF. They appear to be

hypereosinophilic inclusions in the cytoplasm of proximal tubules in

hematoxylin-eosin, dark positive dots in periodic acid-methionine

silver, and electron-dense large phagolysosomes by electron

microscope. Since the proximal tubule epithelial cells are the

physiologic catabolism area for LCs, the acute or chronic signs of

tubular injury should be well demonstrated morphologically by

the pathologist to differentiate the physiologic process.

Fanconi syndrome, which is characterized by electrolyte wasting

and aminoaciduria, is most frequently accompanied by LC

proximal tubulopathy with crystals (Figure 2) [19,20].

TIN is quite rare in the plasma cell dyscrasia-associated

disease group. Acute renal failure is the most common clinical

manifestation. The histomorphological findings are identical

to the other inflammatory TIN findings and the morphological

diagnosis is very easy; however, it is difficult to establish the

connection with plasma cell dyscrasia in terms of etiology and

the rate of establishing the linkage between the two diseases

is very low. Linear monotypic light staining throughout the

tubular basement membranes sometimes can be helpful (Figure

3) [6,21,22,23].

Amyloidosis is a group of misfolded protein disorders and has

the structure of fibrils. Amyloidosis with plasma cell dyscrasia

is an important systemic problem. Amyloid light-chain (AL)

amyloidosis stains with Congo red just like the amyloid A type

and exhibits yellow/orange/green colors under polarized light

[24]. Amyloid fibrils consist of generally light (most frequently λ)

and sometimes heavy chain fragments. Renal involvement is

not infrequent (70%-80%) and it mostly affects the glomeruli

and vessels. Although renal involvement is common, cardiac

involvement is important for early mortality. Amyloidogenic

LCs are directly toxic to the myocardium and rapid suppression

is crucial for mortality. It is important to emphasize that

amyloidosis patients mostly have a low-grade plasma cell clone

and only 20% of patients meet the criteria for MM (Figure 4)

[25,26,27,28,29].

MIDDs are systemic like amyloidosis and are observed

in approximately 3%-5% of myeloma cases. Although

immunoglobulin is stored in many organs, the kidneys are

most commonly affected. Except for a few cases, usually the

immunoglobulin depositions consist of LCs. Most light-chain

deposition disease patients have proteinuria, with 50% of them

Figure 2. Light chain proximal tubulopathy: A) dark positive dots

are a clue for the accumulation of light chain protein in proximal

tubules (PAMS; 400 x ); B) cytoplasmic granular staining with direct

immunofluorescence for lambda light chain (fluorescein, 400 x ).

Figure 3. Light chain-related tubulointerstitial nephritis: A-B)

interstitial lymphocyte-rich inflammation (hematoxylin and eosin;

100 x and 200 x ); C) very few cellular cast formations may be the only

clue for monoclonal gammopathy (hematoxylin and eosin; 400 x ).

Figure 4. AL amyloidosis. Acellular brick red mesangial deposits with Congo red in the glomerulus (A) and also a vessel wall (B) (400 x ).

C) Yellow-orange birefringence in glomerulus and vessels under polarized light with Congo red (200 x ). D) Mesangial lambda light chain

amyloid deposits with direct immunofluorescence (fluorescein, 400 x ).

284



being in the nephrotic range and about 30% of cases having

acute renal failure in the diagnosis. In more than half of the cases,

clinical findings meet the criteria for MM. However, it should be

kept in mind that a significant number of cases have normal

bone marrow biopsy results. Most light microscopic findings are

nodular glomerulopathy and monotypic LC deposition in the

glomerular-tubular basement membrane and in vessels walls

with immunofluorescence (Figure 5) [18,22,30,31,32,33].

A new term, monoclonal gammopathy of renal

significance (MGRS), was recently proposed by the International

Kidney and Monoclonal Gammopathy Research Group for renal

lesions in monoclonal gammopathy. All renal lesions related

to monoclonal gammopathy were reclassified under the title

of MGRS and it was emphasized that the kidneys of patients

whose clinical symptoms do not meet the criteria for MM,

Waldenström macroglobulinemia (WM), chronic lymphocytic

leukemia (CLL), or other plasma/B-cell proliferative disorders

[12] should be monitored (Schema 1). Cast nephropathy and WM

(both of which are well recognized for therapy by nephrologists

and hematologists) causing acute renal failure are excluded

from this description, directly related to high tumor load.

Furthermore, as our experience supports, different patterns

with different levels can be seen in the same case. If the patient

has acute renal failure due to cast nephropathy, hematologists

should immediately determine whether the patient has a clinical

Figure 5. C3 glomerulopathy with monoclonal gammopathy:

A) mesangial matrix expansion (hematoxylin and eosin; 400 x );

B) positivity of the mesangial complement C3c with direct

immunofluorescence (fluorescein, 400 x ).

Schema 1. MGRS-associated renal lesions (modified from

Bridoux et al. [36]).

AH: Immunoglobulin heavy chain amyloidosis, AHL: immunoglobulin heavy

and light chain amyloidosis, AL: immunoglobulin light chain amyloidosis, GN:

glomerulonephritis, HCDD: heavy chain deposition disease, LCDD: light chain

deposition disease, LHCDD: light and heavy chain deposition disease, MGRS:

monoclonal gammopathy of renal significance.

diagnosis of MM or not, because they know very well that MM

is characterized by high tumor mass and high monoclonal Ig

level.

However, in MGRS, the patient has a small clone of

lymphoplasmacytic cells and the structural and biological

features rather than the amounts of immunoglobulin are

especially important for renal disease.

Renal dysfunction is a common complication (approximately

20%-25%) in active MM cases and the grade of renal

dysfunction is probably correlated with myeloma cell load. Half

of patients (50%) even show an improvement in the renal

dysfunction with the treatment of myeloma, but the remaining

ones have some degree of persistent chronic kidney disease

and 2%-12% of these patients need renal transplantation. The

high tumor burden and excessive monoclonal protein secretion

are the main courses of the renal lesions in this group and the

most common manifestation is acute renal failure due to cast

nephropathy [34,35]. In MGRS cases, however, tumor burden

is not high and the determination of the latent renal injury is

based on the physicochemical property of paraprotein [12,36].

As shown in Schema 1, MGRS-associated renal lesions mainly

affect the glomerulus.

Fibrillary glomerulonephritis is characterized by fibril

structures, resembling amyloidosis. However, these fibrils do not

create the amyloid form and do not react with Congo red. Very

few are cases associated with monoclonal gammopathy and

most cases show a membranoproliferative glomerulonephritis

(MPGN) pattern under light microscope and IgG4, IgG1, and

polyclonal LC positivity in immunofluorescence. The fibrils are

non-branching and randomly oriented, similar to the amyloid

fibrils ultrastructurally in electron microscopic assessment, but

the fibril diameters are thicker (12-25 nm in diameter) than the

amyloid fibrils (8-12 nm in diameter) [37,38,39].

Immunotactoid glomerulopathy is characterized by

microtubule organization consisting of monoclonal Ig. More

than half of such patients have CLL or a small lymphoma

and rarely a low-grade plasma cell clone. In renal biopsy,

membranous nephropathy and the MPGN pattern are mostly

seen by light microscope and generally monocytic IgG, C3, and

LC positivity are seen in immunofluorescence. Similarly to other

fibrillary lesions, electron microscopic assessment is very critical

for the diagnosis [36,37,40,41].

Type I cryoglobulinemia arises from monoclonal immunoglobulin.

In several diseases in which the entire Ig is secreted, such as

MGUS, WM, or other B-cell lymphoid disorders, the cryoglobulins

can be detected. Renal involvement is more common in the

IgG type of cryoglobulinemia and about 30% of patients are

affected. Despite episodes of acute renal failure or nephrotic

syndrome, the essential process is a chronic glomerular injury.

285



Endocapillary glomerulonephritis or MPGN patterns are the

most common morphological findings with the hyaline-protein

thrombi in the glomerulus. Monoclonal heavy chains and LCs

(most frequently IgG-k) with complements (C3/C4/c1q) are

detected in immunofluorescence [42,43,44].

Proliferative glomerulonephritis with monoclonal IgG deposits is

a new entity that shows non-organized glomerular deposits

most commonly consisting of IgG3-k. Membranoproliferative/

endocapillary proliferative and atypical membranous are

the most commonly observed patterns by light microscope.

Unlike in other deposit diseases (Randall-type heavy chain

deposition disease/light and heavy chain deposition disease),

monoclonal protein consists of the entire Ig and is detected

only in glomeruli by immunofluorescence. Because the light

and immunofluorescence microscopic appearances resemble

immune complex glomerulonephritis, if immunoglobulin

subgroups are not examined by immunofluorescence routinely,

monoclonal accumulation may be missed by the pathologist. The

possibility of detection of the circulating monoclonal IgG3 is

low compared to IgG1/IgG2 and that is another challenge in

diagnosis [33,34,35,45,46,47,48].

C3 glomerulopathy with monoclonal gammopathy is a

different entity among the other MGRS lesions. Although

the manifestation of the disease is glomerulonephritis, there

are not any Ig deposits in the kidneys. C3 glomerulopathy

is characterized only by C3 deposits in the glomeruli by

immunofluorescence and it can be divided into two main

groups according to the ultrastructure of the deposit under an

electron microscope. One of them is C3 glomerulonephritis and

it is characterized by subendothelial/subepithelial or mesangial

granular deposits upon electron microscopy. The other is dense

deposit disease and it is characterized by “sausage-like” highdensity

deposits in GBM [49,50]. The dysregulation of the

alternative complement pathway is the main problem in both

diseases. Some patients with C3 glomerulopathy have plasma

cell dyscrasia and circulating monoclonal Ig. There are several

hypotheses about the relationship between C3 glomerulopathy

and monoclonal gammopathy [36]. This topic is not the

subject of this article; however, it is important to emphasize

that patients with the diagnosis of C3 glomerulopathy proven

by biopsy should be investigated by the clinician in terms of

monoclonal gammopathy (Figure 5).

Crystal-storing histiocytosis is a very rare disease associated with

MM or MGRS. Although several sites can be affected, including

the kidneys, perirenal adipose tissue, and the lungs, bone

marrow involvement is most typical. Regardless of the site of

involvement, the histiocytes with intralysosomal accumulation

of Ig as crystals are determined by light microscope and most of

the kappa LC cases are identified by DIF. We have no experience

in the kidneys for crystal-storing histiocytosis [51,52].

Treatment Approach

If the patient has a diagnosis of MM and has renal impairment,

the treatment is more standard. The immediate start of

antimyeloma therapy, dialysis (high cut-off hemodialysis),

supportive care, high fluid intake, and avoidance of nephrotoxic

agents are parts of standard of care. The value of plasma

exchange is controversial; some studies showed encouraging

results. These approaches may result in improvement of renal

disease. Independence from dialysis is an important prognostic

factor for survival. With the development of new agents such

as thalidomide, bortezomib, and lenalidomide, the results are

much better. Proteasome inhibitor (PI) (bortezomib)-based

regimens are cornerstones in this setting. For eligible patients,

autologous stem cell transplantation (ASCT) with 100-140 mg/

m 2 melphalan is feasible [7].

In settings without overt myeloma patients, the decision about

the necessity of anti-myeloma therapy may be difficult. However,

MGRS is associated with high morbidity due to renal disease and

sometimes systemic findings may occur based on the monoclonal

immunoglobulin. MGRS regroups all renal disorders caused by

monoclonal proteins secreted by a nonmalignant B-cell clone

(AL amyloidosis, cryoglobulinemia, MIDDs, Fanconi syndrome).

In AL amyloidosis, the decision for myeloma-like therapy can

be easily made, because the disease is usually systemic and has

clinical features like a malignant disease rather than benign

disorders. On the other hand, there are sufficient studies and

data in this area, because it is a more common disorder, and

standard care includes high-dose dexamethasone + melphalan

or bortezomib-based regimens and ASCT in eligible patients [7].

In MIDDs, hematologic responses are best achieved with ASCT

or PI-based therapies and are associated with improved renal

outcomes [53]. Renal transplantation is feasible for MGRS,

but to avoid recurrence after transplantation, control of the

responsible B-cell clone is important [7,54]. However, it is not

clear that small B-cell clones are truly curable; thus, the risk of

disease recurrence cannot be eliminated totally.

Early diagnosis is important; if treatment is begun while renal

functions are still preserved, long-term outcome results are

better. MGRS is a heterogeneous and relatively rare entity and

more collaborative studies and efforts of nephrologists and

hematologists are required to improve its management.

Important Aspects

- Paraprotein can cause injury to the kidneys independent of its

concentration.

- MGRS patients have a small B-cell clone and a low level of

circulating paraprotein. Therefore, serum electrophoresis may

not be sufficient and more detailed investigations may be

286



required to detect monoclonal protein, like immunofixation

and serum free LC assays, especially if the pathologist has any

suspicion.

- The suppression of nephrotoxic monoclonal proteins in MGRS

is very important for renal and also patient survival.

- Regular monitoring of the renal function, proteinuria, or

hematuria can be very helpful for the early detection of the

MGRS renal lesion with biopsy and the early effective treatment

of cases with the diagnosis of monoclonal gammopathy with or

without clinical importance for hematologists.

Authorship Contributions

Concept: Ş.M.Ü, H.Ö., S.S.; Design: Ş.M.Ü, H.Ö., S.S.; Data

Collection or Processing: Ş.M.Ü, H.Ö., S.S.; Analysis or

Interpretation: Ş.M.Ü, H.Ö., S.S.; Literature Search: Ş.M.Ü, H.Ö.,

S.S.; Writing: Ş.M.Ü, H.Ö., S.S.

Conflict of Interest: The authors of this paper have no conflicts

of interest, including specific financial interests, relationships,

and/or affiliations relevant to the subject matter or materials

included.

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288

COMMENTARY

DOI: 10.4274/tjh.2017.0296


Time to Cure Hairy Cell Leukemia

Tüylü Hücreli Löseminin Kür Edilme Zamanı

Ilana Levy 1 , Tamar Tadmor 2,3

1

Bnai Zion Medical Center, Internal Medicine B Department, Haifa, Israel

2

Bnai Zion Medical Center, Hematology Unit, Haifa, Israel

3

Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel

To the Editor,

Major advances in the treatment of hairy cell leukemia (HCL)

occurred during the 1980s when the purine analogs (PAs)

cladribine [1] and pentostatin [2] were introduced. These

agents dramatically altered the clinical course and outcome

of this disease, achieving a 10-year overall survival (OS) rate

of approximately 90%. However, in the last 30 years, first-line

therapy for patients with HCL and even second-line therapy have

not been substantially changed.

In the current issue of this journal, Öngören et al. [3] report a

retrospective analysis of 67 patients treated for classic HCL and

compare 3 different first-line treatment modalities. Among them,

31 patients received cladribine therapy, 19 received interferonalpha

(IFN-α), 16 underwent splenectomy, and 1 was treated

with rituximab monotherapy. Patients treated with a PA as firstline

therapy achieved the highest overall response rate (ORR)

and significantly longer progression-free survival (PFS) with the

lowest relapse rate, but had a similar OS rate when compared to

other treatment modalities. However, in terms of therapy-related

complications, there was a high rate of infections, which were

mostly bacterial, with the highest rate reported in the cladribinetreated

group. These results are in line with previous reports

indicating that IFN-α and splenectomy are much less frequently

used now.

Pentostatin and cladribine are both equally recommended as

first-line therapy, achieving equivalent efficacies. Both are

associated with low rates of relapse or refractory disease (R/R)

[4,5,6,7,8], indicating that HCL is potentially curable [5,7,8].

Nevertheless, HCL patients do relapse after PA therapy, and

the rate or timing of relapse is associated with both complete

remission (CR) and minimal residual disease (MRD) status [6].

Indeed, patients with HCL and MRD positivity have shorter

treatment-free intervals than those in CR after PA therapy [8].

Moreover, for each consequent relapse, the response rate to

retreatment with PA decreases [5,7].

Taking all the above into consideration, in the future we should

encourage the development of novel combinations or the use

of consolidation therapies after the first response with PA is

achieved, particularly in those patients who are MRD-positive, in

an attempt to achieve more durable responses.

Another disadvantage that should be carefully considered is the

high rate of PA toxicity, which includes bone marrow suppression

associated with neutropenia, lymphopenia, T-cell dysfunction,

or stem cell toxicity with the development of hypoplasia and

aplasia [5,6,9,10]. Similar complications were also described in

the study by Öngören et al. [3].

Novel agents with a lower toxicity profile are currently being

tested as alternative therapy or in combination with PA. Rituximab,

a monoclonal antibody, has been studied as monotherapy or in

combination with PA, both in the frontline setting or at relapse

[11]. Results are encouraging, and rituximab seems to be well

tolerated while side effects are quite rare.

Other agents are also being used now for R/R HCL patients. These

drugs are not used as frontline therapy but have a favorable

toxicity profile. These include recombinant immunotoxins

targeting CD22 (BL22, moxetumomab pasudox) [12] or the BRAF

inhibitor (vemurafenib) [4] and have shown positive results

in R/R disease [5]. Finally, the Bruton tyrosine kinase inhibitor

ibrutinib appears to shorten the survival of hairy cells and block



Address for Correspondence/Yazışma Adresi: Tamar TADMOR M.D.,

Bnai Zion Medical Center, Hematology Unit, Haifa, Israel

Phone : +972 483 594 07

E-mail : tamar.tadmor@b-zion.org.il ORCID-ID: orcid.org/0000-0002-3435-8612

Received/Geliş tarihi: August 07, 2017


289

Levy I and Tadmor T: Time to Cure Hairy Cell Leukemia


cell proliferation and intracellular signaling in vitro [13], and it

is effective in some preliminary reports of therapy in refractory

disease [14]. However, in this respect, due to the rarity of the

disease, data are still incomplete and mostly based on small

case series, retrospective studies, or phase I clinical trials

[12,13,14,15].

Based on the recently published HCL consensus by Grever et

al., [6] PAs remain the only first-line therapy for HCL, while

vemurafenib is recommended in some cases with uncontrolled

infection prior to therapy with PAs due to its ability to improve

low blood counts. However, this agent should be replaced by a

PA as soon as the infectious status is controlled. In regard to

relapsed disease, both vemurafenib monotherapy and rituximab

in combination with PA may be recommended, although

repeated PA therapy is preferred in patients who achieve

long first remissions of more than 60 months [6]. Other novel

therapies such as ibrutinib or immunotoxin conjugates are still

mostly used in clinical trials and are not included in current

guidelines.

In conclusion, as reported by Öngören et al. [3], PAs remain

the most effective treatment for classic HCL in terms of PFS

and ORR when compared to earlier therapies such as IFN-α

or splenectomy, and this approach has not been changed over

the past 30 years. Perhaps the time has arrived to challenge

this approach and improve the outcome of HCL by using PAs in

combination with some of the available biological agents, either

as frontline or consolidation therapy for patients with classical

HCL, in an attempt to cure this chronic neoplastic disorder.

Keywords: Hairy cell leukemia, Purine analogs, Cladribine,

Pentostatin

Anahtar Sözcükler: Tüylü hücreli lösemi, Pürin analogları,

Kladribin, Pentostatin


Surgical and Medical Practices: I.L., T.T.; Concept: T.T.; Design: T.T.

Data Collection or Processing: I.L., T.T.; Analysis or Interpretation

I.L., T.T.; Literature Search I.L., T.T.; Writing: I.L., T.T.




included.

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290

RESEARCH ARTICLE

DOI: 10.4274/tjh.2016.0443


Retrospective Evaluation of Hairy Cell Leukemia Patients Treated

with Three Different First-Line Treatment Modalities in the Last

Two Decades: A Single-Center Experience

Geçmiş İki Dekatta Üç Farklı Birinci Basamak Tedavisi Alan Tüylü Hücreli Lösemi

Hastalarının Geriye Dönük Değerlendirmesi: Tek Merkez Deneyimi

Şeniz Öngören 1 , Ahmet Emre Eşkazan 1 , Selin Berk 1 , Tuğrul Elverdi 1 , Ayşe Salihoğlu 1 , Muhlis Cem Ar 1 , Zafer Başlar 1 , Yıldız Aydın 1 ,

Nükhet Tüzüner 2 , Teoman Soysal 1

1

İstanbul University Cerrahpaşa Faculty of Medicine, Department of Hematology, İstanbul, Turkey

2

İstanbul University Cerrahpaşa Faculty of Medicine, Department of Pathology, İstanbul, Turkey

Abstract

Objective: In this study, we retrospectively analyzed the clinical

outcome, treatment responses, infectious complications, and survival

rates of 71 hairy cell leukemia (HCL) cases.

Materials and Methods: Sixty-seven patients received a first-line

treatment and 2-chlorodeoxyadenosine (cladribine-2-CdA) was

administered in 31 cases, 19 patients received interferon-alpha

(INF-α), splenectomy was performed in 16 cases, and rituximab was

used in one.

Results: Although the highest overall response rate (ORR) was

observed in patients receiving 2-CdA upfront, ORRs were comparable

in the 2-CdA, INF-α, and splenectomy subgroups. Relapse rates were

significantly lower in patients who received first-line 2-CdA. The

progression-free survival (PFS) rate with 2-CdA was significantly

higher than in patients with INF-α and splenectomy, but we found

similar overall survival rates with all three upfront treatment

modalities. Infections including tuberculosis were a major problem.

Conclusion: Although purine analogues have improved the ORRs and

PFS, there is still much progress to make with regard to overall survival

and relapsed/refractory disease in patients with HCL.

Keywords: Cladribine, Hairy cell leukemia, Interferon, Splenectomy

Öz

Amaç: Bu çalışmada, geriye dönük olarak 71 tüylü hücreli lösemi (SHL)

olgusunu klinik sonuçlar, tedavi yanıtları, enfeksiyon komplikasyonları

ve sağkalım oranları açısından analiz ettik.

Gereç ve Yöntemler: Altmış yedi hasta birinci basamak tedavi almıştı

ve 2-klorodeoksiadenozin (kladribin-2-CdA) 31 olguya uygulanmış, 19

hasta interferon-alfa (INF-α) almış, splenektomi 16 olguda uygulanmış

ve rituksimab ise bir hastada kullanılmıştı.

Bulgular: En yüksek toplam yanıt oranı (TYO) birinci basamak 2-CdA

alan hastalarda görülmüş olsa da, TYO’lar 2-CdA, INF-α ve splenektomi

alt gruplarında benzerdi. Nüks oranları birinci basamak 2-CdA alan

hastalarda anlamlı olarak daha azdı. Progresyonsuz sağkalım (PS) oranı

2-CdA alanlarda INF-α ve splenektomi hastalarına göre anlamlı olarak

daha yüksek olmakla birlikte, her üç birinci basamak tedavi yaklaşımı

ile toplam sağkalım (TS) oranları benzer olarak bulundu. Tüberkülozun

da dahil olduğu enfeksiyonlar önemli bir problemdi.

Sonuç: Her ne kadar pürin analogları TYO ve PS’yi iyileştirmiş olsa da,

SHL hastalarında TS ve nüks/dirençli hastalık açısından daha yapılması

gereken çok şey vardır.

Anahtar Sözcükler: Kladribin, Tüylü hücreli lösemi, İnterferon,

Splenektomi

Introduction

Hairy cell leukemia (HCL) is a rare mature B-cell neoplasm

characterized by the accumulation of atypical lymphocytes

with prominent cytoplasmic projections in the bone marrow

and spleen, resulting in pancytopenia and splenomegaly [1,2].

Most patients eventually require therapy owing to worsening

cytopenias, frequent and life-threatening infections, and/

or symptomatic splenomegaly. Several treatment modalities,

including splenectomy and immunotherapy with interferonalpha

(INF-α), were used with various clinical and hematologic



Address for Correspondence/Yazışma Adresi: Şeniz ÖNGÖREN, M.D.,

İstanbul University Cerrahpaşa Faculty of Medicine, Department of Hematology, İstanbul, Turkey

Phone : +90 532 334 16 64

E-mail : senizongoren@hotmail.com ORCID-ID: orcid.org/0000-0002-2809-5510

Received/Geliş tarihi: November 10, 2016

Accepted/Kabul tarihi: July 26, 2017

291

Öngören Ş, et al: First-Line Treatment in Patients with HCL


responses until the introduction of the purine nucleoside

analogues 2-chlorodeoxyadenosine (cladribine, 2-CdA) and

2′-deoxycoformycin (pentostatin) [3,4,5]. Purine analogues

have resulted in higher complete response (CR) rates and

durable remissions, and they have become the treatment of

choice in most cases [6]. Monoclonal antibodies (i.e. rituximab)

and immunotoxins are currently recommended for relapsed/

refractory (R/R) cases [7,8,9]. Also among the target-oriented

therapeutic options, the BRAF inhibitor vemurafenib can be

used in patients with R/R HCL [10,11].

In this study, we retrospectively analyzed the clinical outcome,

treatment responses, infectious complications, and survival rates

of HCL patients treated in our institution with three treatment

modalities (splenectomy, INF-α, and 2-CdA) as first-line therapy

between 1991 and 2014.

Materials and Methods

Patients

A total of 71 patients with HCL, who were diagnosed and

followed in our clinic over the past 20 years, were included in this

study. Diagnosis of HCL was established by morphological, flowcytometric,

and immunohistochemical analysis of peripheral

blood, bone marrow, and/or spleen specimens. Information on

the patients’ characteristics, presenting signs and symptoms,

treatment modalities and outcomes, and infections were

retrospectively taken from the patients’ files. While defining the

patient cohort, we excluded cases with variant HCL.

Treatment Modalities

Patients were divided into 3 subgroups according to the

first-line treatments (i.e. splenectomy, INF-α, and 2-CdA)

that they had received. Splenectomy was performed either

laparoscopically or via open surgery. 2-CdA was given either

by continuous intravenous infusion at a dose of 0.1 mg/kg/

day over 7 consecutive days, or by 2-h intravenous infusion

at a dose of 0.1 mg/kg once a week for 7 consecutive weeks,

depending on whether the patients received it as an inpatient or

outpatient treatment, respectively. While on 2-CdA, the patients

were given cotrimoxazole prophylaxis against Pneumocystis

jirovecii pneumonia. INF-α was administered subcutaneously at

a starting dose of 3 MU 3 times a week and maintained with

subsequent toxicity-based dose adjustments. Rituximab was

administered at the conventional dose of 375 mg/m 2 weekly for

4 consecutive weeks, as suggested before [7].

Definition of Response and Survival

Response to treatment was assessed using the criteria described

in the consensus resolution of 1987 [12]. Accordingly, CR was

defined as the morphological absence of hairy cells (HCs)

in the blood and the bone marrow in addition to complete

disappearance of hepatosplenomegaly and cytopenias.

Normalization of peripheral blood counts together with an at

least 50% reduction in the size of organomegaly and the volume

of bone marrow HCs, plus <5% circulating HCs, was designated

as partial response (PR). Presence of CR or PR was defined as

overall response (OR), and any response other than a CR or PR

was considered as no response. During response evaluation, a

bone marrow biopsy was performed 3 months after finishing

2-CdA treatment. Furthermore, relapse after CR was defined as

the reappearance of HCs in the peripheral blood or bone marrow,

development of cytopenias, and/or splenomegaly on physical

examination. Relapse after PR indicated a >50% increase of

residual disease.

Overall survival (OS) denotes the time from the first treatment

until the time of death or last follow-up. The duration of

progression-free survival (PFS) was calculated from the onset

of any first-line treatment until the date of progression. Time to

next treatment (TTNT) was calculated as the time from the end

of the previous treatment to the institution of the next therapy.

Statistical Analysis

Student’s t-test was used for the comparison of the quantitative

variables. Qualitative variables between groups were compared

using the chi-square test. The Kaplan-Meier method was used

for survival analysis [13]. Survival rates were compared by

using the log-rank test. All tests were two-sided, and p<0.05

was considered as statistically significant. All analyses were

performed with SPSS 13.0 for Windows (SPSS Inc., Chicago, IL,

USA).

Results

Sixty-two patients (87%) were male and the median age was 49

years (range: 31-76 years). There were 53 patients (75%) with

splenomegaly, and the numbers of patients with hepatomegaly

and lymphadenopathy were 32 (45%) and 27 (38%), respectively.

The demographic features of the entire cohort including median

leukocyte and platelet counts and hemoglobin levels at diagnosis

are displayed in Table 1. The median duration of follow-up was

57 months (range: 1-217 months). At diagnosis, all patients

had bone marrow biopsy, and flow cytometric evaluation was

performed for 47 patients (66%) from peripheral blood and/

or bone marrow. At diagnosis, hematoxylin and eosin staining,

reticulin staining, tartrate-resistant acid phosphatase staining,

and immunohistochemistry for CD20 and annexin A1 were

performed from the bone marrow aspiration and trephine biopsy.

Treatments and Outcomes

Among the patient cohort, there were 4 patients who did not

receive treatment. Two of them were lost to follow-up, one

patient died due to severe infection, and one had an acute

myocardial infarction (AMI) and died before any treatment was

292



initiated (Figure 1).

First-Line Treatment

Sixty-seven (94%) patients received a first-line treatment.

2-CdA was administered for 31 patients (46%), 19 patients

(28%) received INF-α, splenectomy was performed in 16 cases

(24%), and rituximab was used in one case (2%) (Figure 1).

Although patients in the splenectomy arm were younger than

those in the other 2 arms, the differences were not statistically

significant (Table 2). The 3 treatment subgroups were equally

balanced regarding sex distribution, median hemoglobin levels,

and leukocyte and platelet counts (Table 2). There were 30, 16,

and 13 patients who achieved OR after the first-line treatment

with 2-CdA, INF-α, and splenectomy, respectively (Figure 1). The

OR rates (ORRs) in the 2-CdA, INF-α, and splenectomy subgroups

were 97%, 84%, and 81%, respectively, and although the ORR

of 2-CdA treatment was superior to those of the other 2 groups,

the differences were not significant (Figure 2). During the firstline

treatment, 2 patients (one on 2-CdA and one on INF-α) died

due to infection (Figure 1). No postoperative complications were

observed in patients with splenectomy.

Five of the 31 patients who received 2-CdA as the first-line

therapy required a second-line treatment after a median TTNT

of 23 months (range: 3-58 months) (Table 2). There were 10

patients in the first-line INF-α subgroup who progressed and

needed a second-line treatment following a median TTNT of 21

months (range: 1-96 months). Eleven of the 16 patients who

underwent first-line splenectomy received a second-line therapy

due to relapsed disease at a median TTNT of 5 months (range:

2-73 months). Relapse rates were significantly lower in patients

who received first-line 2-CdA than those who were treated with

INF-α or splenectomy (p=0.007 and p<0.0001, respectively).

However, this was not significantly different when the INF-α

and splenectomy subgroups were compared (p=0.339) (Table 2).

Although the patients with first-line splenectomy had a shorter

median TTNT than those with 2-CdA and INF-α, the difference

did not reach statistical significance (Table 2). The only patient

who received rituximab as frontline treatment remained

refractory to that therapy, and she needed further treatment.

Second-Line Treatment

Figure 1. The distribution of first-line treatment modalities and

outcomes.

2-CdA: Cladribine, HCL: hairy cell leukemia, INF-α: interferon-alpha, RTX:

rituximab, SPL: splenectomy, ORR: overall response rate, NRR: non-response rate.

*See text for details.

Table 1. The baseline characteristics of the patients.

Parameter

Entire cohort

(n=71)

Sex - male/female, n (%) 62 (87)/9 (13)

Median age, years (min-max) 49 (31-76)

Median leukocyte count, x10 9 /L (min-max) 3.0 (0.5-9.4)

Median hemoglobin level, g/dL (min-max) 10 (4-17)

With a median TTNT of 13 months (range: 1-96 months), 27

patients required a second-line treatment due to R/R disease.

Twelve patients received INF-α, 11 patients were treated with

2-CdA, 3 patients underwent splenectomy, and one received

fludarabine.

Eight and 2 of the 12 patients who received second-line

INF-α had been initially treated with splenectomy and 2-CdA,

Median platelet count, x10 9 /L (min-max) 51.5 (21-124)

Splenomegaly - yes/no, n (%)

Mean spleen size, cm (min-max)*

53 (75)/18 (25)

11.4 (2-26)

Hepatomegaly - yes/no, n (%) 32 (45)/39 (55)

Lymphadenopathy - yes/no, n (%) 27 (38)/44 (62)

Median follow-up duration,

months (min-max)

57 (1-217)

*Spleen size was measured by physical examination and stated as the distance from

the left costal margin in cm.

Figure 2. Response rates according to the different first-line

treatment modalities.

*2-CdA vs. INF-α; **2-CdA vs. splenectomy; ***INF-α vs. splenectomy.

ORR: Overall response rate, NRR: non-response rate, 2-CdA: cladribine, INF-α:

interferon-alpha.

293



respectively. In the remaining 2 patients the previous treatment

was also INF-α. The ORR was 100% in patients who received

second-line INF-α, with only 3 patients requiring a third-line

treatment due to relapsed disease.

In patients who had 2-CdA as a second-line treatment, 7 had

received INF-α as the first-line therapy, and 2 had splenectomy.

The other 2 had been treated with rituximab and 2-CdA previously.

All the patients responded to second-line 2-CdA (the ORR was

100%), and only one relapsed after a follow-up of 48 months.

Splenectomy was performed in 3 patients as a second-line

treatment. All of them initially responded to splenectomy;

however, 2 needed further treatment due to relapsed disease.

The patient who received fludarabine as a second-line therapy

achieved PR but was then lost to follow-up after 9 months.

Third-Line Treatment

There were 6 patients who needed third-line treatment. INF-α,

2-CdA, and rituximab were used for 3, 2, and one of them,

respectively, following a median TTNT of 36 months (range:

8-63 months). Among patients receiving third-line INF-α, one

achieved and maintained CR throughout the entire follow-up.

The other 2 remained refractory to INF-α treatment; one died,

and the other patient proceeded to fourth-line treatment. One

of the 2 patients who received 2-CdA as a third-line treatment

achieved and maintained CR and the other patient died due to

refractory disease.

The patient who received third-line weekly rituximab could not

complete the 4 th week of treatment due to an allergic reaction,

which had happened following the 3 rd dose of the drug, and had

to proceed to fourth-line therapy.

Table 2. Patient characteristics and treatment outcomes in patients with 3 first-line treatment options.

First-Line Treatment (n=67) †

Parameter

2-CdA

INF-α

Splenectomy

p value

(n=31)

(n=19)

(n=16)

Sex - male/female, n (%) 27 (87)/4 (13) 18 (95)/1 (5) 14 (88)/2 (12) 0.387*

0.969**

0.453***

Median age, years (min-max) 52 (33-75) 50 (33-76) 47.5 (31-64) 0.564*

0.094**

0.061***

Median leukocyte count, x10 9 /L (min-max) 3.1 (1.6-7.2) 3.0 (0.5-9.4) 3.4 (0.5-9.1) 0.601*

0.510**

0.392***

Median hemoglobin level, g/dL (min-max) 11 (5-17) 10 (4-14) 9 (5-15) 0.098*

0.053**

0.721***

Median platelet count, x10 9 /L (min-max) 62 (22-108) 42.5 (21-124) 56 (29-81) 0.624*

0.552**

0.888***

Patients needing further treatment(s) due to R/R disease, n (%) 5 (16) 10 (53) 11 (69) 0.007*

<0.0001**

0.339***

Median TTNT, months (min-max) 23 (3-58) 21 (1-96) 5 (2-73) 0.902*

0.370**

0.190***

Median follow-up, months (min-max) 53 (5-213) 57 (2-154) 83 (15-217) 0.739*

0.120**

0.230***

†

One patient received rituximab as a first-line treatment, *2-CdA vs. INF-α; **2-CdA vs. splenectomy; ***INF-α vs. splenectomy.

R/R: Relapsed/refractory, TTNT: time to next treatment, 2-CdA: cladribine.

294



Fourth-Line Treatment

There were only 2 patients who received a fourth-line

treatment. One of them was treated with INF-α and the other

with rituximab. The patient who received INF-α died due to

refractory disease, and the patient who was given rituximab

achieved CR.

Infections

In 47 patients, 76 infectious episodes were noted during the

entire follow-up period (Table 3). Bacterial infections were the

leading cause, and 64 bacterial infection episodes were observed.

Of these 65 episodes, 14 were observed after diagnosis prior to

the initiation of any anti-HCL treatment, whereas 50 episodes

were noted during treatment (Table 3). The infections were

diagnosed by means of cultures, radiological imaging techniques,

and tissue biopsies as indicated. There were 18 patients who

required hospitalization (mostly due to neutropenic fever), and

in 17 cases infections occurred within 30 days after completion

of treatment (all patients received 2-CdA).

There were 7 infection episodes caused by Mycobacterium

tuberculosis in 6 patients (5 pulmonary cases, 2 disseminated).

In 2 patients tuberculosis was diagnosed synchronously with

HCL, and 5 episodes occurred during/after anti-HCL treatment.

In patients for whom tuberculosis was diagnosed prior to

treatment initiation, one had pulmonary tuberculosis and he

experienced disseminated disease 3 months after completing

2-CdA. The other patient had disseminated tuberculosis at

diagnosis, which was diagnosed via splenectomy. That patient

then received INF-α due to relapse without any recurrence

of tuberculosis. None of the patients with tuberculosis had

drug-resistant disease, and none of them had any known

comorbidities including diabetes. There were also 2 patients

with invasive pulmonary aspergillosis (IPA).

Six of the 10 episodes of viral infections were related to flu. One

of these patients had influenza A virus subtype H1N1 infection

(swine flu) and had to be followed in the intensive care unit.

Overall, 3 patients died due to infection, and one of them died

because of a febrile neutropenic episode prior to treatment

initiation. In the remaining 2 cases, infections occurred during/

after treatment; IPA was the reason for death in one patient

receiving first-line INF-α and the other patient died due to

sepsis following frontline 2-CdA administration. The distribution

of the infections and outcomes are shown in Table 3.

Survival

During the follow-up, 10 patients (12%) died (7 due to refractory

disease and/or infections, 3 due to AMI and sudden cardiac

death) and 2 patients were lost to follow-up. PFS and OS rates

for the entire cohort after a median follow-up of 57 months

were 62% (Figure 3A) and 83% (Figure 3B), respectively. With

regard to the first-line treatment, the PFS rate was significantly

higher for patients who received 2-CdA than those for patients

who were treated with INF-α and splenectomy (p=0.01 and

p<0.0001, respectively). However, PFS did not statistically differ

between patients who were treated with INF-α and those who

underwent splenectomy (p=0.213) (Figure 4A). There was no

statistically significant OS difference between these 3 treatment

modalities, although survival rates achieved with first-line

2-CdA and INF-α seemed to be superior to those achieved with

splenectomy (Figure 4B).

Figure 3. The progression-free survival (A) and overall survival (B) of the entire cohort.

2-CdA: Cladribine, INF-α: interferon-alpha.

295



Table 3. The distribution of infections among the patient cohort.

Type of infection

Viral (n=10)

Flu

Herpes labialis

HBV

Number of

events, n

6

2

1

Timing of infection, n Type of treatment* Outcome of

infection (resolved/

death), n

Prior to

treatment

initiation

5

0

0

During/after

treatment

1

2

1

2-CdA (n=1)

2-CdA (n=1)

IFN (n=1)

10/0

HCV

1

0

1

2-CdA (n=1)

IFN (n=1) [SPL, n=1]

Bacterial (n=64)

Pneumonia

13

7

6

2-CdA (n=4) [IFN, n=1]


13/0

Tuberculosis

7

2

5


2-CdA (n=2)

RTX (n=1)

7/0

Febrile neutropenia

21

3

18

2-CdA (n=15) [IFN, n=3; RTX, n=1]


RTX (n=1)

20/1

Soft tissue infection

6

1

5

2-CdA (n=1) [RTX, n=1]


6/0

Dental infection

3

0

3


2-CdA (n=2) [IFN, n=1]

3/0

Paronychia

2

0

2


2/0

Sepsis

4

0

4

2-CdA (n=3) [IFN, n=1]


3/1 (2-CdA)

Conjunctivitis

2

1

1

2-CdA (n=1)

2/0

Septic arthritis

1

0

1

IFN (n=1)

1/0

Meningitis

1

0

1

IFN (n=1)

1/0

Urinary tract infection

Splenic abscess

3

1

0

0

3

1

2-CdA (n=2) [RTX, n=1]

RTX (n=1)

IFN (n=1)

3/0

1/0

Fungal (n=2)

Invasive pulmonary

aspergillosis

2 0 2 IFN (n=1)

2-CdA (n=1)

2/1 (IFN)

*In this column, the number of patients who received the defined most recent therapies is shown in parentheses, whereas the number of patients with a previous anti-HCL therapy is

displayed in square brackets.

2-CdA: Cladribine, HBV: hepatitis B virus, HCV: hepatitis C virus, INF-α: interferon-alpha, SPL: splenectomy, HCL: hairy cell leukemia.

Discussion

In this study we retrospectively evaluated the demographic

features of patients with HCL and assessed the efficacy and

tolerability of the main first-line treatments together with the

subsequent therapy options and outcomes. We also identified

the infections associated with the course of the disease. HCL

occurs more frequently in males, and the median age of diagnosis

is 52 years [14]. Our patient cohort showed characteristics that

were similar to the previously reported literature with a median

age of 49 years and a male/female ratio of approximately 7:1.

In our cohort, we identified peripheral and/or visceral

lymphadenopathy in 27 patients (38%). In the most recent

296



consensus guidelines for the diagnosis/management of patients

with classic HCL, performing imaging studies for the detection of,

e.g., lymphadenopathy was optional [15]. It was recommended

that these procedures be reserved for patients in a clinical trial or

those with associated symptoms referable to these systems [15]. We

did not routinely perform imaging studies for all of our patients,

but rather only for patients with signs/symptoms for which

ultrasonography and/or computerized tomography scanning were

indicated. Furthermore, patients with HCL may develop infections

(e.g., tuberculosis) or secondary primary tumors [16], where

lymphadenopathy could be observed in the course of the disease.

Most probably, in some of the patients, these enlarged lymph nodes

are reactive or are due to other conditions, including infections.

After the introduction of the purine analogues, the treatment

algorithm of HCL evolved greatly [5]. Before that, splenectomy

and IFN-α were the mainstays of the treatment [3,4]. The

management of HCL in Turkey also changed from the first

years of the 2000s onwards with the availability of 2-CdA in

the country, and prior to that time, patients with HCL were

receiving mainly INF-α and splenectomy upfront. Thereafter,

2-CdA became the standard choice of treatment for most

patients with HCL. Earlier data indicate that patients who

were treated with first-line splenectomy were usually younger

than patients receiving IFN-α. Both splenectomy and IFN-α

have been associated with favorable clinical and hematologic

responses. However, the median survival with these treatment

modalities was approximately 4 years [3,4]. These earlier findings

were confirmed by our results indicating high but not durable

ORRs with first-line splenectomy and IFN-α. We observed

relapse rates as high as 70% and 50% in patients treated with

splenectomy and IFN-α, respectively.

With the upfront-usage of 2-CdA more responses have been

reported to be higher and durable. In line with the literature,

we only noted 16% R/R disease and a median treatment-free

period of 23 months in our patients treated upfront with 2-CdA.

This was quite similar to what was reported by Saven et al. [17],

demonstrating a relapse rate of 26% after a median follow-up of

29 months. However, with longer follow-up, relapse rates tended

to rise to 40% among patients who received 2-CdA upfront [18].

Twenty-seven patients of our cohort (41%) had to be given

a second-line treatment for R/R disease after a median TTNT

of approximately 1 year. This was consistent with the findings

of Zinzani et al. [19], who found that nearly 44% of patients

relapsed after a median of 2.7 years. In our cohort the median

TTNT was shorter than that observed in the cohort of Zinzani et

al. [19], and most probably the reason for this was the higher

percentage of upfront purine analogue usage (85/121, 70%)

among their patients than ours (31/66, 47%). As expected,

patients receiving first-line 2-CdA had significantly lower

relapse rates than those treated with INF-α and splenectomy.

Second-line 2-CdA treatment in our cohort of patients resulted

in excellent response rates (ORR=100%). Relapse was observed

in one patient only, who also received second-line 2-CdA. Retreating

relapsed patients with an additional course of purine

analogues is a reasonable option. Zinzani et al. [19] recommended

repeating the same treatment regimen with purine analogues

in relapse settings, although changing to a different purine

analogue might yield a better result. Unfortunately, 2-CdA is the

only approved and available purine analogue in Turkey, so we

did not have the opportunity of using other purine analogues

like pentostatin in patients who relapsed after 2-CdA.

Figure 4. The progression-free survival (A) and overall survival (B) when patients were divided into 3 groups according to the first-line

treatment.

*2-CdA vs. INF-α; **2-CdA vs. splenectomy; ***INF-α vs. splenectomy.

2-CdA: Cladribine, INF-α: interferon-alpha.

297



Although the PFS rate with 2-CdA was significantly higher than

those with INF-α and splenectomy, we found similar OS rates

with all three upfront treatment modalities. Most probably one

of the reasons for this is the relatively short follow-up duration

of our study. In addition to that, since most of the patients with

HCL may relapse during follow-up, sequentially re-challenging

the previous successful treatment(s) as well as the administration

of alternative effective agents might have a positive impact on

OS. In our cohort, switching to other potent therapies at relapse

could be another reasonable explanation for the comparable OS

rates between these 3 first-line treatment groups.

A median follow-up duration of 57 months might not be enough

to show OS benefit in patients with chronic leukemias such as

HCL. After four lines of treatments with a median follow-up

of approximately 6 years, 10 patients died and 2 were lost to

follow-up, giving an OS rate of 83%, which was consistent with

the OS rate of 87% that was reported in the article by Zinzani

et al. [19].

One of the most important clinical problems in patients

with HCL is the development of severe and sometimes lifethreatening

infections [20]. Gram-positive and gram-negative

organisms, Aspergillus, and other fungi are the most common

pathogens [21], but tuberculosis [22] and herpes zoster [23] can

be observed, as well. In our patient cohort, by far bacterial and

fungal infections were the most common, and 3 patients died

due to severe bacterial infection, sepsis, and IPA. We also had 6

patients with 7 episodes of tuberculosis infection. Two patients

had tuberculosis and HCL at diagnosis, and 5 episodes occurred

during/after anti-HCL treatment. Tuberculosis is an important

issue since it can lead to the misdiagnosis of patients for HCL

relapse [22]. Thus, when a patient with HCL presents with fever,

tuberculosis should always be kept in mind, especially where

tuberculosis is endemic. We had 2 patients with herpes labialis

and 1 patient with influenza A virus subtype H1N1 infection,

which has also been documented in earlier reports on patients

with HCL [24].

Conclusion

In conclusion, with the introduction of the purine analogues,

the treatment of HCL has been greatly changed. Among our

patient cohort, although ORRs were comparable for first-line

2-CdA, INF-α, and splenectomy, patients with frontline 2-CdA

had superior ORRs with more durable responses with a higher

PFS rate than splenectomy and INF-α cases. The OS rate of

the entire cohort was consistent with the current literature.

Infections including tuberculosis were a major problem, which

caused morbidity and mortality.

Although purine analogues improved the CR rates and PFS,

there is still much progress to be made with regard to OS and

R/R disease. In that sense, the addition of monoclonal antibodies

to purine analogues or incorporation of new target-oriented

therapeutic agents such as BRAF, Bruton’s tyrosine kinase, or

phosphoinositide 3-kinase inhibitors into treatment regimens

might help change the prognosis of the disease further,

especially for younger patients and for those who would poorly

tolerate the current therapy options.

Ethics

Ethics Committee Approval: Since the study has a retrospective

design, Ethics Committee approval is not needed.

Informed Consent: Not applicable.


Surgical and Medical Practices: Ş.Ö., A.E.E., S.B., T.E., A.S.,

M.C.A., Z.B., Y.A., N.T., T.S.; Concept: Ş.Ö., A.E.E.; Design: Ş.Ö.,

A.E.E.; Data Collection or Processing: Ş.Ö., A.E.E., S.B.; Analysis

or Interpretation: Ş.Ö., A.E.E.; Literature Search: Ş.Ö., A.E.E.;

Writing: Ş.Ö., A.E.E.




included.

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299

RESEARCH ARTICLE

DOI: 10.4274/tjh.2016.0489


FMS-Like Tyrosine Kinase 3 (FLT3) and Nucleophosmin 1

(NPM1) in Iranian Adult Acute Myeloid Leukemia Patients with

Normal Karyotypes: Mutation Status and Clinical and Laboratory

Characteristics

Normal Karyotipli İran’lı Erişkin Akut Miyeloid Lösemi Hastalarında FMS-Benzeri Tirozin

Kinaz 3 (FLT3) ve Nükleofosmin 1 (NPM1): Mutasyon Durumu ile Klinik ve Laboratuvar

Karakteristikleri

Narges Rezaei 1 , Nargess Arandi 1 , Behnaz Valibeigi 2 , Sezaneh Haghpanah 1 , Mehdi Khansalar 3 , Mani Ramzi 1

1

Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

2

Department of Pathology, Faculty of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

3

Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran

Abstract

Objective: In this study, we evaluated the frequency of FMS-like

tyrosine kinase 3 (FLT3-ITD and FLT3-TKD) and nucleophosmin (NPM1)

mutations in Iranian patients with cytogenetically normal acute

myeloid leukemia (CN-AML). The clinical and laboratory characteristics

were compared between wild-type and mutant cases.

Materials and Methods: Seventy newly diagnosed de novo AML

patients were recruited at the time of diagnosis prior to chemotherapy;

among them, 54 had CN-AML. For detecting mutations, the FLT3 and

NPM1 genes were amplified by the polymerase chain reaction method,

followed by direct sequencing.

Results: Our results showed that the frequencies of FLT3-ITD, FLT3-

TKD, and NPM1 mutations in CN-AML patients were 25.9%, 5.9%, and

20.8%, respectively. The most frequent NPM1 mutation type was the

type A mutation. The FLT3-ITD mutation was seen more frequently

in non-M3 patients compared with M3 patients. No mutation was

observed in either the FLT3-TKD or the NPM1 gene in patients in

the M3 French-American-British group. There was no significant

association between the presence of FLT3-ITD and NPM1 mutations in

CN-AML patients (p>0.05). The frequency of FLT3-ITD, FLT3-TKD, and

NPM1 mutation was higher in CN-AML patients in comparison with

AML patients with cytogenetic aberrations, although the differences

were not statistically significant (p>0.05). There were no significant

differences in mean white blood cell and platelet counts, serum

hemoglobin levels, and bone marrow blast percentages between

patients with wild-type and mutant FLT3-ITD and NPM1 genes

Öz

Öz

Amaç: Bu çalışmada, İran’lı normal sitogenetikli akut miyeloid

lösemi (NS-AML) hastalarında FMS-benzeri tirozin kinaz 3 (FLT3-ITD

ve FLT3-TKD) ile nükleofosmin 1 (NPM1) mutasyonlarının sıklığını

değerlendirdik. Mutant olmayan (yabanıl-wild-type) ve mutant

olgular klinik ve laboratuvar özellikler açısından mukayese edildi.

Gereç ve Yöntemler: Yetmiş yeni tanı de novo AML hastası kemoterapi

uygulanması öncesinde çalısmaya dahil edildi; bunların 54’ü NS-AML idi.

Mutasyonları tespit etmek için, FLT3 ve NPM1 genleri polimeraz zincir

reaksiyonu ile amplifiye edildi ve bu işlemi direkt dizileme takip etti.

Bulgular: NS-AML hastalarında FLT3-ITD, FLT3-TKD ve NPM1

mutasyonlarının sıklıkları sırasıyla %25,9; %5,9 ve %20,8 olarak

bulunmuştur. En sık gözlenen NPM1 mutasyon tipi, tip A mutasyonuydu.

FLT3-ITD mutasyonu M3 hastalarına göre M3-dışı olgularda daha sık

görülmekteydi. Fransız-Amerikan-İngiliz M3 grubundaki hastalarda

FLT3-TKD veya NPM1 genine ait mutasyon tespit edilmedi. NS-AML

hastalarında FLT3-ITD ve NPM1 mutasyonlarının varlığı açısından

anlamlı ilişki yoktu (p>0,05). FLT3-ITD, FLT3-TKD ve NPM1 mutasyon

sıklığı, her ne kadar istatistiksel olarak anlamlı farklılık saptanmasa

da (p>0,05), NS-AML hastalarında sitogenetik aberasyonu olan AML

olgularına göre daha fazlaydı. FLT3-ITD ve NPM1 genleri açısından

mutant olan ve olmayan hastalarda ortalama lökosit ve trombosit

sayıları, serum hemoglobin düzeyleri ve kemik iliği blast yüzdeleri

arasında anlamlı farklılık yoktu (p>0,05). Yaş ve cinsiyete göre FLT3-

ITD veya NPM1 mutasyonlarının sıklıkları açısından farklılık tespit

edilmedi (p>0,05).

Address for Correspondence/Yazışma Adresi: Nargess ARANDI, M.D.,

Hematology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Phone : +98 713 612 22 63

E-mail : arandin@sums.ac.ir ORCID-ID: orcid.org/0000-0001-6489-0979

Received/Geliş tarihi: December 20, 2016

Accepted/Kabul tarihi: March 10, 2017

300


Rezaei N, et al: FLT3 and NPM1 Mutation in Iranian CN-AML Patients

(p>0.05). No difference was observed in the frequency of FLT3-ITD or

NPM1 mutation regarding age or sex (p>0.05).

Conclusion: Given the high stability of NPM1 during the disease

course, it can be used in combination with FLT3 as well as other known

genetic markers to monitor patients, especially for minimal residual

disease detection.

Keywords: Acute myeloid leukemia, Gene mutation, FLT3, NPM1

Sonuç: NPM1 hastalık sürecindeki yüksek kararlılığı nedeniyle,

özellikle minimal kalıntı hastalık tespiti açısından FLT3 veya diğer

bilinen genetik belirteçler ile kombine olarak hastaların izlenmesinde

kullanılabilir.

Anahtar Sözcükler: Akut miyeloid lösemi, Gen mutasyonu, FLT3,

NPM1

Introduction

Acute myeloid leukemia (AML) is the most common hematologic

malignancy, characterized by uncontrolled proliferation of

hematopoietic stem cells resulting in abnormal accumulation

of myeloblasts [1]. Generally, based on the cytogenetic

abnormalities, the prognosis of AML patients is categorized into

three risk groups: good, intermediate, and poor [2]. However,

about 50% of AML patients have the normal cytogenetic feature

(CN-AML), which represents a diverse subset of patients who are

usually classified into an intermediate risk group [3]. Recently,

assessment of molecular abnormalities has proven to be a useful

marker for risk stratification of these patients into good and poor

risk subgroups [3,4,5,6]. In this regard, somatic mutations of the

FMS-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1), and

Wilms’ tumor 1 (WT1) genes have been well studied [3,7,8,9].

FLT3 is a member of the class III receptor tyrosine kinase (RTK)

family, normally expressed in early bone marrow precursors and

playing an important role in the regulation of hematopoietic cell

proliferation and differentiation [10]. Binding of the FLT3 ligand

to its receptor recruits and activates several signaling molecules

affecting cell proliferation, differentiation, and survival [11]. The

FLT3 receptor consists of five extracellular immunoglobulin-like

domains (Ig1-Ig5), a transmembrane domain, a juxtamembrane

domain (JM), and the two intracellular tyrosine kinase domains

(TK1 and TK2) [12,13,14]. FLT3 is one of the most frequently

mutated genes as approximately 30% of all AML patients have a

mutated form of it [15]. Two types of activating mutations have

been identified in the FLT3 gene: internal tandem duplication

(FLT3-ITD) of the region between exon 11 and 12 in the JM

domain (occurring in 20%-25% of AML patients), and a point

mutation at codon 835 of exon 17 in the TK domain (FLT3-TKD,

also known as D835Y, and occurring in 5%-7% of AML patients)

[8,16]. Both mutations contribute to constitutive activation

of the FLT3 receptor [8]. It has been shown that the FLT3-ITD

mutation has an inverse correlation with patient survival and

can be used as an important poor prognostic factor to predict

clinical outcomes in AML patients, especially those with normal

karyotypes. However, data on the correlation between FLT3-TKD

and AML disease outcome are highly limited [3,4,7,17].

The nucleophosmin gene encodes the NPM1 protein, which

functions as a chaperone that shuttles between the nucleus

and cytoplasm [3,5,7,8]. NPM1 regulates different intracellular

processes such as transport of preribosomal particles, responses

to stress stimuli, DNA repair, centromere duplications, and the

activity and stability of tumor suppressor genes like p53 [3].

Mutation within exon 12 of the NPM1 gene, which is the most

frequent mutation in AML patients (about 35% in all adult AML

patients and 50%-60% of CN-AML cases), results in abnormal

expression and localization of the protein within the cytoplasm

[3]. The most common NPM1 mutation (type A mutation,

occurring in 75%-80% of cases) is the insertion of the TCTG

tetranucleotide at position 956-959 in exon 12, but other less

common mutations in exon 12 have also been described [18,19].

There are various reports describing that NPM1 mutation is

mostly associated with FLT3-ITD mutation and it has been shown

that NPM1 can be considered a favorable prognostic marker in

the absence of FLT3-ITD mutation [3,4,7,17].

Accordingly, in this study, FLT3 and NPM1 mutations were

evaluated in adult Iranian patients with de novo CN-AML and

its correlations with clinical and laboratory parameters were

also assessed.


Patient Selection

This study included 70 newly diagnosed adult patients with de

novo AML who were referred to the Shiraz Namazi Hospital,

affiliated to Shiraz University of Medical Sciences, from

November 2014 to May 2016. All patients were recruited at the

time of diagnosis prior to chemotherapy. AML was diagnosed

using morphology, cytochemistry, and immunophenotyping.

Clinical and laboratory data, including French-American-British

(FAB) subclass, complete blood count, blast percentage, and

hemoglobin (Hb) level, were also collected.

All patients received standard induction chemotherapy, which

consisted of daunorubicin at 45 mg/m 2 on days 1 to 3 and

cytarabine at 100-200 mg/m 2 on days 1 to 7, followed by high

doses of a cytarabine-based consolidation phase (cytarabine

at mg/m 2 3 every 12 h for 3 days, repeated for 2 to 3 cycles).

This study was approved by the Ethics Committee of Shiraz

University of Medical Sciences and written informed consent

was obtained from all the participants.

301

Rezaei N, et al: FLT3 and NPM1 Mutation in Iranian CN-AML Patients Turk J Hematol 2017;34:300-306

Cytogenetic Analysis

Karyotypes were analyzed by standard G-banding technique

[20]. Chromosomal abnormalities were tested by reverse

transcriptase polymerase chain reaction (PCR) for AML1-ETO

and CBFB-MYH11. Among the 70 AML patients, 16 had abnormal

karyotypes: one patient had inv (16) translocation, one had

both t (8;21) and inv (16), 12 had t (15;17), and the remaining

two patients had other translocations. The 54 patients who were

negative for these chromosomal abnormalities were considered

as having CN-AML.

Sample Collection

Five milliliters of fresh peripheral blood and/or bone marrow

samples was collected in ethylenediaminetetraacetic acidcontaining

tubes. DNA was extracted with a DNA extraction kit

(GeNet Bio, Korea) and stored at -80 °C.

Detection of the FLT3-ITD Mutation

For detection of the FLT3-ITD mutation, the JM domain between

exons 11 and 12 was amplified using specific forward primer

FLT.11F 5’-GCAATTTAGGTATGAAAGCCAGC 3’ and reverse primer

FLT.12R 5’-CTTTCAGCATTTTGACGGCAACC-3’. The PCR reaction

was performed in a total volume of 50 µL containing 200 ng

of genomic DNA, 10X PCR buffer (100 mM Tris-HCl, pH 8.8, 500

mM KCl), 2 mM MgCl 2

, 200 µM dNTPs, 10 pM of each primer,

and 1 U of Taq DNA polymerase. PCR conditions included initial

denaturation at 95 °C for 5 min followed by 30 cycles of 94

°C for 30 s, 56 °C for 30 s, and 72 °C for 45 s with a final

extension at 72 °C for 5 min. PCR reaction was conducted in a

PCR T100 thermocycler (Applied Biosystems, USA). The 329-bp

PCR products were run on 3% agarose gel stained with DNA

SafeStain Dye and visualized under UV light. Samples with

additional longer PCR products were identified as FLT3-ITD+. All

mutant samples were verified by direct sequencing using the

ABI Prism 3730XL DNA sequencing analyzer. The sequencing

results were analyzed by Chromas software (version 2.4.3).

Detecting of the FLT3-TKD Mutation

For detection of the FLT3-TKD mutation, the specific forward

primer FLT.17F 5’-CCGCCAGGAACGTGCTTG-3’ and reverse

primer FLT.17R 5’-GCAGCCTCACATTGCCCC-3’ were used.

The PCR reaction was performed in a total volume of 15 µL

with similar reagents as used for the FLT3-ITD mutation,

except for the primers. PCR conditions were also the same,

except for the annealing temperature, which was 65 °C for

30 s. The amplification reaction was conducted in a PCR T100

thermocycler (Applied Biosystems). The 119-bp PCR products

were then digested with 2 U of EcoRV at 37 °C for 17 h, run on

3% agarose gel stained with DNA SafeStain Dye, and visualized

under UV light. The presence of an undigested PCR product was

an indication of a mutant sample.

Detection of the NPM1 Mutation

Exon 12 of the NPM1 gene was amplified using specific primer

NPM1-F 5’-TTAACTCTCTGGTGGTAGAATGAA-3’ and NPM1-R

5’-CAAGACTATTTGCCATTCCTAAC-3’. The PCR reaction was

performed in a similar volume as was used for the FLT3-ITD

mutation. PCR conditions included initial denaturation at 95 °C

for 5 min followed by 30 cycles of 94 °C for 30 s, 57 °C for 60

s, and 72 °C for 75 s with final extension at 72 °C for 5 min.

The PCR products were purified and directly sequenced with

reverse primer NPM1-R2 5’-GGCATTTTGGACAACACA-3’ using

the ABI Prism 3730XL DNA sequencing analyzer and analyzed

by Chromas software (version 2.4.3).


The statistical analysis of data was done using SPSS 18 (SPSS

Inc., USA). For comparison of qualitative data between wildtype

and mutant patients, chi-square and Fisher exact tests were

performed. Independent sample t-tests and Mann-Whitney U

tests were used to compare quantitative data between wildtype

and mutant patients. A p-value of less than 0.05 was

considered statistically significant.

Results

This study included 70 newly diagnosed adult patients with de

novo AML (49 males and 21 females, mean age: 47.73±18.64

years, minimum - maximum: 17-87 years). The demographic

and laboratory data of all the patients are shown in Table 1.

Screening for the Mutation of the FLT3 and NPM1 Genes in

CN-AML

The chromatograms of FLT3-ITD and NPM1 sequencing are

shown in Figure 1.

Of all 54 CN-AML patients, 14 (25.9%) had the FLT3-ITD

mutation, while 40 (74.1%) had the normal FLT3 gene. In

Table 1. Demographic and laboratory data of acute myeloid

leukemia patients.

Variables

Sex, number

Male (%)

Female (%)

Values

49 (70%)

21 (30%)

Age, years, mean ± SD 47.73±18.64

Laboratory data, median (minimum - maximum)

WBC count (x10 3 /mL) 9 (0.3-164.6)

Platelet count (x10 3 /mL) 49 (7-300)

Serum Hb (g/dL) 8.6 (4.4-13.4)

Blast count (%) 90 (50-99)

WBC: White blood cells, Hb: hemoglobin, SD: standard deviation.

302



addition, of the 52 patients genotyped for FLT3-TKD mutation

status, 3 (5.9%) were mutant and 48 (94.1%) were normal. One

patient had both FLT3-ITD and FLT3-TKD mutations.

Of the 53 CN-AML patients genotyped for the NPM1 gene, 11

(20.8%) had NPM1 mutation and 42 (79.2%) had wild-type

NPM1. From the 11 patients with mutant NPM1, 8 (72.8%) had

type A, 1 (9.1%) had type C, and 1 (9.1%) had type D mutation.

One patient (AML-20) had a unique mutation pattern that did

not belong to a typical NPM1 mutation type. Of 11 patients

with mutated NPM1, 5 (45.5%) were also positive for FLT3-

ITD, while none had FLT3-TKD mutation. Thirty-three patients

had the wild-type form of both the FLT3-ITD and NPM1 genes.

There was no significant correlation between the presence of

the FLT3-ITD mutation and NPM1 mutation in CN-AML patients

(p>0.05).

FLT3 and NPM1 Mutations and Different Clinical and Laboratory

Parameters in CN-AML

The mean white blood cell (WBC) and platelet counts, serum

Hb level, and percentage of blasts in the bone marrow were

compared between mutant and wild-type groups of CN-AML

patients (Table 2).

As shown in Table 2, there were no significant differences in

mean WBC and platelet counts, serum Hb level, or percentage

of blasts in the bone marrow between patients with wild-type

and mutant FLT3-ITD and NPM1 genes. Moreover, the mean age

of AML patients did not differ between wild-type and mutant

patients for the FLT3-ITD and NPM1 mutations (p=0.287 and

p=0.387, respectively). No significant differences were observed

between male and female patients in cases of FLT3-ITD and

NPM1 mutation frequency (p=0.450 and p=0.545, respectively).

FLT3 and NPM1 Mutation in AML Patients with Different FAB

Groups and Cytogenetic Aberrations

Of 70 de novo AML patients, 17 had FLT3-ITD, 3 had FLT3-TKD,

and 12 had NPM1 mutations. The frequencies of these mutations

in patients with different cytogenetic abnormalities are shown

in Table 3. Although the frequency of FLT3-ITD, FLT3-TKD, and

NPM1 mutation was higher in CN-AML patients in comparison

with AML patients with cytogenetic aberrations, the differences

were not statistically significant (p>0.05, data not shown).

Figure 1. Sequencing results for FLT3-ITD and NPM1 mutation: A

and B are representative of patients with wild-type and mutant

FLT3-ITD gene, respectively. C and D are representative of patients

with wild-type and mutant NPM1 gene, respectively. The arrows

show the mutation site.

Since the AML subtypes of some patients were not defined, AML

patients were divided into M3 and non-M3 groups according

to the FAB classification. As a result, 12 (17.1%) were M3 and

58 (82.9%) were non-M3. The FLT3 and NPM1 mutation status

was analyzed in AML patients according to FAB groups. The

results showed that there were no differences between the

mutation status of the FLT3-ITD, FLT3-TKD, and NPM1 genes in

the M3 and non-M3 FAB subtypes (Table 4). No mutation was

observed in either FLT3-TKD or NPM1 genes in patients of the

Table 2. Comparison of baseline characteristics between wild-type and mutant groups.

FLT3-ITD p-value NPM1 p-value

Clinical characteristics (-) (+) (-) (+)

WBC count (x10 3 /mL) 23.74±36.21 33.19±45.4 0.538 21.44±34.65 42.45±48.07 0.116

Platelets (x10 3 /mL) 80.14±68.3 65.5±46 0.911 76.81±66.54 73±49.28 0.861

Serum Hb (g/dL) 8.69±2.02 8.92±2.02 0.719 8.6±1.75 9.31±2.74 0.429

Blast count (%) 83.17±9.36 82.64±13.22 0.912 81.59±11.9 86.67±8.16 0.347

WBC: White blood cells, Hb: hemoglobin.

303


Table 3. The frequency of FLT3-ITD, FLT3-TKD, and NPM1 mutations in acute myeloid leukemia patients with different

cytogenetic statuses.

Mutation type FLT3-ITD (+) (n=17) FLT3-TKD (+) (n=3) NPM1 (+) (n=12)

Normal cytogenetics 14 3 11

t(8;21) - - -

t(15;17) 3 - -

inv(16) - - -

t(8;21) + inv(16) - - 1

Other mutation - - -

Table 4. FLT3 and NPM1 mutation status in different French-American-British groups.

FLT3-ITD p-value FLT3-TKD p-value NPM1 p-value

FAB subtypes (-) (%) (+) (%) (-) (%) (+) (%) (-) (%) (+) (%)

Non-M3 44 (83%) 14 (82.4%) 0.604 52 (81.3%) 3 (100%) 0.548 45 (79%) 12 (100%) 0.080

M3 9 (17%) 3 (17.6%) 12 (18.7%) 0 (0%) 12 (21%) 0 (0%)

FAB: French-American-British.

M3 FAB group. The FLT3-ITD mutation was more frequent in

non-M3 patients compared to M3 patients (82.4% vs. 17.6%,

respectively; Table 4).

Discussion

Genetic abnormalities are one of the most common features

observed in AML patients, of which genetic variations of the

FLT3, NPM1, DNMT3A, IDH1/2, and WT1 genes have been given

more attention [3,7].

In the current study, we analyzed the frequency of FLT3 and

NPM1 mutation in 54 adult de novo AML patients with normal

karyotypes (CN-AML). The results showed that the frequency of

FLT3-ITD, FLT3-TKD, and NPM1 mutations was 25.9%, 5.9%, and

20.8%, respectively. The most frequent NPM1 mutant type was

the type A mutation. Our results are consistent with previous

studies that described the FLT3-ITD mutation in 25%-35%,

FLT3-TKD mutation in 7%-10%, and NPM1 in 50%-60% of CN-

AML cases [7,21]. In a study of 39 CN-AML patients by Aly et al.

[22], the frequency of FLT3-ITD was reported to be 15.4%, while

Fröhling et al. [23] and Kainz et al. [24] found that the frequency

of FLT3-ITD was 32% and 30% in CN-AML patients, respectively.

In addition, Falini et al. [18] showed that the frequency of

NPM1 mutation was 61.7%, while different mutation rates were

reported by Zhang et al. [25] (14.3%), Döhner et al. [26] (48.3%),

and Boissel et al. [27] (47%). The discrepancy in the frequency of

FLT3-ITD, FLT3-TKD, and NPM1 mutation between our study and

others may be due to different population groups as well as the

number of cases in the abovementioned studies.

Consistent with previous reports, our results also demonstrated

that the frequency of FLT3-ITD, FLT3-TKD, and NPM1 mutation

was higher in CN-AML patients in comparison with AML patients

with cytogenetic aberrations [3,7,28].

No mutation was detected in the FLT3-TKD or NPM1 gene in

patients in the M3 FAB group. FLT3-ITD mutation was more

frequent in non-M3 patients compared to M3 ones. Consistent

with our results, Falini et al. [18], Thiede et al. [19], and Suzuki

et al. [29] reported no NPM1 mutation in the M3 subtype. In

addition, Verhaak et al. [30] reported a lower frequency of NPM1

mutation in M3 and M0 in comparison with other subgroups.

Therefore, it seems that both FLT3 and NPM1 mutations are

generally mostly seen in AML patients with normal cytogenetics.

Evaluation of the clinical characteristics of the patients revealed

that there were no significant differences in mean WBC and

platelet counts, serum Hb level, or bone marrow blast percentage

between patients with wild-type and mutant FLT3-ITD and

NPM1 genes. No difference was observed in the frequency of

FLT3-ITD or NPM1 mutation regarding age or sex. Consistent

with our findings, Dehbi et al. [31] reported no significant

association between FLT3-ITD mutation and WBC and platelet

counts or blast percentage. Bao et al. [32] also did not observe

any differences in FLT3-ITD mutation frequency according to

age or sex. However, higher WBC counts and increased blast

percentages in FLT3-ITD-positive patients were reported by

Fröhling et al. [23]. Moreover, Haferlach et al. [33] showed a

strong association of bone marrow blast percentage with NPM1

and FLT3-ITD mutations. Gale et al. [28] and Döhner et al. [26]

reported that a significant correlation existed between the

presence of the FLT3-ITD mutation and the NPM1 mutation.

However, there was no significant correlation between the

concomitant mutation of both the FLT3-ITD and the NPM1 gene

in our study, which might be due to the different sample sizes

and also the type of AML (CN-AML in our study and unselected

AML patients in the study by Gale et al. [28]).

It has been demonstrated that the FLT3-ITD mutation promotes

constitutive activation of the FLT3 receptor, leading to ligand-

304



independent cell stimulation and subsequent uncontrolled

proliferation of leukemic blasts [3,8]. Mutation in exon 12 of

NPM1 leads to aberrant cytoplasmic accumulation of the NPM1,

which might contribute to leukemogenesis [21]. Association of

the mutation in both of these genes with clinical outcome has

been shown in various studies; NPM1 has been shown to be

associated with good prognosis, especially in the absence of

the FLT3-ITD mutation, while FLT3-ITD has been independently

considered as a worse prognostic factor that significantly

reduces patients’ survival [22,26,28,30,34,35].

According to our findings, the higher incidence of both the FLT3

and the NPM1 mutation in CN-AML patients underscores that

both FLT3 and NPM1 can be used as candidate genetic markers

for predicting the prognosis of CN-AML patients. In line with

these genes, other known prognostic genetic markers like the

DNMT3A and IDH genes should be considered, which are under

further investigation by our group. Due to time limitations, it

was not possible to follow our patients for a longer period of

time in order to conduct survival analysis. However, further

screening of patients for FLT3 and NPM1 mutations could be

useful to verify the clinical significance of these genes for AML

population prognosis, and especially for assessment of the

presence of the remaining clones as minimal residual disease.

In this regard, the value of increasing the number of patients in

the studied population should be taken into account.

Conclusion

In conclusion, given the high stability of NPM1 during the

disease course, it can be used in combination with FLT3 as well

as other known genetic markers to monitor Iranian CN-AML

patients, especially for minimal residual disease detection.

Acknowledgments

The authors wish to thank the Research Consultation Center for

its editorial assistance. This study was financially supported with

funds provided by Shiraz University of Medical Sciences, Grant

Number 93-01-32-8647.

Ethics

Ethics Committee Approval: This study was approved by the

Ethics Committee of Shiraz University of Medical Sciences.

Informed Consent: Written informed consent was obtained

from all the participants.


Surgical and Medical Practices: M.R.; Concept: N.A.; Design: N.A.;

Data Collection or Processing: N.R., B.V., M.K.; Analysis or

Interpretation: S.H.; Literature Search: N.R.; Writing: N.A.




included.

Financial Disclosure: This study was financially supported with

funds provided by Shiraz University of Medical Sciences, Grant

Number 93-01-32-8647.

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RESEARCH ARTICLE

DOI: 10.4274/tjh.2016.0341


Autoantibodies Against Carbonic Anhydrase I and II in Patients

with Acute Myeloid Leukemia

Akut Miyeloid Lösemi Hastalarında Karbonik Anhidraz I ve II Otoantikorları

Ahmet Menteşe 1 , Nergiz Erkut 2 , Selim Demir 3 , Serap Özer Yaman 4 , Ayşegül Sümer 5 , Şeniz Doğramacı 4 , Ahmet Alver 4,6 , Mehmet Sönmez 2

1

Karadeniz Technical University Vocational School of Health Sciences, Program of Medical Laboratory Techniques, Trabzon, Turkey

2

Karadeniz Technical University Faculty of Medicine, Department of Hematology, Trabzon, Turkey

3

Karadeniz Technical University Faculty of Health Sciences, Department of Nutrition and Dietetics, Trabzon, Turkey

4

Karadeniz Technical University Faculty of Medicine, Department of Medical Biochemistry, Trabzon, Turkey

5

Recep Tayyip Erdoğan University Faculty of Health Services, Department of Nursing, Rize, Turkey

6

Recep Tayyip Erdoğan University Faculty of Medicine, Department of Medical Biochemistry, Rize, Turkey

Abstract

Objective: Cancer, one of the principal causes of death, is a global

social health problem. Autoantibodies developed against the

organism’s self-antigens are detected in the sera of subjects with

cancer. In recent years carbonic anhydrase (CA) I and II autoantibodies

have been shown in some autoimmune diseases and carcinomas,

but the mechanisms underlying this immune response have not yet

been explained. The aim of this study was to evaluate CA I and II

autoantibodies in patients with acute myeloid leukemia (AML) and to

provide a novel perspective regarding the autoimmune basis of the

disease.

Materials and Methods: Anti-CA I and II antibody levels were

investigated using ELISA in serum samples from 30 patients with AML

and 30 healthy peers.

Results: Anti-CA I and II antibody titers in the AML group were

significantly higher compared with the control group (p=0.0001 and

0.018, respectively). A strong positive correlation was also determined

between titers of anti-CA I and II antibodies (r=0.613, p=0.0001).

Conclusion: Our results suggest that these autoantibodies may be

involved in the pathogenesis of AML. More extensive studies are now

needed to reveal the entire mechanism.

Keywords: Acute myeloid leukemia, Autoantibody, Cancer, Carbonic

anhydrase

Öz

Amaç: Kanser, dünyadaki başlıca ölüm nedenlerinden birisi olup,

küresel bir toplum sağlığı sorunudur. Organizmanın kendi antijenlerine

karşı gelişen otoantikorlar pek çok kanser hastasının serumunda tespit

edilmiştir. Son yıllarda karbonik anhidraz (KA) I ve II otoantikorlarının

varlığı bazı otoimmün hastalıklarda ve kanser türlerinde gösterilmiştir,

ancak bu immün yanıtın altında yatan mekanizmalar henüz

açıklanabilmiş değildir. Bu çalışmanın amacı, akut miyeloid lösemili

(AML) kişilerde, KA I ve II otoantikorlarının varlığını değerlendirmek

ve hastalığın otoimmün temeline dair yeni bir bakış açısı sağlamaktır.

Gereç ve Yöntemler: Otuz hasta ve 30 sağlıklı kontrolden elde edilen

serum örneklerinde anti-KA I ve II antikor düzeyleri ELISA yöntemiyle

belirlendi.

Bulgular: AML grubundaki anti-KA I ve II antikor düzeyleri kontrol

grubu (p= sırasıyla 0,0001 ve 0,018) ile karşılaştırıldığında anlamlı

derecede yüksek bulundu. Ayrıca KA I ve II otoantikor seviyeleri

arasında güçlü bir pozitif korelasyon saptandı (r=0,613; p=0,0001).

Sonuç: Elde edilen sonuçlar bu otoantikorların AML patogenezinde

rolü olabileceğini düşündürmektedir. Kesin mekanizmayı ortaya

çıkarabilmek için daha kapsamlı çalışmalar gereklidir.

Anahtar Sözcükler: Akut miyeloid lösemi, Otoantikor, Kanser,

Karbonik anhidraz



Address for Correspondence/Yazışma Adresi: Ahmet MENTEŞE, PhD,

Karadeniz Technical University Vocational School of Health Sciences, Program of Medical Laboratory Techniques,

Trabzon, Turkey Phone : + 90 462 377 78 76

E-mail : amentese028@gmail.com ORCID-ID: orcid.org/0000-0003-2036-5317


Accepted/Kabul tarihi: February 28, 2017

307

Menteşe A, et al: Serum Anti-Carbonic Anhydrase Antibodies in AML


Introduction

Cancer is the second most important cause of mortality and

a major public health problem worldwide [1]. Acute myeloid

leukemia (AML) is a complex and particularly heterogeneous

clonal disease involving arrest of differentiation in the myeloid

lineage along with deposition of immature progenitors in

bone marrow, thus concluding in hematopoietic failure

[2]. The pathogenesis of AML involves various disorders, such

as mutations in transcription factors or epigenetic modifiers,

aberrant signaling pathways, excessive expression of the gene

involved in multidrug resistance, abnormal immune function,

and abnormalities in the bone marrow microenvironment

[3]. Malignant diseases progress with the stimulation of

autoimmunity, characterized by the formation of antibodies

against their own antigens. Autoantibodies can be observed

in the sera of patients with solid tumors and hematological

malignancies [4,5]. These autoantibodies are regarded as early

biomarkers for some types of cancer [6,7,8].

Carbonic anhydrases (CAs) are vitally important enzymes

responsible for the regulation of acid-base homeostasis in both

healthy and pathological conditions. Members of the CA family

contain 16 isoenzymes that differ from one another in terms

of tissue distribution, cell localization, catalytic activity, and

resistance to inhibitors. They perform several functions, such

as transport of carbon dioxide, pH regulation, ion transport,

formation of stomach acidity, bone resorption, calcification,

and tumorigenesis during cancer cell development and

invasion [9,10]. CA I and II are both cytosolic enzymes present

in significant numbers in erythrocytes. CA I is the second most

plentiful protein in erythrocytes after hemoglobin. CA II is a

highly active isoenzyme involved in much total CA activity

in a number of tissues. CA I and/or II autoantibodies have

recently been demonstrated in various pathological conditions,

such as autoimmune diseases (systemic lupus erythematosus,

primary biliary cirrhosis, rheumatoid arthritis, and Sjögren’s

syndrome) and carcinomas (lung, colon, and prostate). However,

the mechanisms underlying this immune response have not yet

been explained [11,12,13,14]. The purpose of this study was to

investigate CA I and II autoantibodies in patients with AML and

to provide a novel perspective regarding the autoimmune basis

of the disease.


Study Group

Informed consent was obtained from all patients and controls.

Approval for the study was granted by the local ethics

committee. Thirty patients newly diagnosed with AML were

included as the study group and 30 healthy peers as the control

group. Diagnosis of AML was made and verified by a panel of

hematologists who also classified each case according to the

French-American-British (FAB) classification [15]. The subtypes

of AML according to FAB were as follows: M0: 1 (3.3%); M1:

1 (3.3%); M2: 13 (43.3%); M3: 3 (10%); M4: 9 (30%); M5: 2

(6.6%); M6: 1 (3.3%). Patients were selected from individuals

presenting to the hematology clinic and referred from other

practitioners. The study group consisted of 17 women and 13

men with a mean age of 52.8±6.3 years, and the control group

of 17 women and 13 men with a mean age of 51.9±14.1. Patients

with renal, coronary, or liver failure and chronic inflammatory

diseases or anemia, and subjects receiving chemotherapy or

using oral contraceptives and anticoagulants, were excluded

from the study.

Blood samples of 5 mL from each individual were placed into

vacutainer tubes without anticoagulant. These were then

centrifuged at 1800 x g for 10 min. Serum samples were stored

at -80 °C until being used for measurements. Platelet (PLT),

hemoglobin (Hb), hematocrit (Hct), and white blood cell (WBC)

levels were determined with a Beckman Coulter autoanalyzer.

Determination of Serum Autoantibody to CA I and II

ELISA is frequently used to detect autoantibodies in blood

samples since it is economical, simple, and quick to perform

[16,17]. It has also been widely used for the evaluation of CA I and

II autoantibodies in different pathological conditions in previous

reports [12,13,18,19,20,21]. Serum CA I and II autoantibodies

were therefore determined using the ELISA method as previously

described elsewhere [18]. Briefly, flat-bottomed plates were

coated with CA I or II (10 µg/mL) (Sigma-Aldrich, St. Louis, MO,

USA) in carbonate buffer (pH 9.6). These were then incubated

for 18 h at 4 °C. In the next stage, the wells were washed four

times with phosphate buffer (PBS) (pH 7) before being blocked

with 3% skim milk in PBS at room temperature for 2 h. The wells

were next washed again four times with PBS containing 0.05%

Tween-20 before incubation with 100 µL of 1:200 diluted serum

for 2 h. Following these washing procedures, each individual

well was incubated for 2 h with 100 µL of a 1:2000 solution

of peroxidase-conjugated anti-human IgG anti-serum (Sigma-

Aldrich, St. Louis, MO, USA) in 3% skim milk in PBS. A further five

washes were performed with PBS containing 0.05% Tween-20,

and the wells were then incubated with 100 µL of substrate

solution for 20 min. Reactions were halted by adding 100 µL

of 2 M sulfuric acid to each well. The resulting absorbance

was measured at 480 nm (Molecular Devices, Sunnyvale, CA,

USA). Control wells containing no CA I or II were also employed

for ELISA investigation of each serum studied. All assays were

performed in duplicate. The specific binding of serum antibody

to CA II was calculated as the mean absorbance of the antigencoated

wells minus the mean absorbance of the control wells.

The results were expressed as absorbance units.

308




Data are shown as mean ± standard deviation for normally

distributed and median (interquartile range) for non-normally

distributed variables. Statistical analysis was performed with

SPSS 13.0 (Chicago, IL, USA) and MedCalc (Version 12.3,

Mariakerke, Belgium) statistical software. Compatibility with

normal distribution was determined using the Kolmogorov-

Smirnov test. Differences between the two groups were

analyzed using Student’s t-test for normally distributed data.

Correlation analysis was calculated using Pearson’s correlation

coefficient and the nonparametric equivalent Spearman’s rank

correlation coefficient at a 95% confidence interval. Receiver

operating characteristic (ROC) curves were used to detect the

discriminatory dominance of CA I and II autoantibodies for the

identification of AML. Sensitivity, specificity, negative predictive

value (NPV), and positive predictive value (PPV) were determined

from ROC graphs for autoantibodies of CA I and II. p<0.05 was

regarded as significant.

value of anti-CA I antibody for healthy subjects was 0.092±0.018,

and the absorbance was higher than 0.146. Positive results were

obtained in 23 of the 30 cases of AML (Figure 1A). The mean

absorbance value of the AML group was significantly higher

(p=0.018) than that of the healthy controls (Table 1). The mean

absorbance +3SD of healthy subjects was also positive. The mean

absorbance value of anti-CA II antibody for the healthy subjects

was 0.079±0.024, and the absorbance was higher than 0.151.

Positive results were obtained in 7 of the 30 cases of AML (Figure

1B). We also observed a strong positive correlation between

titers of anti-CA I and II antibodies (r=0.613, p=0.0001).

ROC curve analysis was also used to quantify serum Hb, Hct,

PLT, WBC, and anti-CA I and II levels. Values for cut-off points,

area under the curve, sensitivity, specificity, PPV, and NPV for

individual parameters are shown in Table 2 and Figure 2.

Results

Thirty AML patients and 30 healthy subjects were included in

this study. There was no significant difference in terms of mean

age between the study and control groups. Levels of anti-CA I

and II antibodies in patients with AML and control subjects are

shown in Figures 1A and 1B, respectively.

The mean absorbance value of the AML group was significantly

higher (p=0.0001) than that of the healthy subjects (Table 1).

The mean absorbance +3 standard deviations (SD) of healthy

subjects was determined as positive. The mean absorbance

Figure 1. Anti-CA I (A) and anti-CA II (B) antibodies in sera of

patients with acute myeloid leukemia and healthy controls. The

dotted line indicates plus 3 SD of health control sera (A 480

=0.146

ABSU for anti-CA I antibody, A 480

=0.151 for anti-CA II antibody).

ABSU: Absorbance units, SD: standard deviation, CA: carbonic anhydrase.

Table 1. Clinical characteristics of the two groups.

AML group (n=30) Control group (n=30) p-values

Anti-CA I Ab (ABSU)

0.195 (0.148-0.311)

0.269±0.211

0.091 (0.074-0.112)

0.092±0.018

0.0001*

Anti-CA II Ab (ABSU)

0.105 (0.069-0.146)

0.127±0.092

0.077 (0.059-0.096)

0.079±0.024

0.018*

Hemoglobin (g/dL) 9.90±2.96 14.8±0.680 0.0001

Hematocrit (%) 28.4±8.42 44.0±1.33 0.0001

Leukocytes (cells/µL) 62907±52501 7436±1336 0.0001

Platelets (cells/µL)

47000 (16750-78000)

64567±57865

253000 (226000-277500)

256233±42242

0.0001*

Data were expressed as mean±SD, median (interquartile range for 25-75%).

p shows differences between the control and AML groups using Student’s t test, *p shows differences between the control and AML groups using the Mann-Whitney U test.

ABSU: Absorbance units, AML: acute myeloid leukemia, CA: carbonic anhydrase.

309



Table 2. Receiver operator characteristic curve analysis of hemoglobin, hematocrit, platelet, white blood cell, and anti-carbonic

anhydrase I and II antibody levels and their sensitivity, specificity, positive predictive value, and negative predictive value.

AUC

Cut-off

Point

Sensitivity Specificity PPV NPV

Anti-CA I 0.919 (0.819-0.973) >0.123 80 (61-92) 100 (88-100) 100 (86-100) 83 (67-94)

Anti-CA II 0.658 (0.524-0.775) >0.097 60 (41-77) 76 (58-90) 72 (51-88) 66 (48-81)

Hb 0.906 (0.803-0.966) <12 83 (65-94) 100 (88-100) 100 (86-100) 86 (68-95)

Hct 0.923 (0.824-0.976) <40 90 (73-98) 100 (88-100) 100 (87-100) 91 (76-98)

PLT 0.998 (0.937-1.000) <198000 100 (88-100) 97 (83-99) 97 (83-100) 100 (88-100)

WBC 0.909 (0.806-0.968) >8890 87 (69-96) 97 (83-99) 96 (81-99) 88 (72-97)

Sensitivity, specificity, PPV, and NPV values were expressed as % within a 95% confidence interval.

ROC: Receiver operator characteristic, PPV: positive predictive value, NPV: negative predictive value, AUC: under the curve, CA: carbonic anhydrase, Hb: hemoglobin, Hct: hematocrit,

PLT: platelet, WBC: white blood cell.

Figure 2. ROC curve analysis for all parameters in patients with

acute myeloid leukemia.

CA: Carbonic anhydrase, Hct: hematocrit, PLT: platelet, WBC: white blood cell.

Discussion

Cancer is the second most important cause of mortality, and

millions of people either have or have had the disease. An

estimated 1.68 million new cancer cases and 595690 deaths

from cancer are predicted to have occurred in the United States

in 2016. Leukemia is one of the most common forms of cancer

[1]. AML is a heterogeneous disease with marked malignancy

of hematopoietic progenitor cells committed to the myeloid

lineage. This phenomenon is most common in subjects aged

over 70, and AML constitutes approximately 30% of all cases of

leukemia [1,22]. Several mutated or overexpressed proteins seem

to be processed and presented to the immune system as tumor

antigens, leading to humoral and/or cellular responses [23].

Autoimmunity is well known to be potentially associated with

cancer, and one of the forms of its expression is the development

of autoantibodies and eventually autoimmune disease. Detection

of autoantibodies may therefore be the first sign of cancer [24].

The ideal tumor biomarker would make it possible to detect

cancer with a simple blood test. The serum biomarkers available

today are based on the measurement of cancer antigens, such as

prostate-specific antigen, carcinoembryonic antigen, the cancer

antigens (CA15-3, CA19-9, and CA125), extracellular protein

kinase A, anti-oncoprotein (HER-2/neu), anti-tumor suppression

antigen (p53), anti-proliferation associated antigens (cyclin A,

cyclin B1, and CDKs), anti-onconeural antigens (Hu and Yo),

and anti-cancer/testis antigens (NY-ESO-1 and MAGE-1) [4,25].

There has therefore been considerable research in recent years

into the identification of new biochemical diagnostic markers

for the early detection of AMLs [3,22,26,27]. Analysis of serum

autoantibodies may become a useful tool for clinicians in

screening for cancer and diagnosis of AML. However, these

markers exhibit limited specificity and sensitivity, and their levels

can also rise even under benign conditions or during gestation.

There is therefore an urgent need for novel biomarkers capable

of adoption into routine clinical use in the diagnosis of AML and

other cancer types [25].

Autoantibodies are common in cancer patients. The autoantibody

response in AML patients has been considered in previous studies,

such as Wilms tumor gene product [28], single-stranded DNA [29],

anti-cardiolipin antibodies [30], the M-phase phosphoprotein

11 (MPP11) [31], receptor for hyaluronan acid-mediated motility

(RHAMM) [32], and RHAMM-like protein [33]. This study is the

first report to show an increased autoimmune response to both

CA I and II in the sera of AML patients. The prevalence of CA I and

II autoantibodies in patients with AML in this study was 76.6%

and 23.3%, respectively. The presence of autoantibodies against

CA I and II has been observed in many pathological conditions,

310



such as metabolic syndrome, recurrent pregnancy loss, acute

anterior uveitis, gastric cancer, Graves’ disease, preeclampsia,

and rheumatoid arthritis. The prevalence of CA I autoantibody is

reported in the range of 9.6%-20%, and that of autoantibodies

against CA II in the range of 4.6%-72.5% [13,19,20,34,35]. From

this perspective, our results were consistent with the literature.

Protection of the acid-base balance is of considerable importance

in tumorigenesis. Extracellular hydrogen ion concentrations in

solid tumors are reported to be higher than those in normal

neighboring tissues [11,36]. Tumor cells express ion transport

protein, such as vacuolar-type H + -ATPase, Cl - /HCO 3-

, and Na + /

H + exchangers between the inner and outer regions of the

cell, thus creating a pH gradient. Many tumor cells synthesize

CAs that catalyze the production of H + and HCO 3

-

ions [11].

CAs are currently the subject of significant research into

carcinogenesis and tumor invasion. Studies have recently

shown an incremental expression of specific cytosolic CA

I and II in some carcinomas, including leukemia, and in the

blast cells of AML [11,36,37]. CA I and II interact with various

molecules due to their cellular localizations, functions, and

wide tissue distribution. These proteins are therefore becoming

target molecules in the body. Autoantibodies against CA I

and II have recently been demonstrated in many pathological

conditions, such as cancer, and autoimmune and idiopathic

diseases. Although the mechanisms involved have not been

identified exactly, oxidative stress has been reported to be

potentially significant in the formation of these autoantibodies

[13,14,19,21,38,39]. Oxidative stress results from acceleration

of the rate of free radical formation and/or a decrease in the

rate at which these are eliminated. In either condition a severe

imbalance occurs between free radical formation and the

antioxidant defense mechanism [40]. Increased reactive oxygen

or nitrogen species (ROS) lead to tissue injury and compromise

numerous biomolecules, including proteins, nucleic acids,

structural carbohydrates, and lipids. The reaction of ROS with

lipids causes these molecules to undergo oxidative breakdown.

Malondialdehyde (MDA) is a one-end product of lipid

peroxidation capable of being covalently bound to proteins,

and especially to the Ɛ-amino groups of lysine residues. These

oxidative disturbances may influence the immune system,

resulting in the development of specific autoimmune processes

[41]. The lipid peroxidation end-products 4-hydroxy-2-nonenal

(HNE) and MDA are known to alter proteins and to modify their

antigenic properties [42]. One study of erythrocytes proved that

CA II is the first target of HNE [43]. Numerous anti-MDA-modified

proteins have been detected in systemic diseases, such as

systemic lupus erythematosus, periarteritis nodosa, scleroderma,

atherosclerosis, and rheumatoid arthritis in previous studies. It

has also been suggested that these autoantibodies may be of

predictive value for systemic diseases [41,44,45,46,47]. Studies

in the literature have shown that the levels of such oxidative

stress parameters as MDA, advanced oxidation protein products,

8-hydroxydeoxyguanosine, and protein carbonyl increase in the

sera of patients with AML, while the activities of antioxidant

enzymes such as superoxide dismutase, glutathione peroxidase,

and monoamine oxidase decrease [48]. In light of these data,

we anticipate that oxidative byproducts, including MDA, might

generate the spread of neoantigens and confirm a potential

association between autoimmunity and oxidative stress.

Study Limitations

The major limitation of this study is the relatively small sample

size of the patient and control groups.

Conclusion

In conclusion, CA I and II autoantibody titers were significantly

higher in subjects with AML compared to the controls. More

extensive studies are now needed to reveal the entire mechanism

involved.

Ethics

Ethics Committee Approval: Approval for the study was granted

by the Karadeniz Technical University Faculty of Medicine Ethics

Council under reference no 2016/31.

Informed Consent: Informed consent was obtained from all

individual participants included in the study.


Surgical and Medical Practices: A.M., N.E., A.A., M.S.;

Concept: A.M., N.E., A.A., M.S.; Design: A.M., N.E., S.D., A.S.; Data

Collection or Processing: A.M., N.E., S.D., S.Ö.Y., Ş.D.; Analysis or

Interpretation: A.M., N.E., S.D., A.S.; Literature Search: A.M., N.E.,

S.Ö.Y., Ş.D.; Writing: A.M., N.E., A.A., M.S., S.D.




included.

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313

RESEARCH ARTICLE

DOI: 10.4274/tjh.2016.0214


Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast

and Accurate Human Umbilical Cord Blood Hematopoietic Stem

Cell Assessment

İnsan Göbek Kordon Kanı Hematopoetik Kök Hücre Değerlendirmesinde Hızlı ve Etkin Bir

Değerlendirme Yöntemi: Akım Sitometrik Aldehit Dehidrogenaz Testi

Emine Begüm Gençer 1,2 , Pınar Yurdakul 1,3 , Klara Dalva 4 , Meral Beksaç 5

1

Ankara University Faculty of Medicine, Cord Blood Bank, Ankara, Turkey

2

Ankara University Faculty of Medicine, Biotechnology Institute, Ankara, Turkey

3

TOBB Economics Technology and University Faculty of Medicine, Department of Medical Microbiology, Ankara, Turkey

4

Ankara University Faculty of Medicine, Stem Cell Research Institute, Ankara, Turkey

5

Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract

Objective: Colony-forming units of granulocytes/macrophages

(CFU-GM) analysis is the most widely used method to determine the

hematopoietic stem cell (HSC) content of human umbilical cord blood

(CB) for prediction of engraftment potential. The measurement of

aldehyde dehydrogenase (ALDH) activity is a more recent method for

HSC qualification. Our aim was to correlate phenotypic and functional

assays to find the most predictive method.

Materials and Methods: In this study, flow cytometric quantitation

of CD34 + cells and ALDH positivity along with CFU-GM capacity were

assessed in fresh and post-thaw CB units.

Results: Among 30 post-processing samples, for each CB unit the

mean total number of nucleated cells (TNCs) was (93.8±30.1)x10 7 ,

CD34 + cells were (3.85±2.55)x10 6 , ALDH + cells were (3.14±2.55)x10 6 ,

and CFU-GM count was (2.64±1.96)x10 5 . Among an additional 19 postthaw

samples the cell counts were as follows: TNCs, (32.79±17.27)x10 7 ;

CD34 + , (2.18±3.17)x10 6 ; ALDH + , (2.01±2.81)x10 6 ; CFU-GM, (0.74±0.92)

x10 5 . Our findings showed that in fresh samples TNCs, CD34 + cells,

and ALDH correlated highly with counts of CFU-GM, CFU-erythroids/

granulocytes-macrophages/megakaryocytic cells (GEMM), and burst

forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35,

r=0.41; CD34 + , r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45,

r=0.6, respectively. In terms of post-thaw samples, the correlations

were as follows: TNCs, r=0.59, r=0.46, r=0.56; CD34 + , r=0.67, r=0.48,

r=0.61; and ALDH, r=0.61, r=0.67, r=0.67, for CFU-GM, CFU-GEMM,

and BFU-E, respectively. All correlations were statistically significant.

Öz

Amaç: Granülositer makrofaj koloni oluşturma (CFU-GM) testi kordon

kanı (KK) hematopoietik kök hücre engrafman potensiyelini ölçmek

için kullanılan bir yöntemdir. Aldehit dehidrogenaz (ALDH) enzimi

ölçüm yöntemide hematopoetik kök hücre (HKH) kalitesini belirlemek

amacıyla kullanılan daha yeni bir metottur. Çalışmamızda fenotipik

ve fonksiyonel olarak korelasyon analizi yapılarak HKH ölçümünde en

etkili metodu bulmayı amaçladık.

Gereç ve Yöntemler: Bu çalışmada taze ve donma çözme sonrası

KK ünitelerinde CD34 + ve ALDH + hücrelerle CFU-GM kapasiteleri

araştırılmıştır.

Bulgular: Otuz taze KK ünitesinde her KK için ortalama değerler:

Toplam çekirdekli hücre sayısı (TNC): 93,8±30,1x10 7 , CD34 + :

3,85±2,55x10 6 , ALDH + : 3,14±2,55 x10 6 , CFU-GM: 2,64±1,96x10 5 . On

dokuz KK ünitesinde donma çözme sonrası hücre değerleri: TNC:

32,79±17,27x10 7 , CD34 + : 2,18±3,17x10 6 , ALDH + : 2,01±2,81x10 6 , CFU-

GM: 0,74±0,92x10 5’ dir. Bulgularımız; taze KK’da TNC, CD34 ve ALDH;

CFU-GM, CFU-GEMM ve BFU-E ile korelasyon gösterirken (TNC, r=0,47,

r=0,35, r=0,41; CD34 + , r=0,44, r=0,54 r=0,41; ve ALDH, r=0,63 r=0,45

r=0,6) donma çözme sonrası KK’da korelasyon sırasıyla CFU-GM,

CFU-GEMM, ve BFU-E için, TNC r=0,59, r=0,46, r=0,56, CD34 + r=0,67,

r=0,48, r=0,61 ve ALDH r=0,61, r=0,67, r=0,67 olarak saptanmıştır.

Bütün bulgularımız istatistiksel olarak anlamlı çıkmıştır.

Sonuç: Çalışmamız, ALDH aktivitesi tayin metodu HKH tayininde

geleneksel yöntemlerle özellikle donma çözme sonrası örnekler

açısından korelasyon göstermiştir. Böylelikle hızlı, ucuz bir metod



Address for Correspondence/Yazışma Adresi: Meral BEKSAÇ, M.D.,

Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Phone : +90 0312 595 79 88

E-mail : meral.beksac@medicine.ankara.edu.tr ORCID-ID: orcid.org/0000-0003-1797-8657

Received/Geliş tarihi: June 10, 2016

Accepted/Kabul tarihi: December 01, 2016

314


Gencer EB, et al: Phenotypical Analysis of Umbilical Cord Blood

Conclusion: In our experience, HSC assessment by ALDH activity

yields the highest correlation with conventional analytical methods,

particularly for post-thaw samples. Thus, this fast, inexpensive method

has the potential to overcome the weaknesses of other techniques.

Keywords: Cord blood, Aldehyde dehydrogenase, Colony-forming

unit-granulocyte/macrophage

olarak ALDH diğer HKH belirlemede kullanılan yöntemlere üstün

olabilecek kapasitededir.

Anahtar Sözcükler: Göbek kordon kanı, Aldehit dehidrogenaz, Koloni

oluşturan birim granülositer/makrofaj

Introduction

Recent scientific evidence demonstrates that different subtypes

of CD34 + cells in the cord blood (CB) hematopoietic stem

cell (HSC) niche have different engraftment potentials [1,2].

It is of crucial importance to determine the quality of the

CB particularly following freeze/thaw cycles. Two different

approaches can be used to assess the functionality and

population-forming capacities of CB HSCs along with the gold

standard method of the International Society of Hematotherapy

and Graft Engineering (ISHAGE) [3]. Ex vivo colony-forming unit

(CFU) assays are the most widely used tests for determining HSC

functions, but they possess serious drawbacks such as difficulty

in routine application, lack of standardization, labor-intensive

nature, and long turnaround time [4]. One of the likely reasons

for this is probably the fact that while being predictive of shortterm

re-populating cells, CFU assays could not determine longterm

populating cells effectively. Long-term populating cells

have been shown to provide long-term immune reconstitution

after CB transplantation (CBT); thus, it is of crucial importance

to assess their numbers. The measurement of aldehyde

dehydrogenase (ALDH) activity can therefore be much more

accurate due to the intracellular presence of this enzyme [5].

It was reported that ALDH enzyme expression is high in early

HSCs in the bone marrow and CB [6,7]. A few published

studied correlated high ALDH activity with better permanent

engraftment following HSC transplantation [5,7,8,9,10,11]. In

the first such study by Lioznov et al. [12], it was reported that

ALDH expression is a practical marker to assess HSC activity

for both stem and progenitor cells before bone marrow and

peripheral blood transplantation. There are hardly any data for

CB investigating the phenotypic and functional properties of CB

HSCs and the correlation of ALDH activity with CFU potential

in pre- and post-thaw CB HSCs [5,7,11,13,14]. In this study, we

aimed to correlate phenotypic assays with functional assays to

find the most predictive method for fresh and post-thaw CB.


CB Unit Selection and Processing

A total of 50 CB units from consenting maternal donors collected

at the Ankara University Faculty of Medicine’s Cord Blood Bank

were included in this study. Thirty CB units that met volume and

total number of nucleated cell (TNC) eligibility criteria (>70 mL and

100x10 7 /U, respectively) were processed and used immediately for

the fresh group and 20 non-conforming CB units that had been

reserved for research purposes were included as the post-thaw

group (1 unit was discarded due to CFU culture contamination). CB

units were processed automatically with a Sepax 2 device (Biosafe)

and TNC counts, post-processing CD34 + cell enumeration, and cell

viabilities were assessed for every CB unit.

Post-Thaw Washing

Nineteen non-conforming CB units were thawed in a 37 °C

water bath and samples of 10 mL were taken into conical

tubes. In order to remove DMSO, CB units were washed using a

washing solution (10% dextran 40 (BioFleks), 20% human serum

albumin (Centurion Pharma), and PBS (Lonza) at 4:1:3/8 (v/v),

respectively). Upon thawing of the CB units they were washed

twice with 1:1 (v/v) washing solution After discarding the

supernatant, the pellet was re-suspended in washing solution

by gentle mixing.

Determination of TNC/CD34 Viability and Counts

The number of TNCs for all units (30 fresh and 19 post-thaw

units) was assessed by complete blood counting with an

automated cell counter (Beckman Coulter, LH780). CB unit

CD34 + cell enumeration and detection of cell viability by

7-aminoactinomycin dye was performed using a Stem-Kit upon

the acquisition of the data with an FC 500 instrument (Beckman

Coulter). The analysis was performed using an ISHAGE single

test platform.

ALDH Analysis

The ALDEFLUOR assay (StemCell Technologies) was used for the

detection of ALDH expression in fresh and post-thaw CB HSCs.

ALDH activity was measured by the protocol recommended by

the manufacturer. Briefly, cell suspensions were adjusted to

10 6 cells/mL with 1500 µL of ALDEFLUOR assay buffer after red

blood cell depletion. ALDEFLUOR reagent (10 µL) was added

to each tube, followed by 5 min of centrifugation at 300×g.

Supernatants were stained with FITC-ALDH, APC A-750-CD38,

phycocyanin (PC) 7-CD34, chrome orange-CD45 (Beckman

Coulter), PE-CD73, and PC 5-CD90 (Becton Dickinson) antibodies

and analyzed by flow cytometry (Beckman Coulter FC500).

Diethylaminobenzaldehyde reagent was used to suppress ALDH

activity in control tubes. Using the ALDH activity assay, CB HSCs

were categorized as ALDH + and ALDH - .

315



CFU Assays

CFU assays were implemented according to the manufacturer’s

recommendations (StemCell Technologies CFU Manual,

MA28404) and modified from Lee et al. [19]. First, 100 µL

of CB sample was removed from all CB units, and after the

addition of 80 µL of HetaSep and 300 µL of Iscove’s modified

Dulbecco medium containing 2% fetal bovine serum (StemCell

Technologies), the mixture was incubated at 37 °C in 5% CO 2

for 20 min (Sanyo CO 2

Incubator). Cells (5x10 5 cells/mL) were

transferred to 3 mL of MethoCult Express medium. After

14 days, colonies were counted and different morphologies

as well as numbers of CFUs were recorded using an inverted

light microscope (Olympus/IX51). The number of colonies was

calculated as the mean value for two dishes.


Pearson correlation coefficient tests (if data distribution was

normal) and Spearman rank correlation coefficient tests (if data

distribution was not in the normal range) were used to assess

the correlations. All statistical analyses were performed with

SPSS 15.0.

Results

In this study we aimed to compare three different methods

in terms of efficiency to assess different re-populating HSCs

from CB, both after processing and after thawing. We analyzed

different cellular fractions, namely TNCs; CD34 + , ALDH + , CD34 +

ALDH + , ALDH + CD34 + , and ALDH + CD34 + CD90 + CD38 - cells; and

colony-forming units of granulocytes/macrophages (CFU-GM),

CFU-erythroids/granulocytes-macrophages/megakaryocytic

cells (GEMM), and burst forming units of erythroids (BFU-E),

for both fresh and post-thawed units. Table 1 demonstrates

the mean, median, and minimum-maximum values of the

aforementioned parameters for fresh and post-thawed samples.

Table 2 provides the correlation values for ALDH positivity and

TNC, CD34 + , and CD34 + CD90 + CD38 - cell numbers as well as

CFU-GM, CFU-GEMM, and BFU-E colony counts among all CB

samples. When fresh samples were analyzed, ALDH activity

correlated well with all the cell populations investigated; TNC,

ALDH + , and CD34 + fractions were found to be highly correlated

both with CFU-GM, CFU-GEMM, and BFU-E and with each other.

The correlation coefficients remained significant for fresh and

post-thawed samples, and when post-thaw data were analyzed,

TNCs, CD34 + , and ALDH were also found to be statistically

correlated with CFU-GM, CFU-GEMM, and BFU-E (Table 2).

Among all parameters compared, the most striking correlation

was detected for CFU-GM numbers and ALDH positivity for

fresh CB units (r=0.629, p<0.001); post-thaw analyses also

revealed a correlation for CFU-GM and ALDH + cells when the

same parameters were investigated (r=0.608, p=0.006; Table 2).

When CFU-GM numbers were tested against all parameters for

Table 1. Characteristics of all fresh and post-thaw CB units tested.

Variables (fresh CB units, n=30; post-thaw CB units, n=19) Mean ± SD Median Minimum - maximum

TNC

(x10 7 /U)

CD34 +

(x10 6 /U)

ALDH +

(x10 6 /U)

ALDH + /CD34 +

(x10 6 /U)

CD34 + /ALDH +

(x10 6 /U)

ALDH + /CD90 + /CD34 + /CD38-

(x10 6 /U)

CFU-GM

(x10 5 /U)

CFU-GEMM

(x10 5 /U)

BFU-E

(x10 5 /U)

Fresh 93.8±30.1 85.5 46.8-168.8

Post-thaw 32.79±17.27 31.05 9.2-47.6

Fresh 3.85±2.55 3.6 0.94-11.64

Post-thaw 2.18±3.17 1.18 0.5-14.5

Fresh 3.14±2.55 2.6 0.12-8.48

Post-thaw 2.01±2.81 1.38 0.20-11.3

Fresh 2.97±2.02 2.4 0.11-8.12

Post-thaw 1.82±2.63 1.28 0.17-1.69

Fresh 3.72±2.28 2.9 0.34-8.94

Post-thaw 3.01±4.33 1.65 0.29-15.31

Fresh 0.19±0.19 0.1 0.005-0.795

Post-thaw 0.40±0.55 0.2 0.01-1.92

Fresh 2.64±1.96 2.14 0.25-7.67

Post-thaw 0.74±0.92 0.41 0.02-2.92

Fresh 3.86±2.73 3.6 0.66-11.49

Post-thaw 0.70±0.98 0.36 0-4.27

Fresh 5.30±3.53 5.6 0.12-16.22

Post-thaw 0.44±0.74 0.19 0-2.9

SD: Standard deviation, CB: cord blood, TNC: total number of nucleated cells, ALDH: aldehyde dehydrogenase, CFU-GEMM: colony-forming units - granulocytes-macrophages/

megakaryocytic cells, BFU-E: burst forming units of erythroids, GM: granulocytes/macrophages.

316



Table 2. Correlations between different groups of cells demonstrating hematopoietic activity in fresh and post-thaw cord blood units.

Variables TNC

TNCs

(x10 7 /U)

(x10 7 /U)

CD34 +

(x10 6 /U)

ALDH +

(x10 6 /U)

ALDH +

CD34 +

(x10 6 /U)

CD34 +

ALDH +

(x10 6 /U)

ALDH + CD90 +

CD 34 + CD38 -

(x10 6 /U)

CFU-GM

(x10 5 /U)

CFU-GEMM

(x10 5 /U)

BFU-E

(x10 5 /U)

Fresh r=0.516** r=0.671** r=0.674** r=0.556** r=0.624** r=0.476** r=0.354 NS r=0.580**

Post-thaw r=0.541* r=0.507* r=0.432 NS r=0.474* r=0.414 NS r=0.591** r=0.461* r=0.556*

CD34 +

(x10 6 /U)

Fresh r=0.799** r=0.799** r=0.967** r=0.446* r=0.444* r=0.543** r=0.414*

Post-thaw r=0.935** r=0.931** r=0.971** r=0.889** r=0.665** r=0.476* r=0.610**

ALDH + (x10 6 /U) Fresh r=0.999** r=0.786** r=0.615** r=0.629** r=0.451* r=0.596**

Post-thaw r=0.988** r=0.935** r=0.868** r=0.608** r=0.672** r=0.668**

ALDH + CD34 + (x10 6 /U) Fresh r=0.786** r=0.616** r=0.623** r=0.435* r=0.586**

Post-thaw r=0.931** r=0.877** r=0.611** r=0.673** r=0.634**

CD34 + ALDH + (x10 6 /U) Fresh r=0.431* r=0.426* r=0.558** r=0.375*

ALDH + CD90 + CD34 +

CD38- (x10 6 /U)

Post-thaw r=0.867** r=0.670** r=0.594** r=0.610**

Fresh r=0.559** r=0.393* r=0.678**

Post-thaw r=0.594** r=0.500* r=0.644**

CFU-GM (x10 5 /U) Fresh r=0.567** r=0.683**

CFU-GEMM

(x10 5 /U)

BFU-E

(x10 5 /U)

Post-thaw r=0.561* r=0.510*

Fresh r=0.398 NS

Post-thaw r=0.554*

*p<0.05,**p<0.01, NS: p>0.05.

TNC: Total number of nucleated cells, ALDH: aldehyde dehydrogenase, CFU: colony-forming units - granulocytes-macrophages/megakaryocytic cells, BFU-E: burst forming units of erythroids, GM: granulocytes/macrophages.

317



Figure 1. Correlation values of TNCs, ALDH, and CD34 + cells with CFU-GM and ALDH + CD34 + cells. Graphs A-D denote correlations for

fresh CB units: TNCs and CFU-GM (A); CD34 + cells and CFU-GM (B); ALDH + cells and CFU-GM (C); CD34 + cells and ALDH + cells (D). Graphs

E-H denote correlations for post-thaw samples: TNCs and CFU-GM (E); CD34 + and CFU-GM (F); ALDH + and CFU-GM (G); CD34 + cells and

ALDH + cells (H).

CB: Cord blood, TNC: total number of nucleated cells, ALDH: aldehyde dehydrogenase, CFU-GM: colony-forming units granulocytes/macrophages.

post-thawed samples, an even higher correlation was detected

in CD34 + cells, which were also positive for ALDH (r=0.670,

p=0.002). On the other hand, there was no significant correlation

between TNCs and ALDH + cells, which were also positive for

CD34 (r=0.432, p=0.065). These correlations are shown in Figure

1. Table 2 shows r values for all parameters investigated.

Discussion

Shoulars et al. [11] developed an ALDH-based method to

estimate the post-thaw quality of CB units. The results of their

study, similar to ours, demonstrated that ALDH activity is highly

correlated with CFU counts and can be integrated into routine

CB unit release procedures prior to transplantation. Thus, our

findings, which show the highest correlation between in vitro

CFU counts and ALDH activity compared to TNCs or CD34, are

confirmed by this very recent publication.

One of the recent methods described for the rapid and accurate

detection of functional CB cell fractions is ALDH activity

measurement in CB HSCs. To date, five studies have looked

at ALDH levels in CB units [5,7,11,13,14]. Our study is unique

in terms of having a detailed post-thaw analysis and to our

knowledge it is also the first to determine the capacity of

ALDH + CD90 + CD34 + CD38 - cells, a group of cells that possess

high engraftment capacity. Most of the papers in the literature

have focused on HSCs with high ALDH activity with conflicting

results related to their role in engraftment following HSC

transplantation [11,14,15,16]. An experiment carried out by

Pearce et al. [17] showed that ALDH and CD34 double-positive

cells constitute 63% of lineage-negative cells for TNCs and only

ALDH + cells improved engraftment. Storms et al. [18] classified

HSCs as being CD34 +/- , ALDH + , and ALDH - and demonstrated

that only CD34 + ALDH + cells were efficient in terms of longterm

and short-term re-population capacities.

We aimed to compare three different approaches to assess

different re-populating HSCs from CB, both fresh and after

thawing. In our study, CD34 + cells were found to constitute

0.49±0.26% of all TNCs and 0.35±0.21% of ALDH + cells.

Additionally, 86.98±13% of all CD34 + cells were found to be

ALDH + , and within all ALDH + cells, 94.54±5.3% were CD34 + .

Among all CB units tested, the rate of ALDH + CD34 - cells

was found to be 5.46% and 13.02% were ALDH - but CD34 + .

Different research groups sought to identify different cellular

populations by ALDH staining intensities, but only two of them

compared the ALDH activity of different CB sub-populations

[5,19]. The clinical significance of those populations remains

to be determined. In a study by Lee et al., [7] CD34 cells were

found to constitute 0.14±0.10% of all TNCs. In comparison,

CD34 positivity was seen in 0.49±0.26% of all TNCs in our study.

Unlike our results, Lee et al. [7] detected less ALDH positivity

among CD34 + cells (74.5±13.8%), and of the entire ALDH +

318



population, 69.9±15.5% of cells were shown to express CD34.

Another similar investigation by Storms et al. [18] demonstrated

even higher HSC rates in fresh CB samples: 0.9±0.5% of TNCs

were CD34 + , but 47.9±14.3% of those cells were ALDH + . ALDH +

cells constituted 0.96±0.5% of TNCs and 50.9±18.3% of ALDH +

cells were CD34 + [18]. In Gentry et al.’s [10] study CD34 + cell

count was found to be 0.15±0.08% of all TNCs, and 0.05±0.02%

of TNCs expressed high ALDH.

Attia et al. [14] reported that ALDH activity detection is not only

quick and easy to perform but also it does not affect the cell

viability or re-populating capacity of CB cells, which may be a

serious drawback of some CD34 detection systems [20]. Ikeda

et al. [13] suggested that prior to CBT the ALDH assessment

method could be an alternative approach to the selection of

CB units for unrelated donors [14]. All of the results from the

papers mentioned here are in favor of our findings indicating

the utility of an ALDH-based approach for CBT settings.

Characterization of the sub-populations of CB is crucial because

high cell doses with adequate viability predict the outcome after

CBT. Engraftment is generally ensured when highly CD34 + cells

are used, but occasionally a graft with partially dysfunctional

cells due to freeze/thaw processes can affect the cells’ shortterm

and long-term re-populating capacities [20]. In vitro

manipulations have been shown to interfere with membrane

CD34 expression without hampering HSC functionality [21,22].

With the ALDH analysis approach, HSCs with relatively high

engraftment capacity but with limited or no growth in CFU tests

can easily be detected.

As we sought to determine the correlation levels for TNCs and

ALDH + and CD34 + cells with CFU capacities for both fresh and

post-thaw samples, we demonstrated that ALDH positivity

correlated highly with CFU-GM capacity (r=0.629, p<0.001) in

fresh samples. On the other hand, the highest correlation was

detected for CFU-GM numbers and CD34 + cells for the post-thaw

group of CB units (r=0.655, p<0.001) (Table 2). With a similar

approach, Lee et al. [19] analyzed ALDH + , CD34 + , ALDH + within

CD34 + , and CD34 + within ALDH + cell populations and CFU-GM

and CFU-GEMM capacities in 245 CB units, both fresh and after

thawing. Unlike our results, CFU-GM count was not found to

be correlated with TNCs in their study. In addition to Lee et al.’s

[19] approach, ALDH + CD90 + CD34 + CD38 - cell populations and

BFU-E capacities were also analyzed in our study. Lee et al. [19]

did not provide any data related to post-thaw samples in terms

of TNC/ALDH and TNC/CD34 + ratios, but we demonstrated in our

study that 0.66±0.4% of TNCs were CD34 + cells and 0.37±0.27%

of TNCs were ALDH + cells.

In a phase 1 study by Gentry et al. [10], CD34 + cell counts

were claimed to be the sole post-thaw CB quality predictor,

indicating a lower transplant-related mortality, but post-thaw

comparisons of CFU-GM counts versus ALDH activity were not

conducted. In our study, ALDH + cells correlated well with CFU-

GM for post-thaw samples (Table 2). Similar to our results, a

positive correlation was detected between ALDH + cells and CFU-

GM positivity (r=0.40, p=0.03) in Frandberg et al.’s [5] study, but

their work did not reveal any correlation of CD34 + cells with

total CFU count (r=0.36, p=0.051).

In addition to ALDH positivity, determination of CD90 + CD34 +

CD38 - cells may also be a good predictor of engraftment both

for fresh and post-thaw CB units. The ALDH + CD90 + CD34 + CD38 -

group of progenitors is unique with their high engraftment and

re-populating capacities [23,24,25]. To our knowledge, our study

is the first to examine the ALDH capacity of these particular

cells. When we analyzed ALDH positivity in CD90 + CD34 + CD38 -

cells, CFU-GM, CFU-GEMM, and BFU-E counts were found to

be well correlated for both fresh and post-thaw CB units (Table

2). Putman et al. [9] performed CFU tests for ALDH hi and ALDH lo

cellular populations of CB BFU-E, CFU-GM, and CFU-GEMM. In

our study the highest correlation of ALDH + cells was found with

CFU-GM and BFU-E for fresh samples. Putman et al. [9] found

that the CB ALDH hi population was significantly enriched for

human hematopoietic progenitor function.

Owing to the nature of ALDH as an intracellular enzyme, it

may be less affected by centrifuge force and thus may reflect

the actual HSC population in a more realistic manner. By using

ALDH as a marker of functionality, the disadvantage of a likely

false negativity caused by CD34 + cell counting could also be

overcome. Similar to our results, Shoulars et al. [11] from Duke

University recently demonstrated that ALDH activity measured

from post-thaw segments highly correlated with CFUs. They

suggested that measurement of ALDH br CD34 + cells might

indicate CFU potency and thus engraftment capacity.

Conclusion

In light of our results and other recently published studies,

we propose that ALDH activity determination can substitute

for CFU-GM tests. This fast and inexpensive method has the

potential to overcome the weaknesses of other techniques, such

as the limitations of CD34 counting due to the internalization of

membrane CD34 expression or lack of standardization and long

turnaround time of CFU assays. We are aware of the limitations

in engraftment prediction by phenotype-based analysis. ALDH

measurement, as confirmed by us, has the highest correlation

with in vitro functional assays. Currently CD34 and CFU-

GM assays, accepted as golden standards, are expected to be

replaced by ALDH measurement, which is a fast, reproducible,

and accurate assessment tool.

Acknowledgments

This study was supported by the Scientific and Technological

Research Council of Turkey (TÜBİTAK, 114S829). We are

grateful to Handan Karakaya for her kind assistance in the CB

319



processing lab and to Sema Meriç for her assistance in flow

cytometry analysis. We would like to thank Dr. Sinan Beksaç

and Dr. Doruk C. Katlan for their assistance with CB collection.

This study was presented as a poster at the 8 th National Bone

Marrow Transplantation and Stem Cell Therapy Congress as

“Phenotypical Analysis of Ex Vivo Granulocyte Colony-Forming

Human Umbilical Cord Blood Cells”.

Ethics

Ethics Committee Approval: Ankara University Faculty of

Medicine, Clinical Research Ethics Committee (04-173-

13/11.03.2013).

Informed Consent: This study was based on blood bank data.


Surgical and Medical Practices: M.B.; Concept: M.B., P.Y.;

Design: M.B.; Data Collection or Processing: E.B.G.; Analysis

or Interpretation: E.B.G., K.D.; Literature Search: E.B.G., P.Y.;

Writing: E.B.G.




included.

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320

RESEARCH ARTICLE

DOI: 10.4274/tjh.2016.0118


Effectiveness of Visual Methods in Information Procedures for

Stem Cell Recipients and Donors

Kök Hücre Alıcıları ve Donör Bilgilendirme İşleminde Görsel Yöntemlerin Etkinliği

Çağla Sarıtürk 1 , Çiğdem Gereklioğlu 2 , Aslı Korur 2 , Süheyl Asma 2 , Mahmut Yeral 3 , Soner Solmaz 3 , Nurhilal Büyükkurt 3 , Songül Tepebaşı 3 ,

İlknur Kozanoğlu 3 , Can Boğa 3 , Hakan Özdoğu 3

1

Başkent University Adana Application and Research Center, Department of Biostatistics, Adana, Turkey

2

Başkent University Faculty of Medicine, Department of Family Medicine, Adana, Turkey

3

Başkent University Faculty of Medicine, Adana Adult Bone Marrow Transplantation Center, Adana, Turkey

Abstract

Objective: Obtaining informed consent from hematopoietic stem cell

recipients and donors is a critical step in the transplantation process.

Anxiety may affect their understanding of the provided information.

However, use of audiovisual methods may facilitate understanding. In

this prospective randomized study, we investigated the effectiveness

of using an audiovisual method of providing information to patients

and donors in combination with the standard model.

Materials and Methods: A 10-min informational animation was

prepared for this purpose. In total, 82 participants were randomly

assigned to two groups: group 1 received the additional audiovisual

information and group 2 received standard information. A 20-item

questionnaire was administered to participants at the end of the

informational session.

Results: A reliability test and factor analysis showed that the

questionnaire was reliable and valid. For all participants, the mean

overall satisfaction score was 184.8±19.8 (maximum possible score

of 200). However, for satisfaction with information about written

informed consent, group 1 scored significantly higher than group 2

(p=0.039). Satisfaction level was not affected by age, education level,

or differences between the physicians conducting the informative

session.

Conclusion: This study shows that using audiovisual tools may

contribute to a better understanding of the informed consent

procedure and potential risks of stem cell transplantation.

Keywords: Hematopoietic stem cell, Donor, Informed consent,

Audiovisual method, Bone marrow transplantation

Öz

Amaç: Hematopoietik kök hücre alıcıları ve donörlerden

bilgilendirilmiş onam alınması, nakil sürecinin en önemli basamağıdır.

Görsel yöntemlerden yararlanılması anlamayı kolaylaştırabilir. Bu

prospektif randomize çalışmada sözel ve yazılı bilgilendirmeye ilave

olarak audiovizüel yöntem kullanılmasının standart yönteme göre

etkinliğinin araştırılması amaçlanmıştır.

Gereç ve Yöntemler: On dakikalık kısa bir bilgilendirme animasyonu

hazırlatıldı. Toplam 82 katılımcı rastgele 2 gruba ayrıldı: Grup 1 (görsel

bilgilendirme yönteminin ilave edildiği grup) ve grup 2 (standart

yöntem uygulanan grup). Bilgilendirme işlemi sonunda katılımcılara

20 soruluk bir anket uygulandı. Ayrıca, yeni yöntemin sonuçlarının

kişiler arası farklılıktan etkilenip etkilenmediği test edildi.

Bulgular: Güvenirlik testi ve faktör analizi anketin güvenilir ve geçerli

olduğunu gösterdi. Tüm olgular için genel memnuniyet ortalama

değeri 200 üzerinden 184,8±19,8 olarak bulundu. Yazılı bilgilendirilmiş

onam form memnuniyeti ortalama puanları yönünden grup 1’deki

olguların memnuniyetleri grup 2’ye oranla anlamlı olarak daha

yüksek olduğu saptandı (p=0,039). Sözel olarak bilgi veren doktor ve

bilgilendirme animasyonu memnuniyeti bakımından gruplar arasında

fark saptanmadı. Olguların memnuniyet seviyesi yaş, eğitim durumu

ve bilgilendiren kişiler arası farklılıktan etkilenmedi.

Sonuç: Bu çalışma, görsel yöntemlerin kullanılmasının işlem ve

muhtemel risklerinin daha iyi anlaşılmasına katkı sağlayabileceğini

göstermektedir.

Anahtar Sözcükler: Hematopoietik kök hücre, Donör, Bilgilendirilmiş

onam, Audovizuel yöntem, Kemik iliği nakli



Address for Correspondence/Yazışma Adresi: Can BOĞA, M.D.,

Başkent University Adana Application and Research Center, Department of Biostatistics, Adana, Turkey

Phone : +90 322 327 27 27-2162

E-mail : drcanboga@hotmail.com ORCID-ID: orcid.org/0000-0002-9680-1958

Received/Geliş tarihi: March 23, 2016


321

Sarıtürk Ç, et al: Visual Information for Stem Cell Donation Turk J Hematol 2017;34:321-327

Introduction

Stem cell transplantation (SCT) is a procedure with severe

morbidity and mortality, but it also has the potential for

long-term survival and recovery [1]. An informed discussion

with the patient and his or her relatives and a comprehensive

examination of the patient and the donor that includes

psychosocial aspects are central to pre-transplant preparation

[2]. SCT cannot be performed without collaboration with the

patients and their relatives, as treatment may result in shortand

long-term changes that affect the patient’s life. Therefore,

patient contribution is essential for a detailed educational

discussion and provision of informed consent [3,4,5,6,7].

The rationale, procedure, and potential outcomes for SCT

can be difficult to understand [1,3]. As the patient may

have severe anxiety due to an often difficult diagnosis and

potentially fatal outcome, it is unrealistic to expect the patient

to easily understand this information. Therefore, to overcome

this difficulty, transplant doctors have developed their own

communication methods based on personal experiences.

The reason for SCT is often not clear to donors and recipients,

and the benefits and drawbacks of transplantation may need

to be discussed in detail. The correct timing for transplantation

is another issue. In many situations, transplantation may be

postponed until other therapeutic methods are attempted [3].

Once transplantation becomes feasible, both short- and longterm

adverse events are discussed. Patients are informed clearly

and objectively about potential side effects. The possibility

of procedure-related death and other severe conditions (e.g.,

admission to the intensive care unit or life support) are also

discussed [7]. Requirements for interventional procedures

to evaluate potential side effects are also covered. Nonfatal

side effects (e.g., chronic graft-versus-host disease) are

mentioned as possible long-term effects. Informed consent is

only obtained after this information is clearly communicated

in accordance with laws, regulations, and standards [7].

The goal of patient and donor education is to help them understand

and accurately evaluate the information and risks. Therefore,

there needs to be verification of patient and donor understanding

throughout the educational procedure for SCT [7]. However,

there are a limited number of reports describing the effectiveness

of visual methods in patient/donor education about SCT.

On this study, we investigated the effectiveness of an

informational animation for transplant patients and donors in

pre-transplant education.


Study Design

This study was conducted between June 2013 and July 2014

using a prospective, randomized, cross-sectional singlecenter

design. The sample comprised adult patients who were

scheduled to undergo autologous or allogeneic peripheral SCT

at the Adana Bone Marrow Transplantation Unit of Başkent

University Faculty of Medicine and donors from whom

peripheral stem cell collection for allogeneic transplantation

was planned. The standard operating procedure (SOP: KIT-

KU 005) was applied to patients and donors after the council

decision had been obtained for transplantation and cell

collection, in accordance with JACIE standards. The transplant

coordinator invited donors and patients to participate in the

study. The clinical medical director, transplant doctor, and

transplant coordinator also participated in the informational

meeting, and a transplant nurse participated when necessary.

Donors were asked to attend the session alone in accordance

with the donor privacy principle. However, patients could

request that first-degree relatives attend the session with them.

Participants were randomly selected and divided into two groups.

Group 1 was the study group, exposed to audiovisual information

in addition to standard verbal and written information. Group

2 was the control group and received only standard verbal

and written information. The transplant coordinator obtained

feedback from participants and completed a questionnaire that

measured the quality of the information session. To eliminate

ethical problems, the audiovisual information was provided

for group 2 (control group) after their initial feedback on the

information session. The verbal information in the sessions was

delivered by two separate transplant doctors to test whether the

audiovisual method was affected by interpersonal differences.

The results were evaluated in accordance with the rules stated

in the Clinical Trials section of the JACIE standards by the

Study Board of the Başkent University Adana Bone Marrow

Transplantation Unit. Approval was obtained from the Başkent

University Scientific Research Board.

Verbal and Written Information

Both groups received standard verbal and written information.

This covered disease status, purpose of the treatment, treatment

principles, stem cell collection procedure, pretreatment

assessment, the drugs used and their side effects, infusion of

stem cells, benefits expected from the treatment, treatment

risks and side effects, other treatment options, and disposal

of the cellular product. These topics were prepared locally in

accordance with international standards (FACT-JACIE standards)

to meet donor and recipient information requirements. Patients

and donors were able to ask questions after the information

session had been completed [4,5,6,7]. The information session

lasted up to 30 min.

Information Animation

The information animation was based on the flow of the topics

discussed in the verbal and written information session. Some

topics were covered in movie format and others were shown as

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Sarıtürk Ç, et al: Visual Information for Stem Cell Donation

graphics and images. A Three D Studio Max program used to

prepare the program and the technical support was provided by

a technical company (Teknik Medya, Adana, Turkey). The visual

animation lasted 10 min, and there were Turkish, Arabic, and

English language and caption options.

Obtaining Patient and Donor Feedback

A 20-item questionnaire was used to collect feedback

including demographic data (age, sex, education status, job,

and the institute where the patient was first diagnosed). In

addition, a 20-item scale was prepared to measure participants’

satisfaction. Seven items assessed satisfaction with the written

informed consent form, seven items were about the information

provided by the doctor, and six items focused on the audiovisual

information. After an interactive interview, participants scored

each question from 1 to 10 based on their satisfaction level.

The questions were pre-tested with 10 randomly selected

healthy subjects before the study to confirm intelligibility. The

scores of the 20 questions were summed to give the overall

satisfaction level. The seven questions concerning the written

informed consent tested satisfaction with the information on

the informed consent form regarding issues such as side effects,

stages of treatment, and treatment method. Questions about the

doctor who provided the information evaluated the same issues.

The questions measuring satisfaction with the information

animation also evaluated how the patient understood the

stages of the disease and the treatment process.


Statistical analysis was performed with SPSS 17.0. Categorical

measurements were summarized as number and percentage

and continuous measurements as mean and standard deviation

(median and minimum - maximum where needed). Chi-square

or Fisher’s exact tests were used for comparison of categorical

variables. The inter-rater agreement was analyzed with kappa

statistics. The consistency between questions was evaluated

using Cronbach’s alpha coefficient. The value of Cronbach’s

alpha coefficient reflects the reliability and internal consistency

of the scale (<0.40 indicates that a scale is not reliable, 0.40

to <0.50 indicates very low reliability, 0.50-0.60 low reliability,

0.60-0.70 sufficient reliability, 0.70-0.90 high reliability, and

≥0.90 very high reliability). The reliability of the scale was

tested with factor analysis. The appropriateness of the data

structure for factor analysis was evaluated with the Kaiser-

Meyer-Olkin (KMO) test, where <0.50 indicates that factor

analysis could not be continued, 0.50 and 0.60 are interpreted

as poor, 0.60-0.70 as weak, 0.70-0.80 as moderate, 0.80-

0.90 as good, and above 0.90 as excellent. Bartlett’s test of

sphericity was used to test the association between statements.

Distributions were controlled for inter-group comparisons.

Student’s t-test was used for variables showing parametric

distribution, and the Mann-Whitney U test was used for

variables not showing parametric distribution. A p-value of less

than 0.05 was considered statistically significant.

Results

In total, 92 subjects who were scheduled to undergo autologous or

allogeneic hematopoietic peripheral SCT and peripheral stem cell

donors for allogeneic transplantation were invited to participate

in the study. Of these, 82 (89%) agreed to participate, and 41

participants were assigned to each group. The ten individuals who

did not agree to participate were allogeneic SCT recipients. The

mean age of participants was 47±14 years (range: 15-67). Mean

age of both group 1 and group 2 was 47±14 years (p=0.886).

Nine participants (11%) were sibling donors, all in group 1.

Participants’ demographic characteristics are shown in Table 1. No

statistically significant differences were found between groups

in terms of sex, marital status, educational status, center where

their diagnosis was made, and patient diagnoses (p>0.05, for all).

Before starting the reliability and validity analysis, the

present researchers reviewed the questionnaires to

determine the participants who had repeated the same

answer. This was not found to be a significant problem, and

analysis continued with 82 participants. The questionnaire

used to determine patient satisfaction had a Cronbach’s

alpha coefficient of 0.94 (95% confidence interval

[CI] 0.92-0.96), indicating that it was highly reliable.

We used factor analysis to measure the validity of the scale. The

20-item satisfaction scale comprised three parts. Consistency

of data in factor analysis was measured with KMO sample

sufficiency and Bartlett’s test of sphericity. The KMO value was

0.769 and the Bartlett’s test results were statistically significant

(χ 2 =2216.4, p=0.0001). The results of both tests showed that

factor analysis of satisfaction scale data was appropriate.

In factor analysis of the 20-item satisfaction scale, questions

with sample adequacy below 0.50 were investigated. No

questions were eliminated because no statement showed

a factor load below 0.50. Factor analysis detected three

factors, all of which had eigenvalues of ≥1, and there were

no overlapping expressions. Factor 1 comprised seven items

and explained 25.6% of the total variance, factor 2 explained

15.4% of the variance, and factor 3 explained 25.7%. The

total variance explained by three factors was 66.7% (Table 2).

The Cronbach’s alphas for the satisfaction scale were

0.95 for written consent, 0.91 for the informing

doctor, and 0.90 for the informational animation.

This indicates that all sections were highly reliable.

The first physician conducted information sessions for 41

participants (21 in group 1, 20 in group 2). The second

physician conducted the information sessions for the

remaining 41 participants (19 in group 1, 22 in group 2). Both

physicians had 10 years of experience as transplant physicians.

323


Table 1. Characteristics of responder patients at the time of the information.

Sex

Total Group 2 Group 1

n % n % n % p-value

Female 36 43.9 15 35.7 21 52.5 0.182

Male 46 56.1 27 64.3 19 47.5

Marital Status

Married 67 81.7 35 83.3 32 80.0 0.811

Single 12 14.6 6 14.3 6 15.0

Divorced-Widow 3 3.7 1 2.4 2 5.0

Educational Status

Primary School 47 57.3 23 54.8 24 60.0 0.555

Middle School 9 11.0 4 9.5 5 12.5

High School 13 15.9 9 21.4 4 10.0

Academic 13 15.8 6 14.3 7 17.5

Origin of First Diagnosis

Başkent University 41 50.0 20 47.6 21 52.5 0.825

Other 41 50.0 22 52.4 19 47.5

Diagnosis

NHL 18 22.0 11 26.2 7 17.5 0.149

ALL 9 11.0 5 11.9 4 10.0

Multiple myeloma 26 31.7 12 28.6 14 35.0

CML 1 1.2 1 2.4 0 0

Acute Leukemia (de novo) 2 2.4 1 2.4 1 2.5

Hodgkin’s Lymphoma 2 2.4 2 4.8 0 0

Sickle Cell Disease 2 2.4 1 2.4 1 2.5

MDS 3 3.7 1 2.4 2 5,0

tAML 2 2.4 1 2.4 1 2.5

NHL: Non-Hodgkin’s lymphoma, ALL: acute lymphoblastic leukemia, AML: acute myeloblastic leukemia, CML: chronic myelocytic leukemia, MDS: myelodysplastic syndrome,

tAML: transformed acute myeloblastic leukemia.

The Cronbach’s alpha for participants’ satisfaction with the

patient/donor information session was 0.94 (95% CI 0.92-0.96).

Table 3 shows participants’ satisfaction with the information

session. Satisfaction with the written informed consent,

the informing doctor, and the informational animation was

measured with an overall satisfaction level that was high

(184.8±19.8) compared with the maximum value of 200. There

was no significant difference between groups with regard

to overall satisfaction. However, a statistically significant

difference was found between groups for satisfaction with

the written informed consent form. Patient satisfaction was

greater in group 1 (p=0.039) (Table 3; Figure 1). There were no

significant differences between groups for satisfaction with the

doctor who provided the information and the informational

animation. The level of satisfaction was not affected by sex or

educational status in either group (p>0.05 for all).

Figure 1. Patient satisfaction with the written consent.

CI: Confidence interval.

324



Table 2. Factor analysis.

Satisfaction with the written consent

Factor

loadings

Information in the informed consent is understandable enough 0.851

The treatment plan is understandable in the informed consent 0.700

The benefits of the treatment are understandable enough in the informed consent 0.805

The risks and side effects of the treatment are understandable enough in the informed consent 0.829

The procedure of stem cell transplantation is understandable enough in the informed consent 0.865

The method of stem cell collection from the blood has been described clearly in the informed consent form 0.693

The medications that will be used during the treatment and their side effects are understandable enough in

the informed consent

Satisfaction with the physician

My doctor has provided me enough information about my disease 0.664

My doctor has provided me information about the stages of my treatment 0.601

My doctor has provided me detailed information about the risks and the side effects of the recommended

treatment

My doctor has provided me enough information about the treatment options other than this treatment

method

My doctor has provided me information about the management of the side effects 0.731

My doctor has answered my questions in detail 0.729

I know that I can always reach my doctor when I have some questions in my mind 0.783

Satisfaction with the informational animation

I could find more clear answers to my questions following the animation 0.829

I could better understand the stages of my treatment through the animation 0.521

I could better understand the benefits of the treatment through the animation 0.747

I could better understand the risks and the side effects of the treatment through the animation 0.799

I could better understand how the bone marrow transplantation would be performed through the animation 0.830

I could better understand the stem cell collection procedure through the animation 0.787

0.638

0.597

0.770

Factors

explaining the

variance

25.384

15.374

25.478

Table 3. Participant satisfaction.

Total

Group 1

Group 2

SD: Standard deviation.

Overall satisfaction

Satisfaction with the

written informed consent


informing physician

n 82 82 82 82

Mean ± SD 184.8±19.8 62.3±8.5 65.8±7.4 56.7±6.3

n 42 42 42 42

Mean ± SD 185.7±22.2 64.2±8.5 64.7±8.6 56.8±6.6

n 40 40 40 40

Mean ± SD 183.8±17.2 60.3±8.2 67.0±5.8 56.5±6.2

p 0.671 0.039 0.161 0.827


informational animation

Discussion

This study investigated the effectiveness of audiovisual

information in providing essential information during the informed

consent process for patients and donors. There were differences

in patient/donor satisfaction between the session that included

audiovisual information and that comprising standard verbal/

written explanations. To our knowledge, this is the first study

to investigate the effects of audiovisual materials in providing

information to hematopoietic stem cell recipients and donors.

In general, the use of audiovisual materials facilitates learning and

reduces learning time [8,9]. This observation has been supported

325


physiologically [10,11] and is reminiscent of the Chinese saying

“I forget if I hear, I remember if I see, I learn if I do”. In this

context, our study aimed to develop an information technique

using audiovisual methods and demonstrate its efficacy with

a verification study in the context of an important issue

such as bone marrow transplantation and stem cell donation.

Hematopoietic SCT is an effective treatment that is performed

for many life-threatening diseases; however, it may result in

significant morbidity and mortality [3]. International standards

and national laws and regulations require that informed consent

be obtained from bone marrow recipients and bone marrow and

stem cell donors [7]. These individuals are informed about the

rationale of the procedure, expectations, application technique,

potential difficulties, and other options if the procedure is

not approved. Verbal and written information is provided. The

main goal of this information is to enable the subject to make

accurate risk assessments and provide informed consent. This

is related to correct understanding of the information [12].

A standard operating procedure was produced for the

information methods used in this study. In this procedure, we

determined the information field, the individuals responsible

for patient/donor information, the individuals who would

attend the formal meeting, national laws and regulations, and

the information required for FACT-JACIE standards [7]. The

duration of the informational session and the materials used

in the session and consent process were standardized. Approval

was obtained from parents or custodians for subjects aged

<18 years and those who were not able to give consent (e.g.,

disabled subjects). A special arrangement was made for pediatric

patients or donors (i.e. a psychiatrist joining the interview).

We found no statistically significant difference between groups

for overall satisfaction. However, satisfaction with the written

informed consent form was greater in the study group compared

with the control group. Participants in the study group answered

questions about treatment stages after sequentially being given

written, verbal, and video explanations, and they were satisfied

with the information given about their disease. However, the

control group provided initial feedback after the written and

verbal information session and additional feedback after they

viewed the animation. Our results indicated that the video helped

patients obtain a more accurate understanding of their disease.

Current FACT-JACIE standards (version 6.0) do not require

visual materials in addition to verbal and written information

for hematopoietic stem cell recipients and donors [7]; verbal

and written information is considered sufficient. However, the

results of our study support the theory that there are benefits

to providing information using audiovisual materials, such

as a decrease in perception difficulty arising from language

and cultural differences, intellectual differences, and aging.

In the present study, participants in both groups evaluated

their experiences of the physician who conveyed information

as similar, which may be regarded as a limitation of the study.

However, as bone marrow transplantation is a critical issue, it

is likely that doctors use similar statements to convey essential

information.

Conclusion

In conclusion, using audiovisual materials and standard methods

for providing information to bone marrow recipients and donors

may positively affect patient/donor perception and overcome

unnecessary anxiety. Although we obtained data about patient/

donor satisfaction at the time of providing informed consent,

further studies may reveal whether better understanding of the

transplant/donation procedure would result in better transplant/

donation experience and outcomes.

Ethics

Ethics Committee Approval: Başkent University Adana

Application and Research Center (KA13/165).

Informed Consent: It was taken.


Surgical and Medical Practices: Ç.G., A.K., S.A., M.Y., S.S., N.B.,

C.B., H.Ö.; Concept: Ç.S., İ.K., C.B., H.Ö.; Design: Ç.S., İ.K., C.B.,

H.Ö.; Data Collection or Processing: S.T., İ.K., C.B., H.Ö.; Analysis

or Interpretation: Ç.S., C.B., H.Ö.; Literature Search: Ç.S., Ç.G.,

A.K., S.A., M.Y., S.S., N.B., S.T., İ.K., C.B., H.Ö.; Writing: Ç.S., Ç.G.,

C.B.




included.

References

1. Mohty B, Mohty M. Long-term complications and side effects after

allogeneic hematopoietic stem cell transplantation: an update. Blood

Cancer J 2011;1:e16.

2. Munzenberger N, Fortanier C, Macquart-Moulin G, Faucher C, Novakovitch

G, Maraninchi D, Moatti JP, Blaise D. Psychosocial aspects of haematopoietic

stem cell donation for allogeneic transplantation: how family donors cope

with this experience. Psychooncology 1999;8:55-63.

3. Confer DL. Hematopoietic cell donors. In: Blume KG, Forman SJ, Appelbaum

F (eds). Thomas’ Hematopoietic Cell Transplantation, Volume 1. Malden,

Blackwell, 2004.

4. Atlas LD. The National Marrow Donor Program in 2006: constants and

challenges. Transfusion 2006;46:1080-1084.

5. Anderlini P, Rizzo JD, Nugent ML, Schmitz N, Champlin RE, Horowitz MM;

IBMTR Statistical Center of the International Bone Marrow Transplant

Registry, Medical College of Wisconsin, Milwaukee, WI, USA; EBMT, Kiel,

Germany. Peripheral blood stem cell donation: an analysis from the

International Bone Marrow Transplant Registry (IBMTR) and European

Group for Blood and Marrow Transplant (EBMT) databases. Bone Marrow

Transplant 2001;27:689-692.

326



6. National Marrow Donor Program. Who We Are: About the National Marrow

Donor Program. Available at https://bethematch.org/about-us/.

7. FACT-JACIE International Standards For Cellular Therapy, 6th ed. Product

Collection, Processing and Administration. Version 6.0. Omaha, Foundation

for the Accreditation of Cellular Therapy, 2015.

8. Rowbotham MC, Astin J, Greene K, Cummings SR. Interactive informed

consent: randomized comparison with paper consents. PLoS One

2013;8:e58603.

9. Wong ST, Pérez-Stable EJ, Kim SE, Gregorich SE, Sawaya GF, Walsh JM,

Washington AE, Kaplan CP. Using visual displays to communicate risk of

cancer to women from diverse race/ethnic backgrounds. Patient Educ

Couns 2012;87:327-335.

10. Alsius A, Munhall KG. Detection of audiovisual speech correspondences

without visual awareness. Psychol Sci 2013;24:423-431.

11. Torsvik T, Lillebo B, Mikkelsen G. Presentation of clinical laboratory results:

an experimental comparison of four visualization techniques. J Am Med

Inform Assoc 2013;20:325-331.

12. Kish A, Lenfoff S, Bengtsson M, Bolmsjö I. Potential adult sibling stem cell

donors’ perceptions and opinions regarding an information and care model.

Bone Marrow Transpl 2013;48:1133-1137.

327

RESEARCH ARTICLE

DOI: 10.4274/tjh.2016.0343


Influence of L-Carnitine on Stored Rat Blood: A Study on Plasma

L-Karnitinin Depolanmış Sıçan Kanı Üzerine Etkisi: Plazmada Yapılan Bir Çalışma

Carl Hsieh, Vani Rajashekharaiah

Jain University, Center for Post Graduate Studies, Department of Biotechnology, Bangalore, India

Abstract

Objective: Plasma acts as a good indicator of oxidative stress in blood.

L-Carnitine is an antioxidant that reduces metabolic stress in cells,

thereby providing a protective effect against oxidative stress (OS).

L-Carnitine as an additive in storage has not been explored. Thus, this

study attempts to analyze the role of L-carnitine in blood storage

solution, citrate phosphate dextrose adenine (CPDA)-1, through

OS markers including antioxidant enzymes, lipid peroxidation, and

protein oxidation.

Materials and Methods: Blood was collected from male Wistar rats

and stored in CPDA-1 solution with L-carnitine (10 mM, 30 mM, and

60 mM: groups LC 10, LC 30, and LC 60, respectively) and without

L-carnitine (control group). Plasma was isolated every 5 th day and the

OS markers were analyzed.

Results: Superoxide dismutase (SOD) and sulfhydryl (SH) increased

over storage in controls, LC 30, and LC 60. Catalase increased in LC

30 and LC 60 during storage. Thiobarbituric acid reactive substances

(TBARS) and protein carbonyl (PrC) levels in all groups increased

initially and reduced towards the end of storage. SOD and SH levels

were maintained while TBARS and PrC levels increased in LC 10.

Conclusion: L-Carnitine was beneficial in terms of increased

antioxidant capacity and SH and decreased lipid peroxidation. This

forms the basis for further studies on L-carnitine as a constituent in

storage solutions.

Keywords: L-Carnitine, Plasma, Antioxidant enzymes, Lipid

peroxidation, Protein oxidation

Öz

Amaç: Plazma kanda oksidatif stresin iyi bir göstergesi olarak görev

yapar. L-karnitin hücrelerde metabolik stresi azaltan böylece oksidatif

strese (OS) karşı koruyucu etki sağlayan bir antioksidandır. L-Karnitinin

depolamada katkı maddesi olarak kullanımı araştırılmamıştır. Bu

nedenle, bu çalışmada kan depolama solüsyonu olan sitrat fosfat

dekstroz adenin (CPDA)-1 ‘e eklenen L-karnitinin rolü antioksidan

enzimler, lipid peroksidasyonu ve protein oksidasyonunu içeren OS

göstergeleri aracılığı ile analiz edilmektedir.

Gereç ve Yöntemler: Wistar sıçanlarından kan örneği alındı ve CPDA-

1 solüsyonunda L-karnitin ile (10 mM, 30 mM, ve 60 mM: Sırasıyla

LC 10, LC 30 ve LC 60 grupları) ve L-karnitinsiz (kontrol grup) olarak

depolandı. Plazma her 5. günde izole edildi ve OS göstergeleri analiz

edildi.

Bulgular: Süperoksit dismutaz (SOD) ve sülfidril (SH) kontroller de

(LC30 ve LC60) depolama boyunca arttı. Katalaz LC30 ve LC60 da

depolama sırasında arttı. Tiyobarbitürik asit reaktif maddesi (TBARS)

ve protein karbonil (PrC) düzeyleri tüm gruplarda başlangıçta arttı

ve depolama sonuna doğru azaldı. SOD ve SH düzeyleri LC 10’da

korunurken TBARS ve PrC düzeyleri arttı.

Sonuç: L-Karnitin antioksidan kapasite ve SH artışı ile lipid

peroksidasyonu azalması açısından faydalı idi. Bu durum, L-karnitinin

depolama solüsyonlarında bileşen olarak ileri çalışmaları yapılması için

bir temel oluşturmaktadır.

Anahtar Sözcükler: L-Karnitin, Plazma, Antioksidan enzimler, Lipid

peroksidasyonu, Protein oksidasyonu

Introduction

L-Carnitine (L-3 hydroxy-4-N-N-N-trimethylaminobutyrate)

is an essential nutrient that the body uses to convert fat into

energy. It is required for the transport of fatty acids from the

cytosol into the mitochondria during breakdown of lipids via

β-oxidation. It acts as an antioxidant that reduces metabolic

stress in cells, thereby providing a protective effect against

lipid peroxidation and oxidative stress (OS) in the phospholipid

membrane and the myocardial and endothelial cells [1].

Disturbances in the redox state can cause OS, which is an

imbalance between the production of reactive oxygen species

(ROS) and the biological system’s natural ability to detoxify

these intermediates or repair the resulting damage caused

by them [2]. OS is also induced during storage of blood. The



Address for Correspondence/Yazışma Adresi: Vani RAJASHEKHARAIAH, PhD,

Jain University, Center for Post Graduate Studies, Department of Biotechnology, Bangalore, India

Phone : +91 988 6178 584

E-mail : tiwari.vani@gmail.com ORCID-ID: orcid.org/0000-0002-4155-0960


Accepted/Kabul tarihi: December 28, 2016

328


Hsieh C and Rajashekharaiah V, Influence of L-Carnitine on Stored Rat Blood

constituents of plasma change during storage due to cell

metabolism and the activation of proteolytic activity [3]. These

changes can be attributed to the depletion of glucose levels in

plasma, cationic pump failure, hemoconcentration, and leakage

of cell constituents and metabolites in erythrocytes [4].

Plasma reflects the overall OS environment in whole blood as

it holds all the cellular components in suspension. Thus, plasma

serves as a good candidate for assessing the changes occurring

during whole blood storage. The study of d’Almeida et al. [5]

reported that 1 week of rat blood storage is equivalent to 4

weeks of human blood storage. Thus, rat blood was used for

studying storage lesions.

The ability of L-carnitine to combat OS was studied by Li et

al. [1], where it was found that L-carnitine could protect

hepatocytes through its antioxidant effect. L-Carnitine could

reduce free radical-induced oxidative damage of intermittent

hypoxia exposure, thus delaying muscle fatigue [6,7].

Various studies have reported on the effects of L-carnitine on

other blood components (erythrocytes and platelets) [8,9,10,11].

Although there are reports on plasma storage [12,13,14,15],

very few have focused on plasma isolated from stored blood.

However, the influence of Carnitine on stored blood is still

unclear. Thus, this study attempts to analyze the role of

L-carnitine as a constituent in blood storage solution through

OS markers (antioxidant enzymes, lipid peroxidation, and

protein oxidation) in plasma.


Animal care and maintenance was in accordance with the

Ethical Committee regulations (841/b/04/CPCSEA).

Chemicals

Epinephrine, thiobarbituric acid (TBA), and bovine serum

albumin (BSA) were purchased from Sigma-Aldrich Chemicals

(St. Louis, MO, USA). All other chemicals used were of reagent

grade and organic solvents were of spectral grade.

Blood Sampling

Animals were lightly anesthetized with ether and restrained in

dorsal recumbency as described earlier [16]. In brief, the syringe

needle was inserted just below the xiphoid cartilage and slightly

to the left of the midline. Blood was carefully aspirated from the

heart into polypropylene collection tubes with citrate phosphate

dextrose adenine (CPDA)-1.

Experimental Design

Blood was drawn from 4-month-old male Wistar rats and stored

over a period of 20 days at 4 °C in CPDA-1. The samples from 20

animals were divided into 4 groups of 5 animals each: i) controls,

ii) LC 10 (samples with L-carnitine at a concentration of 10 mM),

iii) LC 30 (samples with L-carnitine at a concentration of 30

mM), and iv) LC 60 (samples with L-carnitine at a concentration

of 60 mM). Plasma was isolated from whole blood at regular

intervals of 5 days and biomarkers, i.e. antioxidant enzymes, lipid

peroxidation, and protein oxidation products, were assessed.

Plasma Separation

Plasma was isolated in microcentrifuge tubes by centrifuging

for 20 min at 1000 × g. The plasma was removed and stored in

isotonic phosphate buffer at -20 °C for further assays [17].

Antioxidant Enzymes

Superoxide Dismutase [EC 1.15.1.1]: Superoxide dismutase

(SOD) was measured by the method of Mishra and Fridovich [18].

Plasma was added to carbonate buffer (0.05 M). Epinephrine

was added to the mixture and absorbance was measured at 480

nm. SOD activity was expressed as the amount of enzyme that

inhibited oxidation of epinephrine by 50%.

Catalase [EC 1.11.1.6]: Catalase (CAT) was determined by the

method of Aebi [19]. Briefly, plasma with absolute alcohol was

incubated at 0 °C. An aliquot was taken up with 6.6 mM H 2

O 2

and the decrease in absorbance was measured at 240 nm. An

extinction coefficient of 43.6 M cm -1 was used to determine

enzyme activity.

Lipid Peroxidation: Thiobarbituric Acid Reactive Substances

Thiobarbituric acid reactive substances (TBARS) content was

determined by the method of Bar-Or et al. [20]. Plasma with

0.9% sodium chloride was incubated at 37 °C for 20 min, and

then 0.8 M HCl containing 12.5% trichloroacetic acid (TCA) and

1% TBA was added and samples were kept in a boiling water

bath for 20 min and cooled at 4 °C. Centrifugation was carried

out at 1500 × g and absorbance was measured at 532 nm. TBARS

content was calculated by using the extinction coefficient of

1.56x10 5 M -1 cm -1 .

Protein Oxidation

Protein carbonyls: Protein carbonyl (PrC) content was

determined by the method of Reznick and Packer [21]. PrC

content was measured by forming a labeled protein hydrazone

derivative using 2,4-dinitrophenyl hydrazine (DNPH), which was

then quantified spectrophotometrically. Briefly, after precipitation

of protein with an equal volume of 1% TCA, the pellet was

resuspended in 10 mM DNPH. Samples were kept in the dark for 1

h. An equal volume of 20% TCA was added and left on ice for 10

min and then centrifuged at 3000 × g, and the pellet was washed

with an ethanol-ethyl acetate mixture (1:1) to remove the free

DNPH and lipid contaminants. The final pellet was dissolved in 6

M guanidine HCl in 133 mM Tris and absorbance was measured

329

Hsieh C and Rajashekharaiah V, Influence of L-Carnitine on Stored Rat Blood Turk J Hematol 2017;34:328-333

at 370 nm. PrC content was calculated by using the extinction

coefficient of 20,000 M -1 cm -1 .

Protein Sulfhydryls

The protein sulfhydryl (P-SH) concentration in the proteins

was measured as described by Habeeb [22]. In brief, 0.08

mol/L sodium phosphate buffer containing 0.5 mg/mL Na 2

-

ethylenediaminetetraacetic acid and 2% SDS was added to

the sample in each assay tube, and then 0.1 mL of 5,5’-DTNB

was added and the solution was vortexed. Color was allowed

to develop at room temperature and absorbance was measured

at 412 nm. P-SH was calculated from the net absorbance and

molar absorptivity, 13,600 M -1 L -1 cm -1 .

Protein

Protein was determined in the plasma by the method of Lowry

et al. [23] using BSA as the standard.

Catalase

Changes in CAT were significant with storage in all groups.

Control and LC 10 CAT levels were maintained over storage. LC

30 and LC 60 levels were significantly increased at days 15 and

20 (Figure 2).

Lipid Peroxidation - Thiobarbituric Acid Reactive Substances

Changes in TBARS were significant in controls during storage.

TBARS peaked on day 15 in controls. An increase in TBARS was

observed in LC 10 over storage. TBARS content was maximum

on day 10 in LC 30 and LC 60 samples (Figure 3).

Protein Oxidation

Protein carbonyls: Changes in PrC were significant in all groups

with storage. PrC levels were increased at days 15 and 20 in


Results are represented as mean ± standard error. The

Kolmogorov-Smirnov test was performed for suitability of the

data. Values between the groups (storage period) and subgroups

(antioxidants) were analyzed by two-way ANOVA and differences

were considered significant at p<0.05. The Bonferroni post-test

was performed using GraphPad Prism 6 software.

Results

Superoxide Dismutase

Significant changes in SOD were observed in all groups with

storage. SOD in controls increased during storage. SOD was

maintained in LC 10 throughout the storage period. LC 30

and LC 60 samples showed increments in SOD over storage (Figure 1).

Figure 2. Catalase activity in plasma isolated from stored blood.

LC 10: L-carnitine 10 mM, LC 30: L-carnitine 30 mM, LC 60: L-carnitine 60 mM.

Values are mean ± SE of five animals/group. Two-way ANOVA was performed

between the groups and subgroups to analyze catalase activity followed by the

Bonferroni post-test, using GraphPad Prism 6 software. Changes between the

groups are represented in upper case. Changes within the groups are represented

in lower case. Those not sharing the same letters are significantly different.

Figure 1. Superoxide dismutase activity in plasma isolated from

stored blood.



between the groups and subgroups to analyze superoxide dismutase activity

followed by the Bonferroni post-test, using GraphPad Prism 6 software. Changes

between the groups are represented in upper case. Changes within the groups

are represented in lower case. Those not sharing the same letters are significantly

different.

Figure 3. Thiobarbituric acid reactive substances in plasma

isolated from stored blood.



between the groups and subgroups to analyze thiobarbituric acid reactive

substances followed by the Bonferroni post-test, using GraphPad Prism 6

software. Changes between the groups are represented in upper case. Changes

within the groups are represented in lower case. Those not sharing the same

letters are significantly different.

330



controls. PrC was significantly higher than in the other groups

throughout the storage period in LC 10. PrC peaked on day 15

in LC 30 and LC 60 samples (Figure 4).

Protein sulfhydryls: P-SH levels varied significantly over

storage. P-SH increased gradually throughout the storage

period in controls. P-SH was maintained in LC 10 samples over

the storage period. P-SH was significantly higher in LC 30 and

LC 60 than controls and LC 10 (Figure 5).

Discussion

This study assessed whole blood through plasma, as it holds all

the blood components in suspension. It gives an overall view of

the OS microenvironment during storage.

SODs are a group of enzymes that catalyze the conversion of

superoxide into H 2

O 2

and O 2

. Increase in the activity of SOD is

Figure 4. Protein carbonyls in plasma isolated from stored blood.



between the groups and subgroups to analyze protein carbonyl followed by




Figure 5. Protein sulfhydryls in plasma isolated from stored blood.



between the groups and subgroups to analyze protein sulfhydryl followed by




generally a sign of increased formation of superoxide radicals

and thus elevated OS [24]. This was evident in our results

of increased SOD activity during storage. L-Carnitine is a

scavenger of free radicals and protects the cells from OS [25].

Similar results were observed in our study, where L-carnitine

upregulated SOD [1,26]. SOD increased on day 10 and peaked on

day 15 in controls, due to maximum ROS [27]. The decrease on

day 20 in controls can be attributed to ROS overwhelming the

antioxidant enzyme capacity and thus inactivating the enzyme.

A similar trend was observed in LC 10 from day 15. SOD varied

from day 10 onwards in LC 30, which can be attributed to the

modulation of the enzyme activity by L-carnitine in proportion

to superoxides.

CAT degrades H 2

O 2

to H 2

O and O 2

. H 2

O 2

can also be scavenged

by glutathione peroxidase (GPX) [28]. CAT activity was low

initially and increased over storage. This may be due to the

activity of GPX scavenging H 2

O 2

at lower concentrations, while

CAT decomposes H 2

O 2

only at high concentrations [29,30]. CAT

levels were highest on day 15 in controls, similar to SOD, due to

maximum ROS being produced on that day [23]. CAT expression

in LC 10 was in accordance with the SOD levels, where the levels

dropped on day 15. Li et al. [1] and Cao et al. [26] showed that

L-carnitine increased CAT expression, which was also observed

in our results. The increase in CAT in LC 30 and LC 60 can be

attributed to L-carnitine’s ability to upregulate antioxidant

enzyme activity.

TBARS, a biomarker of lipid peroxidation, is a reasonable reflection

of a nonlipophilic peroxidation product, malondialdehyde [26].

The peak on day 15 in controls may be attributed to greater

amounts of ROS generated [27]. L-Carnitine at concentrations

of 30 mM and 60 mM reduced TBARS over storage. This may

be due to L-carnitine’s ability to scavenge ROS and upregulate

antioxidant enzymes at higher concentrations. L-Carnitine

also has the property of preventing the accumulation of lipid

peroxidation end products, hence causing the decrease in TBARS

[31]. This was evident in our results of lipid peroxidation.

Oxidative cleavage of the protein backbone, oxidation of

amino acids, or binding of aldehydes produced from lipid

peroxidation produces PrC. It is formed early and circulates

in the blood for longer periods as it is more stable than lipid

peroxidation products [32]. Protein oxidation products are

effective biomarkers of OS due to their long half-lives [33]. PrC

increased in controls over storage, indicative of oxidative insult

and protein damage. Addition of L-carnitine at 30 mM and 60

mM did not alter the PrC levels significantly, suggesting that

L-carnitine could not alter the formation of carbonyls.

P-SH gets oxidized to disulfides, which is a reversible reaction.

It is mainly present in the cysteine components of proteins

and generally at lower concentrations in glutathione [34].

331

Hsieh C and Rajashekharaiah V, Influence of L-Carnitine on Stored Rat Blood Turk J Hematol 2017;34:328-333

P-SH increased over storage in controls, which indicates that

the endogenous antioxidant system could combat OS during

storage. The increase in P-SH with L-carnitine indicates that

it could protect sulfhydryl groups against oxidation or was

effective in catalyzing the reversible change of disulfides to

sulfhydryls [26].

Arduini et al. [11] reported L-carnitine to be beneficial at 5 mM

in terms of increased ATP concentrations and reduced hemolysis

over storage. However, our study showed that L-carnitine at 10

mM could not prevent protein oxidation and lipid peroxidation,

but LC 30 and LC 60 had reduced oxidative damage through

reduced TBARS and elevated P-SH and antioxidant enzymes

(SOD and CAT).

Conclusion

In conclusion, antioxidant enzymes in plasma could combat

the ROS generated during storage. Our study showed that

L-carnitine at higher concentrations can be further explored as

a constituent of storage solutions as it significantly upregulated

the antioxidant capacity of plasma and reduced oxidative

damage during storage. Therefore, L-carnitine is a promising

constituent in blood storage solutions.

Acknowledgments

The authors acknowledge Dr. Leela Iyengar, Ms. Soumya

Ravikumar, Mrs. Manasa K, and Jain University for their support.

Ethics

Ethics Committee Approval: The Committee for the Purpose of

Control and Supervision of Experiments on Animals (841/b/04/

CPCSEA).

Informed Consent: N/A.


Surgical and Medical Practices (Sample collection): C.H.;

Concept: V.R.; Design: V.R.; Data Collection or Processing: C.H.;

Analysis or Interpretation: C.H.; Literature Search: C.H.; Writing:

C.H., V.R.




included.

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3. Ayache S, Panelli M, Marincola FM, Stroncek DF. Effects of storage time and

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4. Boyanton BL, Blick KE. Stability studies of twenty-four analytes in human

plasma and serum. Clin Chem 2002;48:2242-2247.

5. d’Almeida MS, Jagger J, Duggan M, White M, Ellis C, Chin-Yee IH. A

comparison of biochemical and functional alterations of rat and human

erythrocytes stored in CPDA-1 for 29 days: implications for animal models

of transfusion. Transfus Med 2000;10:291-303.

6. Vani R, Shivashankar Reddy CS, Asha Devi S. Oxidative stress in erythrocytes:

a study on the effect of antioxidant mixtures during intermittent exposures

to high altitude. Int J Biometeorol 2010;54:553-562.

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333

RESEARCH ARTICLE

DOI: 10.4274/tjh.2016.0469


Antioxidants Attenuate Oxidative Stress-Induced Hidden Blood

Loss in Rats

Antioksidanlar Sıçanlarda Oksidatif Stres ile Oluşan Gizli Kan Kaybını Zayıflatır

Hong Qian 1 , Tao Yuan 2 , Jian Tong 3 , Wen-shuang Sun 1 , Jiajia Jin 4 , Wen-xiang Chen 5 , Jia Meng 2 , Nirong Bao 2 , Jianning Zhao 2

1

Southeast University Nanjing General Hospital of Nanjing Military Command, Clinic of Orthopedics, Nanjing, China

2

Nanjing University Faculty of Medicine, Jinling Hospital, Clinic of Orthopedics, Nanjing, China

3

Nanjing University Faculty of Medicine, Nanjing General Hospital of Nanjing Military Command, Clinic of Orthopedics, Nanjing, China

4

Southeast University Nanjing General Hospital of Nanjing Military Command, Department of Respiratory Medicine, Nanjing, China

5

Southern Medical University Faculty of Medicine, Department of Orthopedics, Nanjing, China

Abstract

Objective: Hidden blood loss (HBL), commonly seen after total knee

or hip arthroplasty, causes postoperative anemia even after reinfusion

or blood transfusion based on the visible blood loss volume. Recent

studies demonstrated that oxidative stress might be involved in HBL.

However, whether the antioxidants proanthocyanidin (PA) or hydrogen

water (HW) can ameliorate HBL remains poorly understood. The aim of

this study was to evaluate the effects of PA and HW on HBL.

Materials and Methods: A rat HBL model was established through

administration of linoleic acid with or without treatment with PA

or HW. The levels of hemoglobin (Hb), red blood cell (RBC) count,

superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-PX)

activity, malondialdehyde (MDA), and ferryl Hb were measured.

Results: RBC and Hb values as well as the activity of SOD and GSH-

PX were reduced after administration of linoleic acid, which was

ameliorated by treatment with PA or HW. In addition, the quantity of

MDA was significantly decreased with the administration of PA or HW.

Conclusion: PA and HW could ameliorate HBL in a rat model by

reducing oxidative stress, suggesting that they might be used as a

novel therapeutic approach in the prophylaxis or treatment of HBL

in clinics.

Keywords: Hidden blood loss, Antioxidants, Proanthocyanidin,

Hydrogen water

Öz

Amaç: Total diz veya kalça artroplastisinden sonra yaygın olarak

görülen gizli kan kaybı (GKK), görülebilir kan hacmi kaybına dayanan

reinfüzyon veya kan nakli sonrasında bile postoperatif anemiye neden

olur. Son yıllarda yapılan çalışmalar oksidatif stresin GKK’yla ilişkili

olabileceğini göstermiştir. Bununla birlikte, proantosiyanidin (PA) veya

hidrojenli su (HS) antioksidanlarının GKK’yi iyileştirip iyileştirmediği

anlaşılamamıştır. Bu çalışmanın amacı PA ve HS’nin GKK üzerindeki

etkilerini değerlendirmektir.

Gereç ve Yöntemler: PA veya HS ile muamele edilmiş veya edilmemiş

olan linoleik asit uygulaması ile bir sıçan GKK modeli oluşturulmuştur.

Hemoglobin (Hb), kırmızı kan hücresi sayısı (RBC), süperoksit

dismutaz (SOD) aktivitesi, glutatyon peroksidaz (GSH-PX) aktivitesi,

malondialdehit (MDA) ve ferril Hb düzeyleri ölçüldü.

Bulgular: PA veya HS verilen linoleik asit uygulaması ile RBC ve Hb

değerlerinin yanı sıra SOD ve GSH-PX aktiviteleri azaltıldı. Buna ek

olarak, PA veya HS uygulaması ile MDA miktarı önemli ölçüde azaldı.

Sonuç: PA ve HS bir sıçan modelinde oksidatif stresi azaltarak GKK’yi

düzeltebildiğinden, kliniklerdeki GKK profilaksisi veya tedavisinde yeni

bir terapötik yaklaşım olarak kullanılabileceklerini düşündürmektedir.

Anahtar Sözcükler: Gizli kan kaybı, Antioksidanlar, Proantosiyanidin,

Hidrojenli su



Address for Correspondence/Yazışma Adresi: Jianning ZHAO, M.D.,

Nanjing University Faculty of Medicine, Jinling Hospital, Clinic of Orthopedics, Nanjing, China

Phone : +90 532 325 10 65

E-mail : zhaojianning.0207@163.com ORCID-ID: orcid.org/0000-0001-7169-8117



334


Qian H, et al: Antioxidants, Novel Therapeutics for Hidden Blood Loss

Introduction

Artificial joint replacements are widely employed to alleviate

pain and improve the quality of patients’ lives [1]. The rates

of primary and total hip arthroplasty (THA) and total

knee arthroplasty (TKA) are estimated to increase by 174%-673%

by 2030 as the population ages [2]. However, hidden blood loss

(HBL) predominantly occurs after artificial joint replacement,

such as in cases of TKA and THA [3]. The consequential acute

anemia and transfusions are major concerns for joint surgeons.

The pathogenesis of HBL is very complicated, involving several

factors. A recent study demonstrated that free fatty acids

(FFAs) generated from fatty emboli in the blood circulation are

responsible for HBL through peroxidation injury of membrane

molecules of red blood cells (RBCs) and hemoglobin (Hb) [4].

In addition, antioxidants administered intra- or postoperatively

are predicted to play a protective role in erythrocyte

oxidation and potentially reduce the volume of HBL after

arthroplasty, suggesting that oxidation might be involved in the

pathogenesis of HBL. Consistent with this, our previous study

also demonstrated that FFAs can induce RBC and Hb damage via

reactive oxygen species (ROS) toxicity in vivo [5]. As a natural

antioxidant extract from grape seeds, proanthocyanidin (PA)

possesses a wide range of bioavailability [6]. PA exhibits higher

protective effects against DNA damage and lipid peroxidation

induced by ROS compared with β-carotene, vitamin C,

and vitamin E [7]. PA is a safe and effective bioavailable

antioxidant and ROS scavenger, which is used for the

treatment of ischemia/reperfusion injuries of multiple organs,

malignant tumor progression, carcinogenesis, gastrointestinal

disorders, and Parkinson and Alzheimer disease [6].

As a new antioxidant, hydrogen water (HW) has also been applied

to prevent and treat oxidative stress-associated illnesses using

the establishment of animal models [8,9,10]. HW has been proven

to selectively remove strong oxidants including peroxynitrite

and hydroxyl radicals. Alternatively, ROS play a physiological

role in preventing cells from experiencing oxidative stress [11].

Considering the role of oxidative stress in the pathogenesis of

HBL, whether PA and/or HW as antioxidants ameliorate HBL

remains poorly understood. The objective of this study was to

evaluate the effect of PA and HW on HBL as well as to compare

their protective effects by measuring the levels of Hb, RBC

count, superoxide dismutase (SOD), glutathione peroxidase

(GSH-PX), malondialdehyde (MDA), and ferryl Hb.


Animals

Forty 10-week-old male Sprague-Dawley rats weighing 250±20

g were obtained from the Nanjing University Model Animal

Research Center. All animals were fed daily with rat feed and

potable water or HW under appropriate laboratory conditions at

24 °C with a 12-h light/dark cycle. The animals were randomly

assigned into four groups (n=10 per group). Experimental

procedures were performed strictly according to the Guide

for the Care and Use of Laboratory Animals proposed by the

National Research Council in 1996. All animals were properly

monitored. Animal ethics approval was obtained for this

research. All experimental procedures conducted complied with

the guidelines of the National Institutes of Health Guide for

the Care and Use of Laboratory Animals and the Institutional

Care and Use Committee of Nanjing University. Preoperatively,

all animals were anesthetized via ether inhalation.

Instruments and Reagents

Instruments used included a hematology analyzer (SYSMEX XE-

5000, Kobe, Japan), centrifuge (Hermle Universal Centrifuge

Z323, Gosheim, Germany), microplate reader (Bio-Rad 680,

Hercules, CA, USA), polarizing microscope (Nikon Eclipse 50I,

Tokyo, Japan), spectrophotometer (Hewlett Packard 8453 UVvisible

diode array spectrophotometer, Palo Alto, CA, USA), HWgenerating

apparatus (Bio Coke Laboratory, Tokyo, Japan), and

hydrogen sensor (DHS-001, ABLE, Tokyo, Japan).

The concentration of MDA and the activities of SOD and GSH-PX

were measured with commercially available assay kits (Nanjing

Jiancheng Bioengineering Institute, Nanjing, China). Linoleic

acid was purchased from Sigma-Aldrich (St. Louis, MO, USA).

PA was purchased from Shanghai Aladdin Bio-Chem Technology

Institute (Shanghai, China). HW was prepared by dissolving H 2

gas in drinking water under high pressure of 0.4 MPa using the

HW-generating apparatus. Rats were supplied with HW (0.7 mM)

through a closed glass vessel (300 mL) equipped with an outlet

line containing 2 ball bearings to prevent water degassing. The

H 2

concentration of HW was detected with a hydrogen sensor

(Unisense, Aarhus, Denmark).

Experimental Protocol and Drugs

The procedures below were performed on the rats in all four

groups and the dose used was selected as previously described.

The control group (CON) rats were given potable water and

injected with ethanol alone (0.5 mL, 20%) via intravenous

administration into the tail vein after 2 weeks of feeding.

The linoleic acid (LIN) group animals (receiving LIN)

received potable water and were injected with 0.5 mL of 60

mmol/L linoleic acid diluted in 20% ethanol by intravenous

administration into the tail vein after 2 weeks of feeding.

The LIN+PA group received a 100 mg/kg dose of PA diluted with

potable water daily [12,13] and was injected with 0.5 mL of

60 mmol/L linoleic acid diluted in 20% ethanol by intravenous

administration into the tail vein after 2 weeks of feeding [5].

The LIN+HW group received HW daily and was injected with 0.5 mL

of 60 mmol/L linoleic acid diluted in 20% ethanol by intravenous

administration into the tail vein following 2 weeks of feeding [5].

335

Qian H, et al: Antioxidants, Novel Therapeutics for Hidden Blood Loss Turk J Hematol 2017;34:334-339

During all treatments, rats were monitored daily and were

weighed one to six times per day until the end of the experiment.

None of the rats had any notable discomfort throughout the

experiment.

Routine and Biochemical Analysis of Blood

Blood samples were taken from the caudal vein under anesthesia

(0.5 mL each time) at the beginning of the injection and 24, 48, and

72 h following administration. RBC, hematocrit, and Hb levels were

detected with a hematology analyzer immediately after sampling

collection. Morphological changes of blood cells were observed

following Wright’s staining under a polarizing microscope.

The remaining blood samples were centrifuged and stored at

80 °C for subsequent biochemical analysis. MDA, T-SOD, and

GSH-PX activities were measured by spectrophotometer. The

absorbance values were detected at 532 nm, 550 nm, and 412 nm

wavelengths [12]. Spectral changes of Hb in the LIN and LIN+PA

groups were quantitatively measured by spectrophotometer. Hb

at a concentration of 10 mM was mixed with 0.1 M sodium

phosphate buffer containing 100 mM DTPA. All experimental

procedures were conducted at 25 °C [14].


Hb values were reduced by (0.66±0.34)×10 12 /L and 16.3±8.25

g/L, and in the LIN+PA group those values were decreased by

(0.35±0.1)×10 12 /L and 9.1±4.01 g/L, respectively. A significant

difference was noted in the changes between the LIN and

LIN+PA groups. After 48 h of administration, the changes of RBC

and Hb levels of the LIN group and the LIN+PA group were still

significantly different. In the LIN+HW group, we found the RBC

and Hb values decreased by (0.45±0.22)×10 12 /L and 10.7±3.56

g/L after 24 h, respectively, with a tendency of alleviation of

the reduction of RBC and Hb levels. After 48 h, the decreases

of RBC and Hb (respectively (0.72±0.23)×10 12 /L and 18.2±5.85

g/L) in the LIN+HW group were significantly different compared

to those of the LIN group (respectively (1.15±0.48)×10 12 /L and

25.7±8.38 g/L).

Oxidative Stress Markers

The activities of SOD and GSH-PX in the LIN group significantly

declined after 24 h of administration, reached the lowest levels

after 48 h, and had mild increases after 72 h. Both the LIN+PA

and the LIN+HW group showed a similar variation tendency in

these two markers. However, the SOD and GSH-PX activities

Statistical analysis was performed using SPSS 19.0. All data

were expressed as mean ± standard deviation. The Kolmogorov-

Smirnov test was performed and we concluded that the observed

data were from a population specified by normal distribution.

One-way analysis of variance (ANOVA) was performed followed

by the Tukey test. p<0.05 was considered statistically significant.

Results

Daily consumption of water and body weight among all groups

were monitored. Rats in the CON group consumed 20.0±3.5

mL of potable water daily, while the LIN group consumed

21.0±2.7 mL of potable water daily. In the LIN+PA group, daily

consumption of PA solution was 22.0±2.4 mL, while the LIN+HW

group consumed 24.0±3.4 mL of HW daily. Water consumption

and body weight did not significantly differ among the four

groups.

Routine Blood Tests

Before linoleic acid administration, no significant differences

were observed in RBC and Hb levels among the four groups.

After administration of a dose of 0.5 mL of 60 mmol/L linoleic

acid, RBC and Hb levels significantly changed compared with

the control group (Figure 1), which showed that an in vivo HBL

model had been established successfully. We further analyzed

the RBC and Hb levels of the LIN+PA and LIN+HW groups

compared to those of the LIN group. After 24 h of administration,

the Hb and RBC levels had decreased to different extents in

the three experimental groups. In the LIN group, the RBC and

Figure 1. Changes of hemoglobin and red blood cell levels with

time between control (sham) group and experimental groups.

Values are presented as the mean ± standard deviation, n=10 for

all groups.

*Compared with the control group, p<0.05, #Compared with the

linoleic acid group, p<0.05.

LIN: Linoleic acid, PA: proanthocyanidin, HW: hydrogen water, RBC: red blood cell,

Hb: hemoglobin.

336



in groups LIN+PA and LIN+HW were both obviously higher

than those of the LIN group at each time point (Figure 2).

The MDA concentration in the LIN group reached a

peak after 24 h and then started to decrease slowly.

The LIN+PA and LIN+HW groups also displayed a similar

changing pattern in MDA level. However, both SOD and

GSH-PX activities in groups LIN+PA and LIN+HW were

consistently lower than those of the LIN group (Figure 2).

Ferryl Hb was present and formed by reacting with H 2

O 2

, which

was confirmed by the characteristic absorbance band around

620 nm via the reaction with sulfide ions [15]. The effect of

linoleic acid upon the hemolysis of RBCs, either by itself or in

conjunction with ROS, can be utilized to assess the severity

of oxidative injury of erythrocytes [16]. Blood samples were

collected from these three groups before administration and

every 24 h thereafter. Absorbance peak values were detected

at a wavelength of approximately 425 nm, consistent with the

Soret peak of ferryl Hb (Figure 3).

Histologic Investigations

In the LIN group, a number of shrunken, deformed, and ruptured

blood cells were seen compared with the control group and

the morphological changes were the most distinct after 24

h of administration. In contrast, we could also identify some

shrunken and deformed RBC in groups LIN+PA and LIN+HW, but

ruptured blood cells could hardly be found in those two groups

(Figure 4).

Figure 3. Changes in absorbance at 425 nm among the linoleic

acid, linoleic acid+proanthocyanidin, and linoleic acid+hydrogen

water groups. Values are presented as the mean ± standard

deviation, n=10 for all groups.

*Compared with the control group, p<0.05.

LIN: Linoleic acid, PA: proanthocyanidin, HW: hydrogen water.

Figure 2. Changes in T-superoxide dismutase, glutathione

peroxidase, and malondialdehyde among the linoleic acid, linoleic

acid+proanthocyanidin, and linoleic acid+hydrogen water groups.

Values are presented as the mean ± standard deviation, n=10 for

all groups.

*Compared with the control group, p<0.05.

LIN: Linoleic acid, PA: proanthocyanidin, HW: hydrogen water, SOD: superoxide

dismutase, GSH-PX: glutathione peroxidase, MDA: malondialdehyde.

Figure 4. Protective effects of proanthocyanidin and hydrogen

water on red blood cells. Blood samples were collected before

administration and then every 24 h thereafter. Stains were added

to the blood smears to observe erythrocyte morphological changes.

Images are magnified at 400 x . After 24 h, cell morphology was

obviously changed in the linoleic acid group (A-D) with a large

number of red blood cells shrunken (black arrows), deformed

(blue arrows), and even ruptured (red arrows); in contrast, in the

linoleic acid+proanthocyanidin group (E-H) and LIN+HW group

(I-L), ruptured cells could hardly be identified.

LIN: Linoleic acid, PA: proanthocyanidin, HW: hydrogen water.

337

Qian H, et al: Antioxidants, Novel Therapeutics for Hidden Blood Loss Turk J Hematol 2017;34:334-339

Discussion

Many recent studies have investigated the pathophysiological

mechanisms and therapeutic strategies for HBL [17,18,19], but to

our knowledge, our team provided the first evidence that oxidative

stress can lead to HBL [10] and that antioxidant treatment with

PA or HW ameliorated HBL, suggesting they may represent a

possible therapeutic choice for HBL in clinical practice.

HBL is a severe complication after TKA and THA [20]. Although

several theories concerning the mechanisms of HBL have

been proposed, no theory is convincing enough to explain the

pathological mechanism. Pattison et al. [21] proposed that

hemolysis may partly contribute to postoperative loss, but

they did not provide a pathological mechanism. Faris et al.

[22] demonstrated that hemolysis was detected after reinfusion

with an average volume of 1.3 L of blood, but hemoglobinuria

did not occur due to the activity of Hb. In contrast, Shen et

al. [23] showed that no statistical significance was observed

in HBL between the reinfusion and non-reinfused groups. Li

et al. [24] reported that administration of a tourniquet could

significantly increase HBL in their study, but as much as 600 mL

of HBL can be detected without using a tourniquet. Moreover,

the theory of the “third compartment” was proposed to explain

the mechanism underlying HBL. Erskine et al. [25] reported that

unexplained blood loss was completely due to perioperative

bleeding, probably into the tissue compartments. However, it is

not reasonable that the bleeding would be “pressing” into tissue

compartments because of commonly used techniques, such

as negative pressure drainage and pressure dressing. Therefore,

subsequent investigation is required to fully unravel the

mechanisms underlying HBL.

The increased intramedullary pressure in TKA and THA plays

a vital role in the pathogenesis of fatty metabolism [26,27]. In

addition to the clinical association between fatty emboli and

cardiopulmonary function, the metabolites of fatty emboli,

FFAs, can stimulate ROS production in neutrophils [28] and exert

a negative biological effect on erythrocytes. After ROS were

stimulated and the oxidants accumulated, osmotic fragility of

RBCs increased through oxidizing polyunsaturated fatty acids

derived from the RBC membranes [29] and cytosolic ferrous Hb

[30].

Given the critical role of ROS in the damage or injury of RBCs,

this study investigated the antioxidant effect of PA and HW

on linoleic acid-induced oxidative stress by measuring GSH-

Px, SOD, and MDA. Our results showed that SOD and GSH-

Px activities were increased in the experimental groups with the

use of PA or HW, and the SOD and GSH-Px activities of each

experimental group were significantly decreased after 24 h of

LIN administration, indicating that linoleic acid plays a vital role

in promoting oxidation responses in the body and reducing SOD

and GSH-Px activity. In the LIN+PA and LIN+HW groups, SOD

and GSH-Px showed significantly elevated activities compared

with the LIN group. These findings can be interpreted as PA and

HW exhibiting a positive effect on SOD and GSH-Px activity. The

amount of MDA was significantly decreased due to the effect

of linoleic acid, suggesting the presence of oxidative stress in

the culture medium. In this study, the quantity of MDA was

significantly decreased with the administration of PA or HW [9,12],

consistent with previous studies showing that the elevation of

MDA level induced by lipid peroxidation was counteracted by the

administration of PA or HW. Although the present study indicates

that PA possesses higher anti-HBL effects compared with HW, no

significant variation occurred in our study considering the dosage

and duration of PA administration.

Oxidative injury changes the structure and function of Hb, leading

to Hb denaturation and precipitation. The resultant product is

known as methemoglobin [19]. Hydrophilic hydrogen peroxide is

capable of directly penetrating the RBC membrane and oxidizing

Hb into ferryl Hb [20]. The heme proteins oxidized into the ferryl

species by peroxides are widely regarded as the initiators of a

variety of lipid peroxidation and lipid pseudo-peroxidase responses

[21]. Hypochlorous acid can oxidize glutathione and membrane

protein-SH groups and elevates the osmotic fragility. In addition,

it also induces cell membrane deformation via lipid oxidation

[3]. Multiple investigations have indicated that ferrous Hb can

be oxidized into ferryl Hb by H 2

O 2

and hypochlorous acid. Ferryl

Hb loses the capacity of carrying oxygen. Nevertheless, GSH-Px is

able to decrease the formation of methemoglobin by 93% when

Hb is oxidized by H 2

O 2

[22], highlighting the pivotal role of linoleic

acid in mediating Hb oxidation and subsequent cross-linking of

the oxidation-reduction responses.

Several limitations have to be acknowledged in this study.

First, our studies suggest that FFAs could cause HBL, which

could be ameliorated through treatment with antioxidant

drugs, but we cannot draw the conclusion that oxidative stress

produced by FFAs leading to the toxicity of RBCs is the only

pathophysiological mechanism underlying postoperative blood

loss. Second, the appropriate therapeutic dose and timing of PA

and HW administration and the combination therapy of these

two drugs need further investigation. The significance of the

current experiment is that HBL induced by ROS increase can be

counteracted by antioxidant therapy.

Conclusion

In conclusion, PA and HW could ameliorate HBL in a rat model

by reducing oxidative stress, suggesting they might be used as

novel therapeutic approaches in the prophylaxis or treatment of

HBL in clinical practice.

338



Ethics

Ethics Committee Approval: Animal studies were approved

by the Ethic Committeee of Jinling Hospital and were strictly

performed following the Institutional Animal Care and User

guidelines.

Informed Consent: N/A.


Surgical and Medical Practices: H.Q., T.Y.; Concept: J.Z.;

Design: N.B.; Data Collection or Processing: J.T., W.C.; Analysis

or Interpretation: W.S:, J.J.; Literature Search: Q.H., J.M.;

Writing: Q.H.




included.

Financial Disclosure: This study was supported by the Clinical

Science and Technology Foundation of Jiangsu Province

(BL2012002), the Natural Science Foundation of Jiangsu Province

(BK2012776), and the National Natural Science Foundation of

China (Grant No. 81000814).

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339

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DOI: 10.4274/tjh.2017.0052


High Infection-Related Mortality in Pediatric Acute Myeloid

Leukemia without Preventive Antibiotics and Antifungals:

Retrospective Cohort Study of a Single Center from a

Middle-Income Country

Çocukluk Çağı Akut Miyeloid Lösemilerinde Koruyucu Antibiyotik ve Antifungal Kullanılmadığında

Yüksek Enfeksiyon İlişkili Mortalite: Orta Gelir Grubundaki Bir Ülkeden Tek Merkezin Retrospektif

Kohort Çalışması

Emine Zengin 1 , Nazan Sarper 1 , Sema Aylan Gelen 1 , Uğur Demirsoy 1 , Meriban Karadoğan 1 , Suar Çakı Kılıç 1 , Selim Öncel 2 , Emin Sami Arısoy 2 ,

Devrim Dündar 3

1

Kocaeli University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Kocaeli, Turkey

2

Kocaeli University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Infectious Diseases, Kocaeli, Turkey

3

Kocaeli University Faculty of Medicine, Department of Microbiology, Kocaeli, Turkey

Abstract

Objective: This study aimed to evaluate infection-related mortality

in patients with acute myeloid leukemia (AML) treated without

preventive antibiotics and antifungals in a middle-income country.

Materials and Methods: Infection-related mortality was evaluated

retrospectively in 49 pediatric patients.

Results: A total of 173 chemotherapy courses were administered as

first-line chemotherapy. Four patients died during induction: one

patient due to intracranial bleeding, two patients due to typhlitis,

and one patient due to invasive fungal infection with pulmonary

vascular invasion and massive bleeding. Another two patients died

with resistant disease. During consolidation there were four infectionrelated

deaths and one death due to cardiotoxicity. In first-line

chemotherapy mortality was 22% (11/49); infection-related mortality

was 14% (7/49). Event-free survival and overall survival at 6 years were

42.9% and 61.2% (95% CI: 44-76 and 66-99 months), respectively.

Conclusion: Due to considerable infection-related deaths,

antibacterial and mold-active antifungal prophylaxis may be tried

during neutropenic periods in pediatric AML.

Keywords: Acute myeloid leukemia, Pediatric leukemia, Febrile

neutropenia, Infection

Öz

Amaç: Orta gelir düzeyindeki bir ülkede koruyucu antibiyotik ve

antifungal kullanılmadan tedavi edilen akut miyeloid lösemi (AML)

tanılı hastalarda enfeksiyona bağlı ölümlerin değerlendirilmesidir.

Gereç ve Yöntemler: Kırk dokuz AML tanılı çocuk hastada enfeksiyona

bağlı ölümler retrospektif olarak değerlendirildi.

Bulgular: İlk basamak tedavi olarak toplam 173 kemoterapi kürü

uygulandı. İndüksiyon sırasında bir hasta beyin kanaması, iki hasta

tiflit ve bir hasta invazif mantar enfeksiyonunun akciğer damar

duvarını hasarlaması sonucu gelişen yoğun kanamayla olmak

üzere toplam dört hasta kaybedildi. İki hasta da dirençli hastalık ile

kaybedildi. Konsolidasyon sırasında beş ölüm vardı; dördü bakteriyemi

ve sepsis, biri kardiyotoksisite ilişkiliydi. İlk basamak tedavide ölüm

oranı %22 (11/49) olup, bunların %14’ü (7/49) enfeksiyonla ilişkiliydi.

Olaysız sağkalım ve genel sağkalım sırasıyla 6 yılda %42,9 ve %61,2

(%95 güven aralığı: 44-76 ve 66-99 ay) bulundu.

Sonuç: Dikkate değer sayıda enfeksiyon ölümleri nedeniyle

antibakteriyel ve küf mantarlarına etkili koruyucu tedaviler, ortagelir

düzeyindeki ülkelerde, AML tanılı çocuk hastaların nötropenik

dönemlerinde kullanılabilir.

Anahtar Sözcükler: Akut miyeloid lösemi, Çocukluk çağı lösemileri,

Nötropenik ateş, Enfeksiyon



Address for Correspondence/Yazışma Adresi: Nazan SARPER, M.D.,

Kocaeli University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Kocaeli, Turkey

Phone : +90 262 303 72 16

E-mail : nazan_sarper@hotmail.com ORCID-ID: orcid.org/0000-0003-1599-774X

Received/Geliş tarihi: February 12, 2017


340


Zengin E, et al: Infection-Related Mortality in Pediatric AML

Introduction

Children and adolescents with acute myeloid leukemia (AML)

are at risk of severe infectious complications as a result of

prolonged neutropenia. It was reported that the main causes

of death during chemotherapy are infections [1]. Studies show

that prophylaxis with antibiotics and antifungals reduced

infections, hospitalization days, and mortality [2,3,4], but the

emergence of resistance, particularly in vancomycin-resistant

enterococci (VRE) and gram-negative bacteria, is another

dilemma [5].

The aim of this study was to document infection-related mortality

(IRM) of patients with de novo AML and myelodysplastic

syndrome (MDS)/AML during first-line chemotherapy courses

and compare the results with the literature data.


This retrospective study was performed in a university hospital’s

pediatric hematology unit. The hospital records of all de novo

AML/MDS patients aged <18 years diagnosed from June 2005

through February 2016 were reviewed by two experienced

hematologists of the unit. Patients with Down syndrome were

also included. Before starting chemotherapy, all the parents/

legal guardians gave informed consent for the treatment and for

the usage of patient data in the research. The ethics committee

of the institution also approved the study.

The United Kingdom Medical Research Council (MRC) AML-10

chemotherapy protocol was used as first-line chemotherapy with

some modifications and no randomizations [1]. A modification

was the substitution of amsacrine with idarubicine 10 mg/m 2

on days 0 and 1 in some patients due to unavailability of the

drug. Patients with Down syndrome were treated with reducedintensity

chemotherapy. Patients stayed in two-bed rooms with

a bathroom and there was no high-efficiency particulate air

(HEPA) filtration. Co-trimoxazole prophylaxis was administered.

Granulocyte colony stimulating factor (G-CSF) was only used

in consolidation phases if there was severe infection with

hemodynamic instability. Patients generally were not discharged

until remission, but in the subsequent chemotherapy courses

they were followed as outpatients if there were no infections.

In the first years of the study, ceftazidime and then piperacillin/

tazobactam or cefepime were used as empirical monotherapy.

Carbapenems and teicoplanin were administered as initial

empirical therapy in hemodynamically unstable patients. If

there were any respiratory symptoms at initial presentation of

febrile neutropenia or if fever persisted longer than 96 h, serum

galactomannan monitoring was started and chest computerized

tomography and abdominal ultrasound imaging were

performed. Empirical mold-active antifungals were introduced

after 96 h. Patients were referred to some other centers when

hematopoietic stem cell transplantation (HSCT) was indicated.


Data were analyzed using SPSS 13. Descriptive statistics

were employed and are reported as absolute frequencies or

percentages for qualitative data and as medians and range

or means and standard deviations for quantitative data.

Comparisons of frequency distribution were analyzed with the

nonparametric statistics of the chi-square test or the Kruskal-

Wallis test. For survival, Kaplan-Meier analysis was performed.

All tests were two-tailed and p<0.05 was considered statistically

significant.

Results

Forty-nine patients (32 boys, 17 girls) with AML were diagnosed

and 173 chemotherapy courses were administered as first-line

chemotherapy. Ten of the patients had acute promyelocytic

leukemia, 4 patients had Down syndrome, and 3 patients had

MDS/AML (one had myelofibrosis). The overall remission rate

with first-line chemotherapy was 85.7%. During induction and

consolidation there were 11 deaths. In first-line chemotherapy

mortality was 22% (11/49); IRM was 14% (7/49) (Figure 1).

In 682 sterile-site cultures, 47 cases of pathogen growth

were observed. The isolated pathogens of the three infectionrelated

deaths were Candida guilliermondii (septicemia

and pneumonia), extended spectrum β-lactamase-positive

Klebsiella pneumoniae (septicemia), and Enterobacter

cloacae (septicemia). Except for the patient with Candida

septicemia who had sudden pulmonary hemorrhage, all the

patients required intensive care. Gram-negative bacteria

isolation was more frequent than gram-positive (67.2% versus

32.7%). Escherichia coli and K. pneumoniae were the most

predominant isolates. Viridans streptococci were rarely isolated;

there were three Streptococcus mitis isolations from this

Figure 1. Outcome of pediatric patients with AML/MDS.

CR: Complete remission, CT: chemotherapy, IFI: invasive fungal infection, ICH:

intracranial hemorrhage, HSCT: hematopoietic stem cell transplantation.

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Zengin E, et al: Infection-Related Mortality in Pediatric AML Turk J Hematol 2017;34:340-344

Table 1. Characteristics of the infectious episodes in pediatric patients with acute myeloid leukemia.

ADE I ADE II MACE** MIDAC

# of chemotherapy courses 49 45 41 38

# of febrile episodes 62 41 30 43

# of febrile episodes per chemotherapy course 1.3 0.9 0.7 1.1

# of episodes with fever of unknown origin (%) 12 (19.4) 13 (31.7) 6 (20) 12 (27.9)

*# of clinically documented infections (%) 38 (61.3) 15 (36.6) 14 (46.7) 16 (37.2)

# of microbiologically documented infections (%) 12 (19.4) 13 (31.7) 10 (33.3) 15 (34.9)

# of discharged patients after chemotherapy courses 5 36 36 32

Mean outpatient days of discharged patients after chemotherapy courses 4.3±7.5 12.1±9 16.2±10 9.5±7.3

Mean period off antimicrobials and antifungals during chemotherapy courses (days) 9.2±9.9 20.2±9.4 21.9±9.8 14.2±7.8

*Clinically documented infections also include possible fungal infections, **MACE or modified MACE.

or vancomycin with oral ciprofloxacin reduced bacterial sepsis

and days of hospitalization. Prophylactic oral voriconazole was

also used in that study but it did not reduce fungal infections.

IRM was 2.5% [2].

Figure 2. Kaplan-Meier analysis of event-free and overall survival.

group. Characteristics of the infectious episodes and identified

pathogens are demonstrated in Tables 1 and 2, respectively.

In the first remission, 4 matched related donor (MRD) and 1

matched unrelated donor (MUD) transplants were performed.

Eight patients had allogeneic HSCT in the second remission (4

MRD, 2 MUD, and 2 haploidentical transplants), and two of these

patients had second transplants. Of the 11 transplanted patients,

8 were alive in a median of 54 (range: 6-84) months (Figure 1).

Event-free survival and overall survival at 6 years were 42.9%

and 61.2% (95% CI: 44-76 and 66-99 months), respectively

(Figure 2).

Discussion

Sixty-one percent of the patients were alive during the

retrospective study period. During the first-line treatment there

were 11 deaths (22%), 7 of which (14%) were infection-related.

In the present study, with less intensive chemotherapy, out of

4 patients with Down syndrome there was only one infectionrelated

death during induction.

In a multicenter study of children also treated with the

MRCAML-10 protocol, IRM was 9.1% [1]. Single-occupancy

rooms with HEPA filtration and more training about hand

washing may reduce infections.

It was reported that prophylactic oral cephalosporins did not

significantly reduce bacterial sepsis, but intravenous cefepime

In a multicenter trial some centers used antibacterial (penicillin

or vancomycin and others), antifungal, and G-CSF prophylaxis.

All the centers used fluconazole prophylaxis. The authors

concluded that antibacterial prophylaxis reduced sterilesite

infections. Prophylactic G-CSF reduced bacterial and

Clostridium difficile infections. Mandatory hospitalization did

not reduce bacterial/fungal infection and nonrelapse mortality

but did increase C. difficile infections [6].

We hospitalized patients until remission but in the subsequent

courses we discharged them. Mandatory hospitalization might

prevent some infection-related deaths. One patient died at home

with infection and another’s admission was delayed. In another

study, vancomycin and ceftazidime or cefepime prophylaxis

was administered in addition to fluconazole, voriconazole, or

micafungin. There was only one infection-related death due to

candidal pneumonia. When induction I was excluded, preventive

antibiotics reduced infections compared to no prophylaxis.

The hospital rooms were all single-occupancies and most of

them had HEPA filtration. However, after emergence of VRE

and C. difficile infections, vancomycin was removed from the

preventive therapy and cefepime mono therapy was started [7].

In the multicenter trial AML-Berlin-Frankfurt-Münster (BFM)

2004, excluding patients with Down syndrome, the infectionrelated

morbidity rate was 3.3 per patient and IRM was only

1.5%. There was a reduction in IRM compared to the previous

AML-BFM 93 study. This was explained by the administration of

anti-mold active prophylaxis (amphotericin B, voriconazole, or

posaconazole) in more than 70% of the chemotherapy cycles

and prompt institution of empiric therapy that should include a

glycopeptide in severely ill patients and regular training courses

in the education of the pediatric hematologists [8].

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Zengin E, et al: Infection-Related Mortality in Pediatric AML

Table 2. Isolated pathogens during chemotherapy courses of pediatric patients with acute myeloblastic leukemia.

ADE I ADE II MACE* MIDAC

# of eligible courses 49 45 41 38

# of gram-positive isolates

Staphylococcus epidermidis 3 1 - 5

Micrococcus luteus 1 - 2 -

Streptococcus mitis - 2 - 1

Enterococcus faecalis 1 1 - -

Kocuria kristinae 1 - - -

# of gram-negative isolates

Escherichia coli 4 2 2 10

Klebsiella pneumoniae - 3 1 5

Pseudomonas aeruginosa - 1 1 1

Enterobacter cloacae 1 2 - 1

Acinetobacter spp. 1 - -

Proteus mirabilis - 1 - -

Elizabethkingia meningoseptica 1 - - -

# of fungal isolates

Candida albicans 1 - 1 -

Candida parapsilosis 1 - - -

Candida guilliermondii 2 - - -

Aspergillus fumigatus - - - 1

Aspergillus niger - - 1 -

*MACE or modified MACE.

Conclusion

In a multicenter study of pediatric AML from Turkey, similar to

our study, overall survival was 58.8% and IRM was 16.6%. The

authors also suggested that better supportive care may improve

outcomes [9].

In light of the above literature we think that, to reduce

IRM, we should try prophylaxis in patients with pediatric

AML. Prophylactic piperacillin/tazobactam or cefepime and

voriconazole can be administered in the induction. After

induction oral ciprofloxacin and voriconazole can be tried in

preventive therapy when the absolute neutrophil count (ANC)

falls below 0.5x10 9 /L and discontinued once the ANC recovers

to >0.1x10 9 /L. Prophylaxis may justify the potential risk from

emerging resistant bacteria.

Ethics

Ethics Committee Approval: Kocaeli University Noninterventional

Clinical Research Ethics Board (approval number:

2016/288 - 2016/18.1).

Informed Consent: Informed consent was obtained from parents

for publication of the patient data at the start of chemotherapy.

Authorship Contribution

Surgical and Medical Practices: E.Z., N.S., M.K., S.A.G., U.D., S.Ç.K.,

D.D., S.Ö., E.S.A.; Concept: N.S.; Design: N.S.; Data Collection or

Processing: E.Z., N.S.; Analysis or Interpretation: E.Z.; Literature

Search: N.S.; Writing: N.S.

Conflict of Interest: The authors of this paper have no conflicts of

interest, including specific financial interests, relationships, and/or

affiliations relevant to the subject matter or materials included.

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Howard SC, Pui CH, Ribeiro RC, Hayden RT, Rubnitz JE. Feasibility, efficacy,

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6. Sung L, Aplenc R, Alonzo TA, Gerbing RB, Lehrnbecher T, Gamis AS. Effectiveness

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8. Bochennek K, Hassler A, Perner C, Gilfert J, Schöning S, Klingebiel T,

Reinhardt D, Creutzig U, Lehrnbecher T. Infectious complications in children

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344

BRIEF REPORT

DOI: 10.4274/tjh.2016.0477


Hematopoietic Stem Cell Transplantation in Primary

Immunodeficiency Patients in the Black Sea Region of Turkey

Primer İmmün Yetmezlikli Hastalarda Hematopoetik Kök Hücre Transplantasyonu; Türkiye’de

Karadeniz Bölgesi Deneyimi

Alişan Yıldıran 1 , Mehmet Halil Çeliksoy 1 , Stephan Borte 2 , Şükrü Nail Güner 1 , Murat Elli 3 , Tunç Fışgın 3 , Emel Özyürek 3 ,

Recep Sancak 1 , Gönül Oğur 4

1

Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Allergy and Immunology, Samsun, Turkey

2

Leipzig University, Translational Centre for Regenerative Medicine, Leipzig, Germany

3

Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Samsun, Turkey

4

Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Genetic, Samsun, Turkey

Abstract

Hematopoietic stem cell transplantation is a promising curative

therapy for many combined primary immunodeficiencies and

phagocytic disorders. We retrospectively reviewed pediatric cases of

patients diagnosed with primary immunodeficiencies and scheduled

for hematopoietic stem cell transplantation. We identified 22 patients

(median age, 6 months; age range, 1 month to 10 years) with various

diagnoses who received hematopoietic stem cell transplantation. The

patient diagnoses included severe combined immunodeficiency (n=11),

Chediak-Higashi syndrome (n=2), leukocyte adhesion deficiency (n=2),

MHC class 2 deficiency (n=2), chronic granulomatous syndrome (n=2),

hemophagocytic lymphohistiocytosis (n=1), Wiskott-Aldrich syndrome

(n=1), and Omenn syndrome (n=1). Of the 22 patients, 7 received

human leukocyte antigen-matched related hematopoietic stem cell

transplantation, 12 received haploidentical hematopoietic stem cell

transplantation, and 2 received matched unrelated hematopoietic

stem cell transplantation. The results showed that 5 patients had graft

failure. Fourteen patients survived, yielding an overall survival rate

of 67%. Screening newborn infants for primary immunodeficiency

diseases may result in timely administration of hematopoietic stem

cell transplantation.

Keywords: Hematopoietic stem cell, Transplantation, Children,

Immunodeficiency

Öz

Birçok kombine primer immün yetmezlik ve fagositer bozukluk için

hematopoetik kök hücre nakli küratif bir tedavidir. Bu çalışmada,

primer immün yetmezlik tanısı alan ve hematopoetik kök hücre nakli

yapılan hastaları retrospektif olarak inceledik. Yirmi iki hasta belirlendi.

Hastaların hematopoetik kök hücre nakli sırasındaki ortanca yaşları

6 ay (minimum-maksimum: 1 ay-10 yaş) idi. Hastaların tanıları ağır

kombine immün yetmezlik (n=11), Cheidak Higashi sendromu (n=2),

lökosit adezyon defekti (n=2), MHC sınıf-2 eksikliği (n=2), kronik

granülomatoz hastalık (n=2), hemofagositik lenfohistiyositoz (n=1),

Wiskott-Aldrich sendromu (n=1) ve Omenn’s sendromu (n=1) idi. Yedi

hastaya tam insan lökosit antijen uyumlu, 12 hastaya yarı uyumlu ve

2 hastaya insan lökosit antijen uyumsuz vericiden hematopoetik kök

hücre nakli yapıldı. Beş hasta da graft başarısız oldu. On dört hasta

hayatta kaldı ve ortalama sağkalım %67 idi. Bu hastalık için yenidoğan

taramaları yapılması ile hematopoetik kök hücre transplantasyonları

zamanında yapılabilir.

Anahtar Sözcükler: Hematopoetik kök hücre, Transplantasyon, Çocuk,

İmmün yetmezlik

Introduction

Primary immunodeficiency (PID) disorders are a group

of heterogeneous diseases, many of which are caused by

monogenic defects, resulting in susceptibility to life-threatening

infections, uncontrolled inflammation, or autoimmunity. In

1968, successful transplantation was performed in two patients,

one with severe combined immunodeficiency (SCID) and one

with Wiskott-Aldrich syndrome (WAS). These cases represented

the first successful hematopoietic stem cell transplantation



Address for Correspondence/Yazışma Adresi: Mehmet Halil ÇELİKSOY, M.D.,

Ondokuz Mayıs University Faculty of Medicine, Department of Pediatric Allergy and Immunology, Samsun, Turkey

Phone : +90 362 312 19 19

E-mail : drmhc@hotmail.com ORCID-ID: orcid.org/0000-0002-4164-4668


Accepted/Kabul tarihi: April 11, 2017

345

Yıldıran A, et al: HSC Transplantation in Patients with PID


(HSCT) procedures, ushering in a new era of curative therapies

for treating PID disorders [1,2,3]. To date, only one report has

described HSCT therapies for PID disorders in Turkey [4]. The

aim of this study was to retrospectively document all pediatric

cases of patients diagnosed with PID disorders and considered

for HSCT therapy at our pediatric transplantation center.


In total, 22 infants were diagnosed with PID; 19 of these patients

underwent HSCT at the Ondokuz Mayıs University Faculty of

Medicine, Department of Pediatrics, Pediatric Transplantation

Unit, between June 2010 and December 2013. One patient died

shortly after diagnosis.

Of the 22 patients, 11 were diagnosed with SCID, 2 with MHC class

2 deficiency, 2 with leukocyte adhesion deficiency (LAD), 2 with

chronic granulomatous disease (CGD), 2 (siblings) with Chediak-

Higashi syndrome (CHS), 1 with WAS, 1 with hemophagocytic

lymphohistiocytosis, and 1 with Omenn syndrome (Tables 1 and

2). All patients met the European Society for Immunodeficiencies

- Pan-American Group for Immunodeficiency diagnostic criteria

for PID disease [5]. In terms of the phenotypic profiles of

patients with SCID, seven displayed T-B-NK+ and four showed

T-B+NK+ profiles. The molecular defects of two patients with

SCID could not be determined. In total, eight patients with SCID

and three without SCID underwent haploidentical CD34+ stem

cell transplantation. Additionally, one patient with CGD and one

patient with WAS underwent HSCT from matched unrelated

donors at another center.

Results

Patient Characteristics

The patients’ ages at diagnosis of SCID ranged from 2 to

8 months (median: 3 months). Parental consanguinity was

determined in seven (64%) patients with SCID. Pneumonia

and diarrhea were common complaints in patients with SCID.

Parental consanguinity was determined in nine (82%) non-

SCID patients. Two patients with SCID were referred by another

center for HSCT 3 months after diagnosis. Only one SCID patient

was found to be positive for cytomegalovirus antigenemia at

the time of diagnosis; therefore, a conditioning regimen was

not administered. No engrafted maternal T cells were detected

in patients with SCID at the time of diagnosis. All patients with

SCID were lymphopenic and had few T cells (CD3+ cells <30%).

The age of the non-SCID patients at the time of HSCT ranged

from 3 to 120 months (median: 13 months). Failure to thrive was

the most common complaint in non-SCID patients. Although

parental consanguinity was determined in 64% of SCID and

82% of non-SCID patients, only six patients (27%) had matched

related donors. The characteristics and transplantation data

from SCID and non-SCID cases are shown in Tables 1 and 2.

Complications

A common complication in our patients was graft failure (40%)

that required repeated transplantations. Grade I-II acute graftversus-host

disease (GVHD) was observed in four patients (three

SCID and one non-SCID) after HSCT. Outcomes and complications

in the SCID and non-SCID cases are shown in Tables 3 and 4.

Discussion

Established in 2009, our bone morrow transplantation center

was the first of its kind in the Black Sea Region of Turkey. We

reviewed all pediatric patients diagnosed with PID who were

scheduled to receive HSCT at Ondokuz Mayıs University between

June 2010 and December 2013 (n=22). A similar recent study in

two Balkan countries reported only 15 SCID cases during a 24-

year period [6]. Cipe et al. [4] reported haploidentical HSCT in

18 patients in the capital city of Turkey during a 10-year period;

however, many of these patients were from other regions of

the country. Although four of our patients came from other

regions (two with SCID, one with Omenn syndrome, and one

with LAD), our patient numbers suggested that the prevalence

of PID disorders should have been higher in the Black Sea

Region of Turkey because of the high rate of consanguinity.

Yorulmaz et al. [7] reported that the parental consanguinity

rate was 37.5% in patients with PID; in another region of the

country, these rates were 84%, 75%, and 73% in patients with

SCID, phagocytic system defects, and common variable immune

disease, respectively. As our prenatal consanguinity rates were

lower than expected, we suggest that newborn screenings for

PID disorders should be mandatory, at least in our region. In the

near future, we plan to apply the screening method developed

by Borte et al. [8], which includes a robust triplex polymerase

chain reaction method for quantitation of T-cell receptor

excision circles and κ-deleting recombination excision circles

using single-punch Guthrie cards. We expect to identify

patients with SCID, X-linked agammaglobulinemia, ataxia

telangiectasia, Nijmegen breakage syndrome, and other severe

immunodeficiency syndromes characterized by the absence of T

or B cells with this method.

Cipe et al. [4] concluded that human leukocyte antigen (HLA)-

haploidentical transplantation from parental donors represents

a readily available treatment option, especially for patients

with SCID, offering a high probability of cure. In our study, 12

patients received haploidentical HSCT, 7 received HLA-matched

HSCT from related donors, and 2 received HLA-matched HSCT

from unrelated donors. There are several concerns regarding the

safety of haploidentical HSCT, as it may cause a delay in the

successful outcome in patients with PID disorders. Our study

and others [4,9] showed that T cell-depleted haploidentical

HSCT is a life-saving treatment in patients with PID disorders.

346



Table 1. Characteristics and transplantation data of severe combined immunodeficiency patients.

Patient Sex C# Symptoms

at presentation

1 FD* M + Pneumonia/FTT/oral

thrush

Diagnosis Age at diagnosis

(months)

Age at HSCT

(months)

Type of

HSCT

Donor/source Graft

failure

Conditioning

regimen

GvHD

prophylaxis

T-B-NK+ 6 7 MRD Brother/BM No None CsA RAG2

2 MK M + Pneumonia/diarrhea T-B-NK+ 5 7 Haplo Father/PBSCT No FLU/BU/ATG CsA RAG1

3 MO M + Diarrhea T-B+NK+ 3 5 Haplo Mother/PBSCT No FLU/BU CsA IL2RG

4 IY** M - Pneumonia T-B-NK+ 2 6 Haplo Father/PBSCT No None CsA RAG1

5 PA F + Pneumonia/diarrhea/

oral thrush

T-B-NK+ 5 6 Haplo Mother/Father/

PBSCT

6 YFK** F + Pneumonia/diarrhea T-B-NK+ 3 7 Haplo Father/PBSCT Yes,

3 trials

Yes,

3 trials

Mutation

None CsA RAG2

None CsA JAK3

7 BY M + Pneumonia/diarrhea T-B-NK+ 3 4 MRD Sister/BM No FLU/BU CsA RAG1

8 ET F - Pneumonia T-B+NK+ 8 8 Haplo Mother/PBSCT Died None CsA JAK3

9 FZS F + Diarrhea T-B+NK- 2 2 ND ND Died ND ND ND

10 EK F - FTT/oral thrush T-B-NK+ 2 3 Haplo Mother/PBSCT No FLU/BU CsA ND

11 GS F - Pneumonia/diarrhea T-B+NK+ 6 8 Haplo Mother/PBSCT Yes,

4 trials

FLU/BU CsA JAK3

*Conley et al. [5], **Referred from another region of Turkey.

B: B cell, BM: bone marrow, BU: busulfan, CsA: cyclosporine, F: female, FTT: failure to thrive, FLU: fludarabine, GvHD: graft-versus-host disease, HSCT: hematopoietic stem cell transplantation, M: male, MRD: matched related donor, ATG:

antithymocyte globulin, NK: natural killer cell, PBSCT: peripheral blood stem cell transplantation, SCID: severe combined immunodeficiency, T: T cell, ND: not done, C#: consanguinity.

Table 2. Characteristics and transplantation data of non-severe combined immunodeficiency patients.

Patient Sex C# Symptoms

at presentation

Diagnosis Age at HSCT

(months)

Type of HSCT Donor/source Graft failure Conditioning regimen GvHD

prophylaxis

1 SA* M + Pneumonia/FTT HLH 57 Haplo Father/PBSCT No None CsA

2 EDT F + Pneumonia/FTT MHC class 2 12 MRD MRD/BM Yes FLU/BU CsA

3 PGC F + Otitis LAD 13 MRD MRD/BM No FLU/BU CsA

4 SGE*** F - Erythroderma/FTT Omenn 3 Haplo Mother/PBSCT Yes

2 trials

FLU/BU CsA

5 MK M - Aspergillosis/FTT CGD 120 Haplo Father/PBSCT No Thio/BU/FLU/Alem CsA

6 TTD*** M + Omphalitis/

diarrhea

LAD 4 MRD Sister/BM No FLU/BU/ATG CsA

7 IB F + Diarrhea/FTT MHC class 2 21 MRD Brother/BM No BU/FLU CsA

8 MNA** F + HSM CHS 4 MRD Mother/BM No BU/FLU/ATG CsA

9 MA** F + HSM CHS 3 1 Ag MM Father/PBSCT No BU/FLU/ATG CsA

10 HCÇ## M + Aspergillosis/FTT CGD 75 MUD - No BU/FLU/Alem CsA

11 YEK## M + Petechiae WAS 48 MUD - No BU/FLU/Alem CsA

*Exome sequencing showed ATM frame-shift mutation after he died, **Siblings, ***Referred from another region of Turkey, ## HSCT performed at Akdeniz University.

CHS: Chediak-Higashi syndrome, BM: bone marrow, Thio: thiotepa, BU: busulfan, CsA: cyclosporine, F: female, FTT: failure to thrive, FLU: fludarabine, GvHD: graft-versus-host disease, HSCT: hematopoietic stem cell transplantation, M:

male, MRD: matched related donor, MM: mismatched, ATG: antithymocyte globulin, Alem: alemtuzumab, NK: natural killer cell, PBSCT: peripheral blood stem cell transplantation, SCID: severe combined immunodeficiency, T: T cell, HSM:

hepatosplenomegaly, ND: not done, C#: consanguinity.

347



Table 3. Outcomes and complications of severe combined immunodeficiency patients.

Patient BCG

activation

CMV

reactivation

Bacterial

infections/cause

Viral infections/cause aGvHD/site cGvHD/site PICU admission/cause Last chimerism/

need for IVIG

1 FD Yes No No No Skin No Hemophagocytosis 90%/yes Dead

2 MK Yes No No No No No No 95%/yes Alive

3 MO No No Catheter/

Acinetobacter

No Skin No No 100%/no Alive

4 IY No No No No No No No 87%/yes Alive

5 PA No No No No No Skin No 17%/yes Alive

6 YFK* No Yes No CMV No No No 0%/yes Dead

7 BY Yes No No No No No No 35%/no Alive

8 ET** No No Yes/staph No No No Yes/pneumothorax ND Dead

9 FZS*** No No Yes/staph Yes/unknown ND ND No ND Dead

10 EK No No No No No Autoimmune anemia No 80%/yes Alive

11 GS Yes No No No Skin No No 98%/no Alive

*She was treated for CMV infection at another center before being sent to our clinic, **She had bilateral pneumothorax and Staphylococcus septicemia before being sent to our clinic, *** She was in sepsis before being referred to our

clinic and died on the same day.

ND: Not done, aGvHD: acute graft-versus-host disease, cGvHD: chronic graft-versus-host disease, CMV: cytomegalovirus, BCG: bacillus Calmette-Guerin, PICU: pediatric intensive care unit.

Patient

status

Table 4. Outcomes and complications of non-SCID patients.

Patient BCG

activation

CMV

reactivation

Bacterial infections/

cause

Viral infections/

cause

aGvHD/

site

cGvHD/

site

PICU admission/cause Last chimerism Patient status

1 SA No No No No No No Yes/hemophagocytosis ND Dead

2 EDT No No S. haemolyticus No No No Yes/sepsis ND Dead

3 PGC No No E. faecium No No No No 100% Alive

4 SGE No No P. aeruginosa No No No No 5% Dead

5 MK No No No No No No Yes/ineffective

ventilation

ND Dead

6 TTD No No No CMV Skin Skin No 90% Alive

7 IB No No Cryptosporidium CMV No No No 100% Alive

8 MNA No No No No No No No 100% Alive

9 MA No No No No No No Yes/hemophagocytosis ND Alive

10 HCC No No No No Skin No No ND Alive

11 YEK No No Unknown CMV No No No 100% Alive

ND: Not done, aGvHD: acute graft-versus-host disease, cGvHD: chronic graft-versus-host disease, CMV: cytomegalovirus, PICU: pediatric intensive care unit.

348



In recent report from Jordan, a country that resembles Turkey

socially, Amayiri et al. [10] concluded that delayed diagnosis

(or referral) and reactivation of bacillus Calmette-Guerin

(BCG) are unique challenges for patients with PID disorders.

Similarly, delayed diagnosis is an important problem in our

region because of the insufficient number of immunologists

and lack of physician awareness about PID disorders [11]. BCG

vaccine reactivation has an important effect on the prognosis

of combined immunodeficiencies, but this vaccine also helps to

identify interferon gamma/interleukin 2 axis defects with BCGitis

[12] and to determine T-lymphocyte function with a positive

tuberculin (purified protein derivative) test.

Epidemiological studies in various countries have shown

that X-linked common gamma-chain deficiency is the most

common type of SCID, affecting almost half of all patients.

In our patients with SCID, 55% had RAG1 and RAG2, 33%

had JAK3, and 11% had IL2RG mutations. The present study

showed that RAG mutations are more prevalent in SCID

cases in Turkey than in Europe and the United States [13].

However, gamma-chain deficiencies are rare in the Greek

population [14]. The higher incidence of RAG mutations in

our region could be related to high parental consanguinity.

We used the CliniMACS method for efficient T-cell depletion

prior to transplantation. After applying this method, we

observed acute (18%-28%) and chronic (9%-18%) GVHD in the

SCID and non-SCID cases. Our patients displayed low infection

rates and BCG activation and less need for treatment in the

pediatric intensive care unit (PICU), which could have been

due to the use of prophylactic antituberculosis treatment.

A previous study addressing the outcomes of and mortalityrelated

risk factors for pediatric patients with PID requiring PICU

admission reported respiratory problems as the leading cause

for hospital admission [15]. In our study, six patients required

PICU admission, mostly due to severe infection and respiratory

problems.

Conclusion

This study showed that PID disorders are common and that the

delayed diagnosis of such disorders is an important problem

in the Black Sea Region of Turkey. Routine screening for these

diseases should be performed in newborn infants.

Acknowledgments

We would like to thank Professor Dr. Aydan İkincioğulları and

Professor Dr. Fikret Arpacı for their material and moral support.

Ethics

Ethics Committee Approval: Retrospective study.

Informed Consent: Retrospective study.


Surgical and Medical Practices: M.H.Ç., A.Y., S.B., Ş.N.G., M.E.,

T.F., E.Ö., R.S., G.O.; Concept: A.Y.; Design: A.Y.; Data Collection

or Processing: A.Y.; Analysis or Interpretation: A.Y.; Literature

Search: M.H.Ç., A.Y.; Writing: M.H.Ç., A.Y.




References

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Clin North Am 2015;35:695-711.

2. Gatti R, Meuwissen HJ, Allen HD, Hong R, Good RA. Immunological

reconstitution of sex-linked lymphopenic immunological deficiency. Lancet

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3. Bach FH, Albertini RJ, Joo P, Anderson JL, Bortin MM. Bone-marrow

transplantation in a patient with the Wiskott-Aldrich syndrome. Lancet

1968;2:1364-1366.

4. Cipe FE, Dogu F, Aytekin C, Yuksek M, Kendirli T, Yildiran A, Bozdogan G,

Karatas D, Reisli I, Dalva K, Arpacı F, Ikinciogullari A. HLA-haploidentical

transplantations for primary immunodeficiencies: a single-center

experience. Pediatr Transplant 2012;16:451-457.

5. Conley ME, Notarangelo LD, Etzioni A. Diagnostic criteria for primary

immunodeficiencies. Representing PAGID (Pan-American Group for

Immunodeficiency) and ESID (European Society for Immunodeficiencies).

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6. Pasic S, Vujic D, Veljković D, Slavkovic B, Mostarica-Stojkovic M, Minic P,

Minic A, Ristic G, Giliani S, Villa A, Sobacchi C, Lilić D, Abinun M. Severe

combined immunodeficiency in Serbia and Montenegro between years 1986

and 2010: a single-center experience. J Clin Immunol 2014;34:304-308.

7. Yorulmaz A, Artaç H, Kara R, Keleş S, Reisli İ. Primer immün yetmezlikli 1054

olgunun retrospektif değerlendirmesi. Astım Alerji İmmünoloji 2008;6:127-

134 (in Turkish).

8. Borte S, von Döbeln U, Fasth A, Wang N, Janzi M, Winiarski J, Sack U, Pan-

Hammarström Q, Borte M, Hammarström L. Neonatal screening for severe

primary immunodeficiency diseases using high-throughput triplex realtime

PCR. Blood 2012;119:2552-2555.

9. Arpacı F, Tezcan İ, Kuzhan O, Yalman N, Uçkan D, Kürekçi AE, İkincioğulları

A, Özet A, Tanyeli A. G-CSF-mobilized haploidentical peripheral blood

stem cell transplantation in children with poor prognostic nonmalignant

disorders. Am J Hematol 2008;83:133-136.

10. Amayiri N, Al-Zaben A, Ghatasheh L, Frangoul H, Hussein AA. Hematopoietic

stem cell transplantation for children with primary immunodeficiency diseases:

single center experience in Jordan. Pediatr Transplant 2013;17:394-402.

11. Yüksek M, İkincioğulları A, Doğu F, Elhan A, Yüksek N, Reisli I, Babacan E.

Primary immune deficiency disease awareness among a group of Turkish

physicians. Turk J Pediatr 2010;52:372-377.

12. Yıldıran A, Ak E, Akyol Ş, Sancak R, Picard C, Dogu F, Ikinciogullari A. Yaygın

BCG enfeksiyonu olan IL12R defekti olgusu. Deneysel ve Klinik Tıp Dergisi

2010;27:85-87 (in Turkish).

13. Buckley RH. Molecular defects in human severe combined immunodeficiency

and approaches to immune reconstitution. Annu Rev Immunol 2004;22:625-

655.

14. Michos A, Tzanoudaki M, Villa A, Giliani S, Chrousos G, Kanariou M. Severe

combined immunodeficiency in Greek children over a 20-year period: rarity

of γc-chain deficiency (X-linked) type. J Clin Immunol 2011;31:778-783.

15. Odek C, Kendirli T, Doğu F, Yaman A, Vatansever G, Çipe F, Haskoloğlu Ş,

Ateş C, İnce E, İkincioğulları A. Patients with primary immunodeficiencies in

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349

IMAGES IN HEMATOLOGY

DOI: 10.4274/tjh.2016.0096


Bullous Pyoderma Gangrenosum in a Patient with Acute

Myelogenous Leukemia as a Pathergic Reaction after Bone

Marrow Biopsy

Akut Miyelojenik Lösemili Olguda Kemik İliği Biyopsisi Sonrası Paterjik Reaksiyon Şeklinde

Büllöz Piyoderma Gangrenosum Gelişmesi

Nur Efe İris 1,2 , Reyhan Diz-Küçükkaya 3 , Mutlu Arat 3 , Zahide Eriş 4

1

İstanbul Bilim University Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey

2

Avrupa Florence Nightingale Hospital, Clinic of Infectious Diseases and Clinical Microbiology, İstanbul, Turkey

3

İstanbul Bilim University Faculty of Medicine, Department of Internal Medicine Division of Hematology, İstanbul, Turkey

4

İstanbul Bilim University Faculty of Medicine, Department of Dermatology, İstanbul, Turkey

Figure 1. Bone marrow biopsy puncture area with 6x4 cm cribriform ulceration with

expanding bullous margin.

A 59-year-old male patient presented with a wound over the

sacral region on a bone marrow biopsy puncture that had been

present for 3 weeks (Figure 1). There was an ulceration of 6x4

cm with a bullous margin. Bullous pyoderma gangrenosum (PG)

was diagnosed by the dermatology consultant. Histopathologic

examination of the biopsy specimen from the ulcer showed

necrosis with an underlying mixed inflammatory cell infiltration

within the dermis extending to the subcutis. Cultures of

skin biopsies were negative for bacteria, fungi, and atypical

mycobacteria. A bone marrow biopsy showed acute myelogenous

leukemia (AML) transformed from myelodysplastic syndrome.

PG is an uncommon neutrophilic ulcerative skin disease. In

contrast to its name, PG is neither an infectious nor a gangrenous

condition. Pathergy is commonly observed, especially after



Address for Correspondence/Yazışma Adresi: Nur EFE İRİS, M.D.,

İstanbul Bilim University Faculty of Medicine, Department of Infectious Diseases and Clinical Microbiology, İstanbul,

Turkey Phone : +90 212 361 88 00

E-mail : nurefeiris@yahoo.coml ORCID-ID: orcid.org/0000-0002-4859-0009


Accepted/Kabul tarihi: June 02, 2016

350


Efe İris N, et al: Bullous PG in a Patient with AML as a Pathergic Reaction after Bone Marrow Biopsy

debridement of a lesion [1,2]. In PG there is an excessive

inflammatory reaction to trauma of the skin by a needle. In this

case there was a pathergic reaction after bone marrow biopsy.

Definitive diagnosis requires both clinical recognition and

exclusion of infectious or neoplastic disorders [3]. PG is usually

associated with an underlying systemic disease [1,4]. Based on

clinical morphology, PG is classified into four variants: ulcerative,

pustular, bullous, and vegetative [5]. Bullous PG is commonly

associated with myeloproliferative diseases [5]. Association with

leukemia signifies a poor prognosis [5].

Our patient was in remission for AML, he underwent allogeneic

hematopoietic stem cell transplantation, and the PG resolved

completely.

Keywords: Acute myelogenous leukemia, Bullous pyoderma

gangrenosum, Pathergy

Anahtar Sözcükler: Akut myeloid lösemi, Büllöz piyoderma

gangrenosum, Paterji

Informed Consent: It was received.




included.

References

1. Fox PL, Geyer AS, Husain S, Grossman ME. Bullous pyoderma gangrenosum

as the presenting sign of fatal acute myelogenous leukemia. Leuk Lymphoma

2006;47:147-150.

2. Bennett ML, Jackson JM, Jorizzo JL, Fleischer AB Jr, White WL, Callen JP.

Pyoderma gangrenosum. A comparison of typical and atypical forms

with an emphasis on time to remission. Case review of 86 patients from 2

institutions. Medicine (Baltimore) 2000;79:37-46.

3. Callen JP, Dubin HV, Gehrke CF. Recurrent pyoderma gangrenosum and

agnogenic myeloid metaplasia. Arch Dermatol 1977;113:1585-1586.

4. Srivastata M, Rencic A, Nousari HC. A rapidly expanding ulcer.

Myelodysplastic syndrome-associated (paraneoplastic) pyoderma

gangrenosum. Arch Dermatol 2003;139:531-536.

5. Powell FC, Su WP, Perry HO. Pyoderma gangrenosum: classification and

management. J Am Acad Dermatol 1996;34:395-409.

351


DOI: 10.4274/tjh.2016.0199


Giant Intracranial Solitary Plasmacytoma

İntrakraniyal Dev Plazmositom

Osman Kara 1 , Tayfur Toptaş 1 , Işık Atagündüz 1 , Süheyla Bozkurt 2 , Önder Şirikçi 3 , Tülin Fıratlı Tuğlular 1

1

Marmara University Faculty of Medicine, Department of Hematology, İstanbul, Turkey

3

Marmara University Hospital Faculty of Medicine, Department of Pathology, İstanbul, Turkey

3

Marmara University Hospital Faculty of Medicine, Department of Biochemistry, İstanbul, Turkey

Figure 1. The mass occupied the left frontoparietal region,

displacing the left lateral ventricle and causing a shift of the midverge

of the brain.

Figure 2. Postoperative cranial magnetic resonance imaging.

A 41-year-old man presented with complaints of severe headache and

vomiting during the last 5 days. Neurological and systemic examination

revealed no abnormality. A mass of 112x49 mm, which occupied

the left frontoparietal parenchymal region, was evident on the T 1

sequence of magnetic resonance imaging (Figure 1). This homogeneous

contrasted mass displaced the left lateral ventricle and caused a shift

of the mid-verge of the brain. The mass was totally removed (Figure 2).

Pathological examination revealed a plasma cell dyscrasia with lambda

monoclonality.

Bone marrow biopsy was consistent with a clonal plasma cell

accumulation of 5%. Two tiny M-protein peaks were detected

on serum protein electrophoresis, which was compatible with

immunoglobulin (Ig) G-and IgA-lambda monoclonal bands

on serum immunofixation electrophoresis (Figure 3). There

were no other plasmacytomas or lytic lesions detected with

positron emission tomography-computed tomography imaging.

The patient was diagnosed with a solitary plasmacytoma and

treated with radiotherapy only. He had no complaints at the

sixth month after diagnosis.



Address for Correspondence/Yazışma Adresi: Osman KARA, M.D.,

Marmara University Faculty of Medicine, Department of Hematology, İstanbul, Turkey

Phone : +90 505 492 69 25

E-mail : dr_osmankara@hotmail.com ORCID-ID: orcid.org/0000-0001-9531-8123



352


Kara O, et al: Giant Intracranial Solitary Plasmacytoma

Keywords: Plasmacytoma, Myeloma, Intracranial

Anahtar Sözcükler: Plazmasitom, Miyelom, İntrakraniyal

Informed Consent: Was obtained from the patient.




included.

Figure 3. M-protein peaks on serum protein electrophoresis

and monoclonal bands on immunofixation electrophoresis are

depicted.

Ig: Immunoglobulin.

353


DOI: 10.4274/tjh.2017.0203


Peculiar Cold-Induced Leukoagglutination in Mycoplasma

pneumoniae Pneumonia

Mycoplasma pneumonia Pnömonisinde Alışılmamış Soğuk-Aracılı Lökoaglütinasyon

Yasushi Kubota 1,2 , Yuka Hirakawa 3 , Kazuo Wakayama 4 , Shinya Kimura 1

1

Saga University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Saga, Japan

2

Saga University Faculty of Medicine, Department of Transfusion Medicine, Saga, Japan

3

Saga University Faculty of Medicine, Department of General Medicine, Saga, Japan

4

Saga University Faculty of Medicine, Department of Clinical Laboratory Medicine, Saga, Japan

Figure 1. A peripheral blood smear showed not only RBC agglutination (A) but also neutrophil aggregates, eosinophil aggregates, and

monocyte aggregates (A-D).



Address for Correspondence/Yazışma Adresi: Yasushi KUBOTA, M.D.

Saga University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, Saga, Japan

Phone : +81-952-34-23 66

E-mail : kubotay@cc.saga-u.ac.jp ORCID-ID: orcid.org/0000-0001-7785-1362



354


Kubota Y. et al. Peculiar Cold-Induced Leukoagglutination in Mycoplasma pneumoniae Pneumonia

An 18-year-old woman was diagnosed with atypical pneumonia

and treated with oral levofloxacin. Skin eruptions also appeared.

On day 6 after admission, laboratory tests revealed the

following: red blood cells (RBCs), 1.76x10 9 /L; hemoglobin, 128

g/L; white blood cells (WBCs), 7x10 9 /L with 56% neutrophils,

27% lymphocytes, 6% monocytes, 10.5% eosinophils, and

1% basophils. A peripheral blood smear showed not only

RBC agglutination but also neutrophil aggregates, eosinophil

aggregates, and monocyte aggregates (Figure 1). After warming

to 37 °C, the agglutination disappeared. The RBC and WBC

counts returned to 4.44x10 9 /L and 9x10 9 /L with 55% neutrophils,

26% lymphocytes, 6% monocytes, 12% eosinophils, and 1%

basophils. Blood chemistry analysis showed total bilirubin

of 0.4 mg/dL and lactate dehydrogenase of 510 U/L. A direct

antiglobulin test showed 1+ anti-C3d and 1+ anti-C3b3d. A

passive agglutination test in paired serum samples revealed

seroconversion of M. pneumoniae antibodies (1:80 to 1:20,480).

Cold agglutinin was detected to a titer of 1:8192.

Cold-induced erythrocyte agglutination is frequently observed

in cases of M. pneumoniae infection, but leukoagglutination is

rare [1,2]. Though the pathomechanism of leukoagglutination is

still uncertain [3], it has been postulated that immunoglobulin

M cold agglutinin directed against I antigens of the leukocyte

membranes is responsible for transient cold-induced

leukoagglutination [4]. A previous series of four pediatric

cases of M. pneumoniae infection, all of which showed

leukoagglutination, reported that eruption, eosinophilia, a high

titer of cold agglutinin, and a high titer of M. pneumoniae

antibodies were observed [5]. When leukocytopenia occurs in

patients with these symptoms, pseudoleukopenia induced by

leukoagglutination should be recognized as one potential cause.

Keywords: Leukoagglutination, Cold agglutinin, Mycoplasma

pneumoniae, Eosinophilia, Pseudoleukopenia

Anahtar Sözcükler: Lökoaglütinasyon, Soğuk aglütinin,

Mycoplasma pneumoniae, Eozinofili, Psödolökopeni

References

1. Berentsen S, Randen U, Tjønnfjord GE. Cold agglutinin-mediated

autoimmune hemolytic anemia. Hematol Oncol Clin North Am 2015;29:455-

471.

2. Gertz MA. Management of cold haemolytic syndrome. Br J Haematol

2007;138:422-429.

3. Glasser L. Pseudo-neutropenia secondary to leukoagglutination. Am J

Hematol 2005;80:147.

4. Pruzanski W, Faird N, Keystone E, Armstrong M. The influence of

homogeneous cold agglutinins on polymorphonuclear and mononuclear

phagocytes. Clin Immunol Immunopathol 1975;4:277-285.

5. Takiguchi M, Iizuka A, Nagao T. White blood cell aggregation in Mycoplasma

pneumoniae infection. Syonika 1981;22:643-648 (in Japanese).

355

LETTERS TO THE EDITOR


Liver Transplantation in a Patient with Acquired

Dysfibrinogenemia Who Presented with Subdural Hematoma: A

Case Report

Subdural Hematom ile Prezente Olan Edinsel Disfibrinojenemi Nedenli Karaciğer Nakli

Uygulanan Hasta: Olgu Sunumu

Şencan Acar 1 , Gökhan Güngör 2 , Murat Dayangaç 3 , Reyhan Diz-Küçükkaya 4 , Yaman Tokat 3 , Murat Akyıldız 1

1

İstanbul Memorial Ataşehir Hospital, Liver Transplantation Unit, İstanbul, Turkey

2

Konya Training and Research Hospital, Department of Internal Diseases, Konya, Turkey

3

İstanbul Bilim University Faculty of Medicine, Department of General Surgery, İstanbul, Turkey

4

İstanbul Bilim University Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey

To the Editor,

Fibrinogen is one of the most abundant proteins in the blood; normal

levels range from 200 to 400 mg/dL. Fibrinogen is synthesized in

the liver and is essential for the clotting of blood. It also binds

to platelets, supports aggregation, and plays an important role

in wound healing. Fibrinogen deficiencies can be caused by

decreased levels (hypo- or afibrinogenemia) or defective function

(dysfibrinogenemia). Dysfibrinogenemia may either be autosomal

dominantly inherited or acquired and it can manifest as bleeding or

thrombotic events, or in some cases both simultaneously. Situations

causing acquired dysfibrinogenemia include chronic liver disease,

malignancies, and autoimmune diseases. Herein, we report a liver

transplant recipient with dysfibrinogenemia who presented with

subdural hematoma due to liver cirrhosis.

A 41-year-old male presented to the emergency department

with headache and sicchasia. Cranial computerized tomography

(CT) imaging showed subdural hematoma and surgical drainage

was planned. Since he had thrombocytopenia, prolonged

prothrombin time (PT), and hyperbilirubinemia, he was

evaluated by a gastroenterology specialist and diagnosed with

decompensated liver cirrhosis. Laboratory findings are shown in

Table 1. Abdominal CT imaging showed liver cirrhosis, ascites,

and splenomegaly.

The neurosurgery specialist suggested conservative treatment

because of the high risk of surgery for the patient who had

decompensated liver cirrhosis. The patient then consulted with

the hematology department. Both the patient and his family had

a negative history of bleeding. The peripheral blood smear was

not consistent with disseminated intravascular coagulopathy

(DIC). Thrombin time (TT) was 26 s (normal range: 16-20 s),

fibrinogen activity was 106 (180-350), PT was 18.7 s (9.8-12.7

s), and activated partial thromboplastin time (aPTT) was 41.7

s (27-38.8 s). Results for the mixing test (the patient’s plasma

was mixed with normal pool plasma), corrected PT, aPTT, TT, and

factors X, V, VIII, and IX were within normal limits. Acquired

dysfibrinogenemia because of liver cirrhosis was diagnosed

based on thromboelastographic findings.

Cryoprecipitate and fresh frozen plasma were administered until

coagulation test results returned to normal, but the patient’s

consciousness deteriorated. Subdural hematoma drainage was

then performed and consciousness dramatically improved.

The patient was placed on the waiting list for cadaveric

liver transplantation due to decompensated liver cirrhosis.

Cadaveric liver transplantation was performed 3 months later.

Posttransplant follow-up coagulation tests were dramatically

improved (Table 2).

Table 1. The patient’s laboratory findings.

Hb: 9.9 g dL -1 aPTT: 41.7 s (27-38.8 s) GGT: 69 U L -1 Fibrinogen activity: 106 (180-350)

WBC: 3560 µL -1 TT: 26 (16-20 s) T.Bil: 3.2 mg dL -1 D-dimer: 1.43 (<0.7)

PLT: 74,000 µL -1 AST: 43 U L -1 D.Bil: 1.77 mg dL -1 Creatinine: 0.7 mg dL -1

INR: 1.7 ALT: 27 U L -1 Albumin: 4 g dL -1 Ammonia: 134 µg dL -1

PT: 18.7 s (9.8-12.7 s) ALP: 26 U L -1 Globulin: 2.5 g dL -1

Hb: Hemoglobin, aPTT: activated partial thromboplastin time, GGT: gamma-glutamyltransferase, WBC: white blood cell, PLT: platelet, INR: international normalized ratio,

PT: prothrombin time.

356



Table 2. The patient’s hematological laboratory findings

upon liver transplantation follow-up.

PT: 10.3 s

aPTT: 31.5 s

Fibrinogen activity: 218

D-dimer: 0.47

PT: Prothrombin time, aPTT: activated partial thromboplastin.

Dysfibrinogenemia can be caused by posttranslational sialylation

of fibrinogen, as is seen in patients with chronic liver disease, and

it can present with bleeding complications. Dysfibrinogenemia

can also cause thrombotic complications if defective fibrinogen

molecules are resistant to plasmin cleavage. In the presented

case, dysfibrinogenemia secondary to liver cirrhosis resulted in

serious bleeding that was cured following liver transplantation.

In cases of portal hypertension and liver cirrhosis, defective

hemostasis can occur due to a decrease in procoagulant and

anticoagulant protein synthesis, a decrease in the destruction

of activated coagulant factors, functionally abnormal

fibrinogen synthesis, thrombocytopenia, and abnormal platelet

function [1,2,3]. Abnormal fibrinogen is found in patients

with diseases characterized by increased sialic acid content.

However, dysfibrinogenemia may also occur in other systemic

diseases such as multiple myeloma, autoimmune disorders,

and malignancy, and with the usage of some medications

(glucocorticoid, isotretinoin, and antileukemic agents) [4,5].

When dysfibrinogenemia is suspected, factor deficiencies and

DIC should be excluded. Normal or high antigen levels and

low functional activity are commonly seen in patients with

dysfibrinogenemia [6].

There is no specific treatment for dysfibrinogenemia and

treatment should be personalized. Fresh frozen plasma or

cryoprecipitate should be given for bleeding [7]. However,

antithrombotic treatment (warfarin) should be offered in cases

of thrombotic complications. Acquired dysfibrinogenemia

may lead to fatal bleeding in cirrhotic patients and liver

transplantation is the only curative treatment in cases of that

rare complication.

Keywords: Dysfibrinogenemia, Liver transplantation, Subdural

hematoma

Anahtar Sözcükler: Disfibrinojenemi, Karaciğer nakli, Subdural

hematom




included.

References

1. Sallah S, Kato G. Evaluation of bleeding disorders. A detailed history and

laboratory tests provide clues. Postgrad Med 1998;103:209-218.

2. Senzolo M, Burra P, Cholongitas E, Burroughs AK. New insights into

the coagulopathy of liver disease and liver transplantation. World J

Gastroenterol 2006;12:7725-7736.

3. Fuse I. Disorders of platelet function. Crit Rev Oncol Hematol 1996;22:1-25.

4. Undas A, Zabczyk M, Iwaniec T. Dysfibrinogenemia: from bleeding tendency

to thromboembolic disorders. Boletim da SPHM 2011;26:5-17.

5. Undas A. Acquired dysfibrinogenemia in atherosclerotic vascular disease.

Pol Arch Med Wewn 2011;121:310-318.

6. Beyan C. Diagnostic management in patients with thrombophilia. Turkiye

Klinikleri J Int Med Sci 2005;1:71-81 (in Turkish with an abstract in English).

7. Soransen B, Bevan D. A critical evaluation of cryoprecipitate for replacement

of fibrinogen. Br J Haematol 2010;149:834-843.

Address for Correspondence/Yazışma Adresi: Şencan ACAR, M.D.,

İstanbul Memorial Ataşehir Hospital, Liver Transplantation Unit, İstanbul, Turkey

Phone : +90 533 769 71 96

E-mail : sencanacar@yahoo.com ORCID-ID: orcid.org/0000-0001-8086-0956


Accepted/Kabul tarihi: May 31, 2017

DOI: 10.4274/tjh.2017.0045

357

LETTERS TO THE EDITOR Turk J Hematol 2017;34:356-381

Chronic Active Parietal Osteomyelitis Due to Salmonella typhi in a

Patient with Sickle Cell Anemia

Orak Hücreli Anemi Hastasında Salmonella typhi Kaynaklı Kronik Aktif Parietal Osteomiyelit

Ahmad Antar 1 , George Karam 2 , Maurice Kfoury 3 , Nadim El-Majzoub 4

1

Almoosa Specialist Hospital, Department of Internal Medicine, Division of Hematology-Oncology, Al-Ahsa, Saudi Arabia

2

Almoosa Specialist Hospital, Department of Neurosurgery, Al-Ahsa, Saudi Arabia

3

Almoosa Specialist Hospital, Department of Diagnostic Radiology, Al-Ahsa, Saudi Arabia

4

American University of Beirut Medical Center, Department of Pathology and Laboratory Medicine, Beirut, Lebanon

To the Editor,

Sickle cell disease (SCD) is a genetic disorder characterized by

marked heterogeneity in clinical and hematologic severity, with

musculoskeletal system manifestations being a major cause

of morbidity and disability [1]. The increased susceptibility

of SCD patients to infections, including osteomyelitis, has

long been recognized with several mechanisms postulated

including impaired splenic function, defects in complement

activation, genetic factors, deficiencies in micronutrients, and

the presence of infarcted or necrotic bone [2]. Salmonella is

the most common cause of osteomyelitis in SCD, followed by

Staphylococcus aureus and gram-negative enteric bacilli;

this prevalence could be related to the fact that intravascular

sickling of the bowel leads to patchy ischemic infarction [3,4].

The most common sites of osteomyelitis are the femur, tibia,

or humerus. Patients usually present with acute onset of pain,

swelling, and tenderness over the affected area in association

with fever and elevated inflammatory markers. However, in

some cases, osteomyelitis has atypical presentations with a

more indolent course and often with abscess formation [5].

Here we present a 50-year-old female patient with sickle cell

anemia (SCA) who developed parietal osteomyelitis with abscess

formation and involvement of the dura due to Salmonella typhi,

who was treated successfully by surgery followed by antibiotics.

A 50-year-old Saudi female patient living in the Eastern Province

of Saudi Arabia, diagnosed with SCA (HgS: 78%) with occasional

vaso-occlusive crisis and no sickle cell-related complications,

presented to us with a 1-month history of a painless right

parietal subgaleal collection increasing in size over time with no

history of trauma and no fever or neurological manifestations.

Laboratory testing revealed an elevated white blood cell count

and a high estimated sedimentation rate level (125 mm/h).

Magnetic resonance imaging of the brain revealed an osteolytic

defect centered on the right parietal bone and sizable subgaleal

complex collection (Figure 1). The patient underwent right

Figure 1. Magnetic resonance imaging of the brain demonstrating

peripheral enhancement of the scalp abscess alongside noticeable

enhancement of the adjacent soft tissues, pathological bone, and

underlying pachymeningeal layer enhanced on a coronal T1W

slice (left) with a clear intra-osseous edematous edema in keeping

with osteomyelitic changes isolating a central bone sequestrum,

communicating with a subperiosteal/subgaleal fluid collection on

a fluid attenuated inversion recovery weighted slice (right).

parietal craniectomy with cranioplasty (removal of the right

parietal subgaleal collection and the corresponding bone in

addition to the invaded dura). Pathology of the specimen

revealed a right parietal subgaleal abscess and right parietal

bone chronic active osteomyelitis. Culture of the specimen grew

Salmonella typhi.

The morbidity of chronic osteomyelitis combined with other

complications of SCD decreases patients’ quality of life. Patients

with SCD are more prone to osteomyelitis. The most common

causative organism is Salmonella. The usual manifestations of

osteomyelitis are pain, swelling, tenderness, and fever. However,

like in our case, sometimes osteomyelitis presents late, as a

more indolent process often with abscess formation and in

unusual and more critical sites. Our case highlights the atypical

presentation of osteomyelitis in a patient with SCD, which could

cause devastating complications if not treated properly, early,

and by a multidisciplinary team approach.

358



Keywords: Sickle cell anemia, Osteomyelitis, Salmonella typhi

Anahtar Sözcükler: Orak hücreli anemi, Osteomiyelit,

Salmonella typhi




included.

References

1. Almeida A, Roberts I. Bone involvement in sickle cell disease. Br J Haematol

2005;129:482-490.

2. Booth C, Inusa B, Obaro SK. Infection in sickle cell disease: a review. Int J

Infect Dis 2010;14:e2-e12.

3. Atkins BL, Price EH, Tillyer L, Novelli V, Evans J. Salmonella osteomyelitis in

sickle cell disease children in the East End of London. J Infect 1997;34:133-138.

4. Burnett MW, Bass JW, Cook BA. Etiology of osteomyelitis complicating

sickle cell disease. Pediatrics 1998;101:296-297.

5. Barrett-Connor E. Bacterial infection and sickle cell anemia. An analysis

of 250 infections in 166 patients and a review of the literature. Medicine

(Baltimore) 1971;50:97-112.

Address for Correspondence/Yazışma Adresi: Ahmad ANTAR, M.D.,

Almoosa Specialist Hospital, Department of Internal Medicine, Division of Hematology-Oncology,

Al-Ahsa, Saudi Arabia Phone : +013 530 70 00

E-mail : a.antar@almoosahospital.com.sa ORCID-ID: orcid.org/0000-0003-1829-197X



DOI: 10.4274/tjh.2017.0094

Acquired Leukocyte Inclusion Bodies Resembling Döhle Bodies

During Acute Cholangitis

Akut Kolanjit Seyrinde Lökositlerde Döhle Benzeri Edinsel İnklüzyonlar

Gökhan Özgür 1 , Musa Barış Aykan 2 , Murat Yıldırım 1 , Selim Sayın 1 , Ahmet Uygun 3 , Cengiz Beyan 4

1

Gülhane Training and Research Hospital, Department of Hematology, Ankara, Turkey

2

Health Sciences University, Gülhane Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey

3

Health Sciences University, Gülhane Faculty of Medicine, Department of Gastroenterology, Ankara, Turkey

4

TOBB University of Economics and Technology Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey

To the Editor,

A 66-year-old woman was admitted to the gastroenterology

department with epigastric pain, nausea, and subicterus.

Her complaints had begun 6 h earlier. Her abdomen was soft

and flat, with localized tenderness on palpation in the right

subcostal area. Laboratory studies revealed a white cell count

of 17.9x10 9 /L, hemoglobin concentration of 14.4 g/dL, and

platelet count of 48x10 9 /L, and they were notable for elevated

serum cholestatic enzymes. The abdominal ultrasound was

remarkable for cholangitis. The patient received broad-spectrum

antibiotics. A peripheral blood smear examination, performed

to evaluate thrombocytopenia, revealed the presence of blue

intracytoplasmic inclusions in neutrophils (Figures 1A-C). On

the 11 th day of treatment, her blood smear was examined once

again and the Döhle body-like inclusions were resolved (Figure

1D).

May-Hegglin anomaly is an uncommon autosomal dominant

abnormality characterized by large, basophilic inclusion bodies

(resembling Döhle bodies) in neutrophils [1,2]. Döhle bodies

Figure 1. A) Inclusion bodies in neutrophils and macrothrombocyte;

B), C) inclusion bodies in neutrophils; D) peripheral blood smear

after treatment.

can be seen in bacterial infections. Hematologic findings of

systemic diseases may be confused with hematological diseases

such as May-Hegglin anomaly. We thought that the granules

were Döhle bodies due to cholangitis. The disappearance of the

inclusion bodies upon treatment is important in differential

diagnosis.

359


Keywords: Cholangitis, Döhle bodies, May-Hegglin anomaly

Anahtar Sözcükler: Döhle cisimciği, Kolanjit, May-Hegglin

anomalisi




References

1. Saito H, Kunishima S. Historical hematology: May-Hegglin anomaly. Am J

Hematol 2008;83:304-306.

2. Gülen H, Erbay A, Kazancı E, Vergin C. A rare familial thrombocytopenia: May-

Hegglin anomaly report of two cases and review of the literature. Turk J

Haematol 2006;23:111-114.

Address for Correspondence/Yazışma Adresi: Gökhan Özgür, M.D.,

Gülhane Training and Research Hospital, Department of Hematology, Ankara, Turkey

Phone : +90 312 304 4107

E-mail : gokhanozgur2010@hotmail.com ORCID-ID: orcid.org/0000-0003-0357-0503



DOI: 10.4274/tjh.2017.0121

Three Novel Calreticulin Mutations in Two Turkish Patients

İki Türk Hastada Üç Yeni Kalretikulin Mutasyonu

Veysel Sabri Hançer 1 , Hüseyin Tokgöz 2 , Serkan Güvenç 3 , Ümran Çalışkan 4 , Murat Büyükdoğan 1

1

İstinye University Faculty of Medicine, Department of Medical Genetics, İstanbul, Turkey

2

Selçuk University Meram Faculty of Medicine, Department of Pediatric Hematology, Konya, Turkey

3

Batman District State Hospital, Clinic of Hematology Batman, Turkey

4

Necmettin Erbakan University Meram Faculty of Medicine, Department of Pediatrics, Konya, Turkey

To the Editor,

Calreticulin (CALR) mutations were first identified exclusively in

JAK2-MPL-negative essential thrombocythemia (ET) and primary

myelofibrosis (PMF) at a rate of 60%-88%, accounting for 1/4 to

1/3 of all patients with ET and PMF [1,2,3]. As of today, more than

55 different types of mutations have been reported. The two most

common mutations accounting for 85% of mutated cases are

either a 52-bp deletion (type 1; c.1099_1150del; L367fs*46; 44%-

53% of cases) or a 5-bp insertion (type 2; c.1154_1155insTTGTC;

K385fs*47; 32%-42% of cases). The remaining 15% include

various other infrequent mutations that are often unique or

found in only a few patients [4,5].

Here we present three CALR mutations in two patients with PMF

and ET that have not been reported before as shown in Figure

1. Known CALR mutations and BCR-ABL, JAK-2 V617F, and MPL

515L/K test results were found to be negative.

Patient 1: The patient was a 46-year-old man with low back

pain. Magnetic resonance imaging scanning of the lumbosacral

region revealed sacroiliitis on the left side and he was referred

to a rheumatologist for further investigations. Anemia (Hb:

10.8 g/dL) and thrombocytosis (700x10 9 /L) with a high lactate

dehydrogenase level (351 U/L) were found in initial tests. The

other tests for a possible rheumatologic disease, including

Figure 1. A) Electropherogram result of the primary

myelofibrosis patient, B) electropherogram result of the essential

thrombocythemia patient.

human leukocyte antigen-B27, were all negative when the

patient was seen. Physical examination was almost normal

with no sign of organomegaly. Spleen size was also normal in

the abdominal ultrasound. The peripheral blood smear showed

dacrocytes, occasional myelocytes (1%), and metamyelocytes

(1%). The bone marrow biopsy showed diffuse grade 3-4

reticulin fibrosis with atypical proliferation of megakaryocytes

and increased cellularity consistent with PMF.

Patient 2: A 9-year-old pediatric patient with thrombocytosis

(2800x10 9 /L) was identified in a routine check-up. Physical

360



examination was normal except for mild splenomegaly.

Complete blood count revealed increased platelet count

(2800x10 9 /L) with normal hemoglobin and leukocyte count.

Platelets were very abundant and clustered in the peripheral

blood smear. Bone marrow aspiration and biopsy examinations

showed tri-lineage hematopoiesis with an increased number and

clusters of megakaryocytes without fibrosis, which is consistent

with ET. She had persistently elevated platelet counts ranging

between 2000x10 9 /L and 2800x10 9 /L without any evidence

of reactive/secondary thrombocytosis such as infections,

medicine, autoimmune disorders, neoplasms, trauma, surgery, or

hematological disorders such as iron deficient anemia, chronic

hemolytic situations, and acute hemorrhages.

Genomic DNA was extracted from whole blood, exon 9 of the

CALR gene was amplified by polymerase chain reaction, and then

the amplified fragments were sequenced. All nucleotide numbers

refer to the wild-type cDNA sequence of CALR (NM_004343) as

reported in Ensembl. Here we report three new CALR mutations

[1-bp deletion; c.1116delA (D373fs*57) and c.1120 A>C] in the

same patient with PMF and c.1108 G>T in a patient with ET. We

performed germline testing from the cheek epithelium and both

patient samples were confirmed as wild-type CALR. These novel

mutations occurred and changed the amino acid sequence of

the C domain amino acid residues, which will interfere with the

calcium-binding capacity of the molecule. It is important to

determine the type of mutation. Type 2-like CALR mutations are

mainly associated with an ET phenotype, low risk of thrombosis,

and indolent clinical course, while type 1-like mutations are

mainly associated with a myelofibrosis phenotype and a high

risk of progression from ET to myelofibrosis. The identification

of new CALR mutations will improve our understanding of the

pathophysiology of myeloproliferative neoplasms.

Keywords: Essential thrombocythemia, Primary myelofibrosis,

Calreticulin

Anahtar Sözcükler: Esansiyel trombositemi, Primer miyelofibroz,

Kalretikulin




included.

References

1. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumie E, Milosevic

JD, Them NC, Berg T, Gisslinger B, Pietra D, Chen D, Vladimer GI, Bagienski

K, Milanesi C, Casetti IC, Sant’Antonio E, Ferretti V, Elena C, Schischlik F,

Cleary C, Six M, Schalling M, Schönegger A, Bock C, Malcovati L, Pascutto C,

Superti-Furga G, Cazzola M, Kralovics R. Somatic mutations of calreticulin

in myeloproliferative neoplasms. N Engl J Med 2013;369:2379-2390.

2. Fu R, Zhang L, Yang R. Paediatric essential thrombocythaemia: clinical and

molecular features, diagnosis and treatment. Br J Haematol 2013;163:295-

302.

3. Guglielmelli P, Nangalia J, Green AR, Vanucchi A.M. CALR mutations in

myeloproliferative neoplasms: hidden behind the reticulum. Am J Hematol

2014;89:453-456.

4. Tefferi A, Wassie EA, Guglielmelli P, Nangat N, Belachew AA, Lasho TL,

Finke C, Ketterling RP, Hanson CA, Pardanani A, Wolanskyj AP, Maffioli M,

Casalone R, Pacilli A, Vannucchi AM, Passamonti F. Type 1 versus Type 2

calreticulin mutations in essential thrombocythemia: a collaborative study

of 1027 patients. Am J Hematol 2014;89:121-124.

5. Giona F, Teofili L, Capodimonti S, Laurino M, Martini M, Marzella D,

Palumbo G, Diverio D, Foà R, Larocca LM. CALR mutations in patients with

essential thrombocythemia diagnosed in childhood and adolescence. Blood

2014;12:3677-3679.

Address for Correspondence/Yazışma Adresi: Veysel Sabri HANÇER, M.D.,

İstinye University Faculty of Medicine, Department of Medical Genetics, İstanbul, Turkey

Phone : +90 533 634 30 14

E-mail : vshancer@yahoo.com ORCID-ID: orcid.org/0000-0003-2994-1077

Received/Geliş tarihi: April 05, 2017


DOI: 10.4274/tjh.2017.0146

361


Imatinib-Induced Interstitial Pneumonitis Successfully Switched

to Nilotinib in a Patient with Prior History of Mycobacterium

tuberculosis Infection

Mycobacterium tuberculosis Enfeksiyonu Öyküsü Olan Hastada Başarılı Bir Şekilde

Nilotinibe Geçilen İmatinib ile Uyarılmış İnterstisyel Pnömonitis

Zhuan-Bo Luo 1 , Ning Xu 1 , Xiao-Ping Huang 1 , Gui-fang Ouyang 2

1

Department of Respiratory Diseases, Ningbo First Hospital, Affiliated Medical School of Ningbo University, Ningbo, China

2

Department of Hematology, Ningbo First Hospital, Affiliated Medical School of Ningbo University, Ningbo, China

To the Editor,

Imatinib mesylate (IM) has been proven to be an effective

treatment of chronic myeloid leukemia (CML) and this drug is

well tolerated [1]. Interstitial lung disease (ILD) associated with

imatinib therapy is rare. We report the case of a patient who

had a prior treatment history of Mycobacterium tuberculosis

infection and developed interstitial pneumonia after 10

months of imatinib for CML and who has not relapsed since

the introduction of the recent tyrosine kinase inhibitor nilotinib.

A 48-year-old Chinese man was diagnosed with chronic-phase

Philadelphia chromosome-positive CML in January 2015. His

medical history was unremarkable, but he had a history of

previous treatment for pulmonary tuberculosis 25 years ago. He

was initially treated with IM at a dose of 400 mg daily, which

was well tolerated. Complete hematological response was

rapidly achieved after 2 months. Following the administration

of imatinib, the patient gradually developed a dry cough and

dyspnea on exertion. In November 2015, he visited the clinic

because of progressing nonproductive cough. He had been

treated with imatinib at a dose of 400 mg/day for 10 months.

On examination, fine crackles were audible, predominantly in

both posterior lower lung fields. No elevations of the acutephase

reactants were detected, and the immunoglobulin E

blood level was within the normal limits. Rheumatoid factor

was negative, and antinuclear antibodies were positive at 1/100

with homogeneous staining. Sputum culture was negative and

no acid-fast bacilli were observed. Lung function estimation

demonstrated mild impairment of gas exchange with diffusing

capacity [carbon monoxide diffusion in the lung (DLCO)] of

5.61 mmol/min per kPa (51.9% predicted) and mild restrictive

impairment with forced vital capacity of 3.30 L (69.1% predicted).

A chest radiograph showed fibrotic scar lesions in the left upper

lung field associated with the previous pulmonary tuberculosis.

A computed tomography scan (Figure 1A) showed significant

extension of the interstitial lung abnormalities, predominantly

in the lower lobes. Bronchoscopy revealed normal airways, and

histopathological analysis of the transbronchial lung biopsy

demonstrated nonspecific interstitial pneumonitis, showing

thickened alveolar septa with modest infiltration of chronic

inflammatory cells and slight interstitial fibrosis (Figures 2A and

2B). As these findings were highly suggestive of imatinib-induced

interstitial pneumonitis, this agent was discontinued and was

replaced by nilotinib. At the same time, prednisone at 30 mg/

day was given during the initial days, and it was slowly tapered

to 10 mg/day over 2 months. Because no signs of recurrence

of pulmonary tuberculosis were detected and the patient was

afraid of the side effects of anti-tuberculosis drugs, we did not

give anti-tuberculosis prophylaxis, but we maintained close

follow-up. This resulted in a gradual improvement in his clinical

condition. Partial radiological resolution was observed after 4

months and further improved at 8 months (Figure 1B). DLCO

improved to 6.78 mmol/min per kPa (62.8% predicted) and

forced vital capacity was 4.50 L (83.3% predicted). The switch

to nilotinib at 800 mg daily was well tolerated and followed by

complete cytogenetic and major molecular response sustained

for 8 months.

IM is a targeted therapy that is highly active in patients with

CML. It acts by inhibition of tyrosine kinase of the BCR-ABL

fusion oncoprotein specific to CML. ILD is a rare adverse event

associated with IM therapy. Few case series have been reported

[2,3,4,5]. In the present case, the diagnosis of IM-induced ILD

was made based on history, clinical symptoms, radiological

findings, and pathological results. Furthermore, other etiologic

factors for ILD were excluded via microbiologic and clinical

studies.

Until recently, there has been a lack of data for specific risk

factors for the development of IM-induced ILD. However, the

incidence of the disease seems higher in patients with preexisting

362



Figure 1. A) Chest computed tomography (CT) shows bilateral

diffuse subpleural nodules, interlobular septal thickening,

reticulation, and peribronchial ground glass opacities in both

lungs. B) Chest CT scan 8 months after the switch to prednisone

and nilotinib: lung abnormalities were decreased.

pulmonary diseases. The largest case series study from Japan,

which analyzed 27 patients with IM-induced ILD [6], revealed

that preexisting lung disease was present in more than 40%

of patients with IM-induced ILD. In this case, we had found

fibrotic change and pleural thickening of the left upper lung

associated with prior infection of Mycobacterium tuberculosis.

Thus, the case raises the possibility of the association between

IM-induced ILD and airway injury related to prior infection of

Mycobacterium tuberculosis. Physicians caring for a patient

presenting with respiratory symptoms while on imatinib therapy

should consider interstitial pneumonitis, especially in patients

with previous lung or airway injuries resulting from prior

infection of Mycobacterium tuberculosis.

Among the other tyrosine kinase inhibitors, similar pulmonary

complications were reported with dasatinib [7] and gefitinib [8]

in Japan, but not with nilotinib. In this case, 8 months after

the introduction of nilotinib, interstitial pneumonitis had not

recurred. Although the mechanistic basis for the absence of

cross-intolerance is not fully understood, second-generation

nilotinib appears to be an option in cases of ILD induced by

other tyrosine kinase inhibitors.

Figure 2. A and B) Histopathological appearance of transbronchial

lung biopsy specimens. Thickened alveolar septa with modest

infiltration of chronic inflammatory cells and slight interstitial

fibrosis are observed. Hematoxylin and eosin stain (H&E), 100 x

(A); H&E stain, 400 x (B).

Keywords: Imatinib mesylate, Interstitial pneumonitis, Chronic

myeloid leukemia, Nilotinib, Tuberculosis

Anahtar Sözcükler: İmatinib mesilat, İnterstisyel pnömonitis,

Kronik miyeloid lösemi, Nilotinib, Tüberküloz




included.

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of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid

leukemia. N Engl J Med 2001;344:1031-1037.

2. Delomas T, Darne C, Besson C. Lack of recurrence of imatinib-induced

interstitial lung disease with nilotinib. Leuk Lymphoma 2012;53:332-333.

363


3. Dao K, Vedy D, Lopez J, Staneczek O, Buclin T, Livio F. Imatinib-induced dosedependent

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patient with chronic myeloid leukemia. Turk J Hematol 2013;30:435-436.

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interstitial lung disease associated with imatinib mesylate. Leukemia

2006;20:1162-1164.

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Giovanelli H, Calvo F, Tazi A, Rousselot P. Lung abnormalities after dasatinib

treatment for chronic myeloid leukemia: a case series. Am J Respir Crit Care

Med 2007;176:814-818.

8. Ando M, Okamoto I, Yamamoto N, Takeda K, Tamura K, Seto T, Ariyoshi

Y, Fukuoka M. Predictive factors for interstitial lung disease, antitumor

response, and survival in non-small-cell lung cancer patients treated with

gefitinib. J Clin Oncol 2006;24:2549-2556.

Address for Correspondence/Yazışma Adresi: Zhuan-Bo LUO, M.D.,

Department of Respiratory Diseases, Ningbo First Hospital, Affiliated Medical School of

Ningbo University, Ningbo, China

E-mail : luozhuanbo2929@163.com ORCID-ID: orcid.org/0000-0003-0684-8363

Received/Geliş tarihi: April 12, 2017


DOI: 10.4274/tjh.2017.0155

Prostate Involvement in a Patient with Follicular Lymphoma

Foliküler Lenfomalı Hastada Prostat Tutulumu

Seda Yılmaz 1 , Sinan Demircioğlu 1 , Özlen Bektaş 1 , Özcan Çeneli 1 , Sıdıka Fındık 2

1

Necmettin Erbakan University Meram Medicine Faculty, Department of Hematology, Konya, Turkey

2

Necmettin Erbakan University Meram Medicine Faculty, Department of Pathology, Konya, Turkey

To the Editor,

While extranodal involvement is observed in 50% of cases

of non-Hodgkin’s lymphoma, prostatic involvement is rare.

Prostatic lymphoma accounts for 0.09% of all prostate

neoplasms and 0.1% of all non-Hodgkin’s lymphomas [1].

Our patient was monitored for 4 years and had stage 4BS

follicular lymphoma (bone marrow involvement; mesenteric

lymph nodes in the abdomen, the largest of which was

measured as 9x4 cm; cervical and mediastinal lymph nodes;

and splenomegaly and B symptoms) at the time of diagnosis.

He received CVP (cyclophosphamide, vincristine, prednisolone),

CHOP (cyclophosphamide, adriamycin, vincristine, prednisolone),

and gemcitabine therapy, respectively, and had lower urinary

tract symptoms during follow-up. A hypertrophic prostate was

palpated during the physical examination. The prostate-specific

antigen (PSA) level was measured to be 8.3 (normal range:

0-4) ng/mL. Urinary analysis showed microscopic hematuria.

Ultrasound examination detected a prostate volume of 60 mL.

Transurethral resection of the prostate (TUR-P) pathology results

showed a diffuse lymphocytic infiltration and positive staining

for CD20, CD10, CD5, and BCL-2 (Figure 1). The symptoms of

the patient regressed after treatment with rituximab plus

bendamustine.

Prostate cancer is the most common cancer among men

worldwide. There were 1,618,000 cases with 366,000 deaths in

2015 [2]. Prostatic lymphoma is a rare condition that accounts for

0.09% of all prostate neoplasms. While extranodal involvement

Figure 1. Diffuse lymphocytic infiltration.

is observed in about 50% of cases of non-Hodgkin’s lymphoma,

prostatic involvement is rare. The usual clinical manifestations

of prostatic involvement in lymphomas are lower urinary tract

symptoms and acute urinary retention. High serum PSA levels

are not typical for prostatic lymphoma. Our patient presented

with high PSA levels.

A study that investigated prostate materials from 4831 subjects

determined lymphoma in 29 subjects (0.6%). Eleven (0.23%)

subjects had a history of concurrent lymphoma [3]. In patients

with prostate cancer, the incidence of non-Hodgkin’s lymphoma

of the prostate was observed to be 0.2% in a series of 4319

radical prostatectomy cases [4] and 1.19% in another series of

1092 cases [5].

364



In conclusion, prostatic lymphoma is clinically difficult to

distinguish from benign prostatic hyperplasia and prostatic

carcinoma as it occurs in the same age group and presents

with similar symptoms; thus, the histopathological and

immunohistochemical findings in TUR-P material are important.

Early and appropriate treatment improves the patient’s quality

and length of life.

Keywords: Follicular lymphoma, Extranodal, Prostatic

involvement

Anahtar Sözcükler: Foliküler lenfoma, Ekstranodal, Prostat

Tutulumu




included.

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C, Barber RM, Barregard L, Bhutta ZA, Brenner H, Dicker DJ, Chimed-

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Hay RJ, Hunter-Merrill R, Huynh C, Hosgood HD, Johnson CO, Jonas JB,

Khubchandani J, Kumar GA, Kutz M, Lan Q, Larson HJ, Liang X, Lim SS, Lopez

AD, MacIntyre MF, Marczak L, Marquez N, Mokdad AH, Pinho C, Pourmalek

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Razek HMA, Malekzadeh R, Malta DC, Marcenes W, Markos D, Melaku YA,

Meles KG, Mendoza W, Mengiste DT, Meretoja TJ, Miller TR, Mohammad

KA, Mohammadi A, Mohammed S, Moradi-Lakeh M, Nagel G, Nand D, Le

Nguyen Q, Nolte S, Ogbo FA, Oladimeji KE, Oren E, Pa M, Park EK, Pereira

DM, Plass D, Qorbani M, Radfar A, Rafay A, Rahman M, Rana SM, Søreide K,

Satpathy M, Sawhney M, Sepanlou SG, Shaikh MA, She J, Shiue I, Shore HR,

Shrime MG, So S, Soneji S, Stathopoulou V, Stroumpoulis K, Sufiyan MB,

Sykes BL, Tabarés-Seisdedos R, Tadese F, Tedla BA, Tessema GA, Thakur JS,

Tran BX, Ukwaja KN, Uzochukwu BSC, Vlassov VV, Weiderpass E, Wubshet

Terefe M, Yebyo HG, Yimam HH, Yonemoto N, Younis MZ, Yu C, Zaidi Z, Zaki

MES, Zenebe ZM, Murray CJL, Naghavi M. Global, regional, and national

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2017;3:524-548.

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lymphomas discovered at the time of prostatectomy and prostate biopsy: a

study of 29 cases. Am J Surg Pathol 2005;29:693-699.

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Marshall FF, Epstein JI, Schoenberg M. Incidental non-Hodgkin’s lymphoma

in patients with localized prostate cancer. Urology 1999;53:175-179.

5. Terris MK, Hausdorff J, Freiha FS. Hematolymphoid malignancies diagnosed

at the time of radical prostatectomy. J Urol 1997;158:1457-1459.

Address for Correspondence/Yazışma Adresi: Sinan DEMİRCİOĞLU, M.D.,

Necmettin Erbakan University Meram Medicine Faculty, Department of Hematology, Konya, Turkey

Phone : +90 555 432 44 74

E-mail : sinandemircioglumd@gmail.com ORCID-ID: orcid.org/0000-0003-1277-5105



DOI: 10.4274/tjh.2017.0181

365


Coexistence of EZH2, NOTCH1, IL7R, and PHF6 Mutations in Adult

T-cell Acute Lymphoblastic Leukemia

Erişkin T-hücre Akut Lenfoblastik Lösemi’sinde EZH2, NOTCH1, IL7R ve PHF6

Mutasyonlarının Birlikteliği

Xilian Zhou 1 , Yan Gu 1 , Qi Han 1 , Mario Soliman 2 , Chunhua Song 2 , Zheng Ge 1

1

Zhongda Hospital, Medical School of Southeast University Department of Hematology Nanjing, China

2

Pennsylvania State University, Department of Pediatrics, Pennsylvania, USA

To the Editor,

Enhancer of zestehomolog 2 (EZH2) mutations are reported

in solid tumors [1,2,3] as well as leukemia, and they are

most commonly detected in early T-cell precursor acute

lymphoblastic leukemia (ETP-ALL) [4,5,6,7,8], which is an

extraordinarily aggressive malignancy of enigmatic genetic

basis [9]. We screened EZH2 mutations in 146 Chinese adult ALL

patients, among which 24.7% (36/146) cases were T-cell acute

lymphoblastic leukemia (T-ALL) and 12.9% (4/31) T-ALL cases

were identified as ETP-ALL. We found three EZH2 mutations

in two patients with T-ALL. One patient had Mu#1:D730fs*1,

a truncation mutation that was previously reported in acute

myeloid leukemia, and the another patient had two new EZH2

mutations, Mu#2:K466T and Mu#3:T467fs*>3 (Figure 1). We

also screened the mutations in other genes (Table 1). Strikingly,

the EZH2 mutations coexisted with mutations of NOTCH1, IL7R,

and PHF6 in the two patients and they responded poorly to

chemotherapy and experienced difficult clinical histories and

inferior outcomes (Table 1). Patient 1 was diagnosed with T-ALL

with myeloid expression based on his bone marrow (BM) smear

and immunophenotypes (Table 1). With the first inductive

therapy (Table 1), the patient achieved complete remission (CR)

with 0.1% blasts in the peripheral blood (PB) and 0.8% in BM.

One year later, the patient relapsed with 90.4% lymphoblasts in

the BM and 1.0% in the PB, and CR was achieved after the first

chemotherapy. During the following treatment, he underwent

an intramedullary and an extramedullary relapse infiltrating his

left tonsil and then endured three more relapses. On the fifth

relapse, the BM blast rate was 50.4%. Although the patient was

treated with nelarabine, no CR was achieved in the subsequent

treatments. Even though the BM blast rate was 5.2%, the

patient died of infection during the BM suppression period

after he received the last chemotherapy. We examined the

EZH2 mutational status in the BM samples of the 1 st relapse, 5 th

relapse, and 6 weeks after his 5 th relapse; the EZH2 and NOTCH1

mutation status remained the same as in the first diagnosis even

after the nelarabine treatment (Figure 1D). Patient 2 presented

with 80.0% lymphoblasts in the PB and 78.0% blasts in the

BM (Table 1). Two somatic mutations, K466T and T467fs*>3 in

Figure 1. Location and sequencing data of the EZH2 mutations.

A) Mutation 1 (Mu#1:D730fs*1), located in exon 19, is a frame

shift-creating insertion; on the protein level, it leads to a

truncated protein with a length of 731 amino acids, which is

located in the conserved catalytic SET domain(amino acids 618-

731). This domain is critical for the methyltransferase activity of

EZH2. The other two mutations (Mu#2:K466T; Mu#3:T467fs*>3)

located within exon 11 are anon-synonymous single-nucleotide

substitution and a frame shift-creating deletion, respectively; on

the protein level, they result in the substitution of EZH2 lysine466

to tyrosine and a truncation of the EZH2 protein, respectively.

Mu#2 and Mu#3 are novel EZH2 mutations; both of them are

located in the SANT domain of the EZH2 protein (amino acids

435-485), which is known to be incharge of the DNA binding.

Mu#1 was detected in patient 1 and the other two in patient

2. Blue, pink, and yellow bars correspond to exons encoding

the SANT domains, the cysteine-rich CXC domain, and the SET

domain, respectively. The red arrows show EZH2 mutations. B-F)

The direct sequencing data of EZH2 mutations (B, D, F) and wildtype

(C, E). B: c.2187_2188insT p.D730fs*1; C: EZH2 exon 19 wildtype;

D: Mu#1 after nelarabine treatment; E: EZH2 exon 11 wildtype;

F: c.1397A>C; 1399delA p.K466T; T467fs*>3.

366



Table 1. Clinical features of patients with EZH2 mutations.

Patient 1 Patient 2

Age (years)/Sex 49/Male 41/Female

WBC, x10 9 /L 9.7 64.9

Hemoglobin (g/L) 128 71

Platelets (x10 9 /L) 96 38

BM/PB blasts (%) 50.4/0 78.0/80.0

Mutation screening Patient 1 (nucleotide/mutant ID) Patient 2 (nucleotide/mutant ID)

Gene ALL (%)

(n=146)

T-ALL (%)

(n=36)

Exon

EZH2 1.4 5.6 Exon 19 2187_2188insT/COSM52999

Exon 11 1397A>C / (new) 1399delA / (new)

NOTCH1 75.0 74.4 Exon 34 7541_7542delCT/COSM13065 7329_7330delinsCCCA / COSM5752017

Exon 27 5033T>A / COSM21907

PHF6 33.3 33.3 Exon 2 134delG+insCC/(new)

IL7R 3.4 9.7 Exon 2 197T>C/COSM149813 197T>C / COSM149813

Exon 4 254G>A/COSM149814 254G>A / COSM149814

Exon 6 755_756ins9 / (new)

Exon 8 1066A>G/rs3194051

FBXW7 4.6 16.7 Exons 5-12 Negative Negative

PTEN 12.1 12.5 Exons 1-9 Negative Negative

CRLF2 27.7 17.2 Exons 1-6 Negative 33C>G / (new)

SH2B3 21.2 16.0 Exons 1-7 Negative Negative

DNM2 14.7 15.2 Exon 6 789G>A/rs199976453

Exon 20 2139T>C/rs2229920 2139T>C / rs2229920

TP53 6.9 11.1 Exons 4-9 Negative Negative

JAK1 7.0 14.8 Exons 13,14,16-19 Negative Negative

Immunophenotype (%) Patient 1 Patient2

CD34 64.3% 91%

CD13 28% 98%

CD33 97% 90%

CD3 32.5% -

CD5 99.4% 38%

CD7 99.5% 63%

Hepatomegaly Negative Negative

Splenomegaly Negative Positive

Lymphadenopathy Negative Positive

IKZF1 deletion Negative Negative

BCR/ABL1 Negative Negative

Complex karyotype Negative Negative

Treatment

1xHyperCVAD+2xIDA+FLAG+1xFLAG

+1xBFM2002-HR-1+1xMOAP+4xCA

G+Methylprednisolone+1xICE+3xNel

arabine+1xDecitabine+0.5xCAG

1 st CR time 42 days to achieve CR Unknown

Relapse time after CR1 21 months Unknown

Total relapse time 5 Lost to follow-up

Outcome Death Lost to follow-up

WBC: White blood cell, BM: bone marrow, PB: peripheral blood, CR: complete remission.

1xHyper-CVAD+1xMA; no nelarabine

treatment

367


EZH2 exon 11, were detected in her BM sample (Figure 1). No

CR was achieved with the first induction therapy. Finally, the

patient was administered methotrexate and cytarabine and

endured a long period of BM suppression. Unfortunately, the

patient was lost to follow-up. Our data indicated the oncogenic

and poor prognostic effect of EZH2 mutations on T-ALL. The

coexistence of EZH2 mutations with mutations in the NOTCH1,

PHF6, and IL7R genes suggested a new mechanism underlying

the tumorigenesis of EZH2 mutations in T-ALL. T-ALL and

particularly ETP-ALL still have largely negative outcomes. In the

past years, the effect of the use of nelarabine for relapsed and

refractory T-ALL seemed to be negligible [10]. In our cohort, the

first patient’s relapse, even after nelarabine treatment, revealed

the insensitivity of patients with multiple mutations to such

treatment. Moreover, our case report suggested that the gene

mutations may be the cause of the failure of the drug treatment

and emphasized the importance of developing more effective

therapies as well as more active and tailored treatments for

aggressive T-ALL.

Acknowledgment

This work was supported in part by the National Natural Science

Foundation of China (81270613,30973376); Jiangsu Province

Key Medical Talents (RC2011077); the Scientific Research

Foundation for the Returned Overseas Chinese Scholars;

State Education Ministry (39 th ); China Postdoctoral Science

Foundation (20090461134); special grade of financial support

from the China Postdoctoral Science Foundation (201003598);

the Six Great Talent Peak Plan of Jiangsu (2010-WS-024); the

Nanjing Municipal Bureau of Personnel (2009); the Fundamental

Research Funds for the Central Universities (2242017K40271,

2242016K40143) (ZG); and the Milstein Medical Asian American

Partnership Foundation Research Project Award in Hematology

(2017) (ZG and CS).

Keywords: EZH2, Adult, T-cell, Acute lymphoblastic leukemia

Anahtar Sözcükler: EZH2, Erişkin, T-hücre, Akut lenfoblastik

lösemi




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Address for Correspondence/Yazışma Adresi: Zheng GE, M.D.,

Zhongda Hospital, Medical School of Southeast University Department of Hematology Nanjing, China

Phone : 86-25-83262468

E-mail : janege879@hotmail.com ORCID-ID: orcid.org/0000-0001-8028-1612



DOI: 10.4274/tjh.2017.0194

368



Circulating Tumor Cells in Neuroblastoma

Nöroblastomada Dolaşan Tümör Hücreleri

Mili Jain 1 , Ashutosh Kumar 1 , Sanjay Mishra 1 , Nishant Verma 2 , Madhu Mati Goel 1

1

King George’s Medical University, Department of Pathology, Uttar Pradesh, India

2

King George’s Medical University, Department of Pediatrics, Uttar Pradesh, India

To the Editor,

A 2-year-old girl presented with fever, hepatomegaly, and

progressively increasing proptosis of the right eye for 1 month.

Abdominal ultrasound revealed a well-defined multi-lobulated

predominantly hyperechoic mass lesion of 10.9x2.5x6.1 cm

with a few foci of coarse calcification and small cystic areas

arising from the right suprarenal region. The lesion was

inferiorly compressing the renal parenchyma; however, the

interface was well maintained. Medially it was crossing the

midline and encasing the aorta and its branches. The features

were of neuroblastoma. The diagnosis was confirmed by Tru-

Cut biopsy from the suprarenal mass showing small round blue

cells with salt and pepper chromatin. Immunohistochemistry

was positive for synaptophysin. Non-contrast computerized

tomography scanning of the head and orbit revealed a right

retro-orbital mass with specks of calcification. The peripheral

blood smears showed a few clusters and rosettes of circulating

neuroblastoma cells. The bone marrow aspirate smears showed

extensive infiltration by neuroblastoma cells dispersed singly, in

clusters as well as in rosettes with central neuropils (Figure 1).

The patient was categorized as stage IV as per the International

Neuroblastoma Staging System.

Neuroblastoma is the most common extracranial solid tumor

in children. The mean age of presentation is 18 months and

the majority of patients (90%) are diagnosed by 5 years of

age [1]. The adrenal gland is the most common primary site.

Approximately 75% of children have metastases to regional

lymph nodes, bone marrow, cortical bones, the orbit, the

liver, and skin at the time of diagnosis. The histology shows

primitive neuroblasts with variable degrees of differentiation

to Schwann cells and ganglion cells. Homer-Wright rosettes,

i.e. neuroblasts surrounding a tangle of neuropils, are seen.

Infants may present with blue-red cutaneous masses, referred

to as blueberry muffin baby. The differential diagnosis includes

alveolar rhabdomyosarcoma (ARMS), Ewing’s sarcoma/primitive

neuroectodermal tumor (PNET), and lymphoma. ARMS cells are

Figure 1. Leishman staining. A, B) Peripheral blood smear (630 x )

showing neuroblastoma tumor cell cluster with nuclear molding

and rosette-like arrangement. C) Bone marrow smear (400 x )

showing Homer-Wright rosette with neuroblasts surrounding

central neuropil.

more pleomorphic with abundant cytoplasm and are positive

for myogenic markers (desmin, myogenin). Ewing’s sarcoma/

PNET patients are usually older; cells show finely stippled

chromatin- and glycogen-filled cytoplasm, with expression of

CD99. Neuroblastoma may clinically mimic acute leukemia [2].

These cells can be differentiated from blasts by expression of

synaptophysin or neuron-specific enolase. The blasts are positive

for leukocyte common antigen (LCA/CD45). Catecholamine

metabolites homovanillic acid and vanillylmandelic acid

are elevated in serum and urine in approximately 95% of

patients. Metastatic disease can be evaluated using iodine-

123-metaiodobenzylguanidine (123-I-MIBG) scanning. N-MYC

amplification is associated with poor prognosis. Morphologically

identifiable circulating tumor cells (CTCs) are a rare finding [3].

Positive CTCs are associated with poor prognosis [4].

Keywords: Neuroblastoma, Circulating tumor cells, Metastasis

Anahtar Sözcükler: Nöroblastoma, Dolaşan tümör hücreleri,

Metastaz

369





included.

References

1. Brodeur G, Hogarty M, Bagatell R, Mosse YP, Maris JM Neuroblastoma.

In: Pizzo P, Poplack D, (eds). Principles and Practice of Pediatric Oncology.

Philadelphia, Lippincott Williams & Wilkins, 2016.

2. Gökçe M, Aytaç S, Ünal Ş, Altan İ, Gümrük F, Çetin M. Acute megakaryoblastic

leukemia with t(1;22) mimicking neuroblastoma in an infant. Turk J Hematol

2015;32:64-67.

3. Moodley V, Pool R. Circulating neuroblastoma cells in peripheral blood. Br J

Haematol 2003;123:2.

4. Kuroda T, Morikawa N, Matsuoka K, Fujino A, Honna T, Nakagawa A, Kumagai

M, Masaki H, Saeki M. Prognostic significance of circulating tumor cells and

bone marrow micrometastasis in advanced neuroblastoma. J Pediatr Surg

2008;43:2182-2185.

Address for Correspondence/Yazışma Adresi: Mili JAIN, M.D.,

King George’s Medical University, Department of Pathology, Uttar Pradesh, India

Phone : +91 979 354 60 90

E-mail : milijain786@gmail.com ORCID-ID: orcid.org/0000-0002-2598-1939



DOI: 10.4274/tjh.2017.0199

Megakaryocytic Emperipolesis Associated with

Thrombocytopenia: Causative or Coincidence?

Trombositopeni ile Birlikte Megakaryositik Emperipolez: Nedensel veya Tesadüf?

Manu Goyal 1 , Sreeja Thandilath Thekkelakayil 2 , Anurag Gupta 2,3

1

AmPath Hyderabad Hospital, Clinics of Hematopathology and Molecular Hematopathology, Telangana, India

2

AmPath Hyderabad Hospital, Clinic of Hematopathology, Telangana, India

3

AmPath Hyderabad Hospital, Clinic of Cytogenetics, Telangana, India

To the Editor,

Phagocytosis, emperipolesis, and entosis are physiological and

pathological phenomena characterized by the engulfment of

one cell into another cell [1]. Emperipolesis is defined as active

penetration of one cell by another, which remains intact [2].

Emperipolesis differs from phagocytosis in that an engulfed cell

exists temporarily within another cell and with an intact normal

structure, while in phagocytosis, the engulfed cell is destroyed

by the proteolytic action of lysosomal enzymes [1,2]. Entosis

is a non-apoptotic cell death mechanism that occurs in cell

populations deprived of matrix attachment [3,4].

A 31-year-old male presented with severe headache to

the emergency department. He was afebrile without any

organomegaly or neurological deficit. An urgent computed

tomography scan of the brain showed subarachnoid

hemorrhage. Complete blood counts revealed hemoglobin of

80 g/L, leukocyte count of 4.9x10 9 /L, platelet count of 5x10 9 /L,

and a few giant platelets on peripheral smear. Prothrombin

time, activated partial thromboplastin time, and fibrinogen

were within the normal ranges. Bone marrow evaluation

performed to assess the cause of severe thrombocytopenia

showed normal erythropoiesis and myelopoiesis with increased

megakaryocytes. These megakaryocytes showed neutrophils

Figure 1. Photomicrograph of the trephine biopsy shows

megakaryocytic emperipolesis containing neutrophils

(hematoxylin and eosin stain, original magnification 630 x ).

with marked emperipolesis (Figure 1). There was no evidence

of malignancy or infiltrate. A working diagnosis of immunemediated

thrombocytopenia was issued and the patient was

treated with steroids and intravenous immunoglobulins. In

view of the marked thrombocytopenia and hemorrhagic

complications, the patient was transfused with multiple units of

single-donor platelets. Despite aggressive medical management,

370



his platelet counts did not improve. He was discharged against

medical advice and lost to follow-up.

Emperipolesis is a hallmark of Rosai-Dorfman disease (RDD);

however, it can also be seen in both malignant hematolymphoid

disorders (like Hodgkin lymphoma, non-Hodgkin lymphoma,

acute myeloid leukemias, myeloproliferative disorders

or myelodysplastic syndrome) and non-hematological

malignancies (neuroblastoma, rhabdomyosarcoma) [1,5].

Emperipolesis can be either megakaryocytic or histiocytic. The

former engulfs erythroblasts, myeloid cells, or neutrophils and

is seen in hematolymphoid disorders, while the latter engulfs

inflammatory cells (lymphocytes and plasma cells) as seen in

RDD [1].

The exact mechanism for megakaryocytic emperipolesis is

unknown. Centurione et al. [6] in their mice model suggested

that abnormality in GATA1 transcription factor (either

due to mutation or deletion) results in thrombocytopenia,

megakaryocytic emperipolesis, and resultant myelofibrosis.

Increased expression of P-selectin is known to mediate neutrophil

sequestration on the outer surface of megakaryocytes, promoting

increased neutrophil-megakaryocyte interactions [6,7]. A few

studies indicated that the release of alpha-granular proteins,

growth factors, and cytokines produced by megakaryocytes as

well as neutrophil protease in the microenvironment induce

emperipolesis [5,8]. The fate could be the cannibalism of the

invading cell, host cell death, transcytosis, or division of both

the invading and recipient cells [4,7]. Further research at the

molecular level is needed to elucidate the underlying specific

mechanisms.

With regards to platelet counts, there have been few case reports

of megakaryocytic emperipolesis associated with thrombocytosis,

rarely in thrombocytopenia associated with myelodysplasia and

none associated with immune-mediated thrombocytopenia [9].

In the present case, whether megakaryocytic emperipolesis was

responsible for the thrombocytopenia or simply a coincidence is

difficult to establish. We present this rare phenomenon so that

similar observations cumulatively would help in resolving this

complex issue.

Keywords: Thrombocytopenia, Megakaryocytic emperipolesis,

GATA1

Anahtar Sözcükler: Trombositopeni, Megakaryositik

emperipolez, GATA1




included.

References

1. Rastogi V, Sharma R, Misra SR, Yadav L, Sharma V. Emperipolesis - a review.

J Clin Diagn Res 2014;8:ZM01-2.

2. Humble JG, Jayne WH, Pulvertaft RJ. Biological interaction between

lymphocytes and other cells. Br J Haematol 1956;2:283-294.

3. Overholtzer M, Mailleux AA, Mouneimne G, Normand G, Schnitt SJ, King

RW, Cibas ES, Brugge JS. A nonapoptotic cell death process, entosis, that

occurs by cell-in-cell invasion. Cell 2007;131:966-979.

4. Xia P, Wang S, Guo Z, Yao X. Emperipolesis, entosis and beyond: dance with

fate. Cell Res 2008;18:705-707.

5. Sable MN, Sehgal K, Gadage VS, Subramanian PG, Gujral S. Megakaryocytic

emperipolesis: A histological finding in myelodysplastic syndrome. Indian J

Pathol Microbiol 2009;52:599-600.

6. Centurione L, Di Baldassarre A, Zingariello M, Bosco D, Gatta V, Rana RA,

Langella V, Di Virgilio A, Vannucchi AM, Migliaccio AR. Increased and

pathologic emperipolesis of neutrophils within megakaryocytes associated

with marrow fibrosis in GATA-1 low mice. Blood 2004;104:3573-3580.

7. Gupta N, Jadhav K, Shah V. Emperipolesis, entosis and cell cannibalism:

demystifying the cloud. J Oral Maxillofac Pathol 2017;21:92-98.

8. Schmitt A, Jouault H, Guichard J, Wendling F, Drouin A, Cramer EM.

Pathological interaction between megakaryocytes and polymorphonuclear

leukocyte in myelofibrosis. Blood 2000;96:1342-1347.

9. Bobik R, Podolak-Dawidziak M, Kiełbiński M, Jeleń M, Wróbel T.

Emperipolesis in megakaryocytes in patients with thrombocytosis in the

course of myeloproliferative disorders. Acta Haematol Pol 1995;26:179-

183.

Address for Correspondence/Yazışma Adresi: Manu GOYAL, M.D.,

AmPath Hyderabad Hospital, Clinics of Hematopathology and Molecular Hematopathology, Telangana, India

Phone : +09 866 969 997

E-mail : dr_manu31@yahoo.com ORCID-ID: orcid.org/0000-0003-1970-4270



DOI: 10.4274/tjh.2017.0211

371


First Observation of Hemoglobin San Diego, a High Oxygen Affinity

Hemoglobin Variant, in Turkey

Türkiye’de Gözlenen İlk Hemoglobin San Diego (Oksijene İlgisi Yüksek Bir Hemoglobin

Varyantı) Olgusu

Ebru Yılmaz Keskin 1 , Ali Fettah 1 , Ana Catarina Oliveira 2 , Şule Toprak 1 , Andreia Lopes 2 , Celeste Bento 2,3

1

Süleyman Demirel University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Isparta, Turkey

2

Coimbra University, Centro Hospital, Clinic of Hematology, Coimbra, Portugal

3

CIAS, Coimbra University, Department of Life Sciences, Coimbra, Portugal

To the Editor,

Congenital erythrocytosis (CE) or congenital polycythemia

represents a rare clinical entity. High oxygen affinity hemoglobin

(Hb) variants are a very rare cause of secondary CE. In 1966,

Charache et al. [1] published the first case of a Hb variant

associated with erythrocytosis, Hb Chesapeake. Since then,

more than 220 variants with high oxygen affinity have been

discovered and the autosomal dominant inheritance has been

confirmed [2].

Many Hb variants have been reported so far from Turkey [3,4,5].

We report herein the first observation of Hb San Diego, a high

oxygen affinity Hb variant, from Turkey in a case of CE.

Case: A 15-year-old female patient residing in Kastamonu,

Turkey, and examined due to the complaints of occasional

headache, fatigue, dizziness, nausea, and chest pain was found

to have an elevated Hb level. Erythrocytosis was also present

in other family members, including her father and paternal

grandmother (Figure 1). Both the father and grandmother had a

history of several phlebotomies.

Laboratory data are presented in Table 1. Serum biochemistry,

abdominal ultrasonography, and echocardiographic

examinations were all unremarkable. In addition to her family

history consistent with a disorder transmitted autosomal

dominantly, the finding of reduced P50 suggested the presence

of a high oxygen affinity Hb. Hb electrophoresis performed with

the high-performance liquid chromatography (HPLC) method

with the device ZIVAK using the Hb Variant Whole Blood HPLC

Analysis Kit yielded no abnormal Hb variant. The examination

was repeated with Trinity Biotech’s Premier Hb9210 resolution

method and displayed the presence of a Hb variant in both the

patient and her father (Figure 1). Sanger sequencing analysis

confirmed the associated mutation in the β-globin gene [Hb San

Diego; β109(G11)Val→Met] (Figure 2).

Figure 1. A) Pedigree of the family with erythrocytosis and

hemoglobin (Hb) San Diego, illustrating dominant mode of

inheritance of erythrocytosis. The propositus is indicated with

an arrow; B) high-performance liquid chromatography (premier

Hb9210 resolution) showing the presence of Hb San Diego.

Figure 2. Identification of hemoglobin San Diego in β-globin

gene by Sanger sequencing analysis in the index case.

HbVar: Hemoglobin variant.

372



Table 1. Laboratory findings of the patient at the time

of admission.

Hemoglobin (g/L) 169 (NR: 120-160)

Hematocrit (%) 50.6 (NR: 36-46)

Red blood cells (10 6 /µL) 5.72 (NR: 4.1-5.1)

Reticulocyte ratio (%) 1.2 (NR: 0.3-1.5)

MCV (fL) 88.5 (NR: 70.6-95.6)

White blood cells (10 3 /µL) 4.4 (NR: 4.5-13.0)

Platelets (10 3 /µL) 258 (NR: 150-400)

Ferritin (ng/mL) 12.4 (NR: 12-150)

Vitamin B12 (pg/mL) 421 (NR: 200-820)

Folic acid (ng/mL) 6.3 (NR: 3.0-7.2)

Homocysteine (µmol/L) 5.8 (NR: 5.0-13.0)

Erythropoietin (mIU/mL) 12.4 (NR: 4.3-20.0)

P50* (mmHg) 15.5 (NR: 22.6-29.4)

*P50 is the oxygen tension at which the hemoglobin molecule is one-half saturated.

NR: Normal range, MCV: mean corpuscular volume.

Erythrocytosis may be the clinical manifestation of the presence

of a high oxygen affinity Hb. Hb San Diego was first reported in

1974 in a Filipino family [6]. Thereafter, it has been described in

subjects of different origins [7,8,9,10,11,12]. Our case represents

the first one of Hb San Diego in Turkey. Although Hb San Diego

was described as electrophoretically silent [6], it could be

clearly identified using the new Trinity Biotech Premier Hb9210

resolution technology.

In their study evaluating 70 patients with CE, Bento et al.

[11] sequenced all the genes described as associated with CE

and erythrocytosis molecular etiology was identified in only

25 (36%) subjects, a high oxygen affinity Hb being the cause

in 14 (56%) of these 25 subjects. Determination of the P50

value, calculated easily from fresh venous blood gas samples,

is a practical and useful test, and a decreased value may direct

clinicians to order examinations regarding a Hb variant [12].

Some high oxygen affinity Hbs are electrophoretically silent but

their identification can be rapidly done by direct sequencing of

the globin genes (HBB and HBA).

Management of CE caused by a high oxygen affinity Hb should

be personalized, and it should primarily depend on smoking

cessation and physical activity. Phlebotomy and platelet

aggregation inhibitors’ prescription should be evaluated

carefully, and blood donation is not advised [2].

Keywords: Abnormal hemoglobins, Hemoglobin San Diego,

Hemoglobin variant

Anahtar Sözcükler: Anormal hemoglobinler, Hemoglobin San

Diego, Hemoglobin varyantı




included.

References

1. Charache S, Weatherall DJ, Clegg JB. Polycythemia associated with a

hemoglobinopathy. J Clin Invest 1966;45:813-822.

2. Mangin O. High oxygen affinity hemoglobins. Rev Med Interne 2017;38:106-

112.

3. Altay Ç. Abnormal hemoglobins in Turkey. Turk J Haematol 2002;19:63-74.

4. Akar E, Akar N. A review of abnormal hemoglobins in Turkey. Turk J

Haematol 2007;24:143-145.

5. Akar N. An updated review of abnormal hemoglobins in the Turkish

population. Turk J Haematol 2014;31:97-98.

6. Nute PE, Stamatoyannopoulos G, Hermodson MA, Roth D. Hemoglobinopathic

erythrocytosis due to a new electrophoretically silent variant, Hemoglobin

San Diego (β109 (G11) Val→ Met). J Clin Invest 1974;53:320-328.

7. González Fernández FA, Villegas A, Ropero P, Carreño MD, Anguita E, Polo

M, Pascual A, Henández A. Haemoglobinopathies with high oxygen affinity.

Experience of Erythropathology Cooperative Spanish Group. Ann Hematol

2009;88:235-238.

8. Loukopoulos D, Poyart C, Delanoe-Garin J, Matsis C, Arous N, Kister J,

Loutradi-Anagnostou A, Blouquit Y, Fessas P, Thillet J, Rosa F, Galacteros F.

Hemoglobin San Diego/beta zero thalassemia in a Greek adult. Hemoglobin

1986;10:143-159.

9. Coleman MB, Adams LG 3rd, Walker AM, Plonczynski MW, Harrell AH, Kark

JA, Schechter GP. Hb San Diego [beta 109 (G11) Val-->Met] in an Iranian:

further evidence for a mutational hot spot at position 109 of the betaglobin

gene. Hemoglobin 1993;17:543-545.

10. Miniero R, Pullano MN, Oliverio AC, Benevento C, Madonna G, Altomare

F, Giancotti L. Two Calabrian children with Hemoglobin San Diego. Child

2012;1:11.

11. Bento C, Percy MJ, Gardie B, Maia TM, van Wijk R, Perrotta S, Della Ragione

F, Almeida H, Rossi C, Girodon F, Aström M, Neumann D, Schnittger S,

Landin B, Minkov M, Randi ML, Richard S, Casadevall N, Vainchenker W,

Rives S, Hermouet S, Ribeiro ML, McMullin MF, Cario H; ECE-Consortium,

Chauveau A, Gimenez-Roqueplo AP, Bressac-de-Paillerets B, Altindirek D,

Lorenzo F, Lambert F, Dan H, Gad-Lapiteau S, Catarina Oliveira A, Rossi C,

Fraga C, Taradin G, Martin-Nuñez G, Vitória H, Diaz Aguado H, Palmblad J,

Vidán J, Relvas L, Ribeiro ML, Luigi Larocca M, Luigia Randi M, Pedro Silveira

M, Percy M, Gross M, Marques da Costa R, Beshara S, Ben-Ami T, Ugo V; ECE-

Consortium. Genetic basis of congenital erythrocytosis: mutation update

and online databases. Hum Mutat 2014;35:15-26.

12. Bento C, Almeida H, Maia TM, Relvas L, Oliveira AC, Rossi C, Girodon F,

Fernandez-Lago C, Aguado-Diaz A, Fraga C, Costa RM, Araujo AL, Silva J,

Vitoria H, Miguel N, Silveira MP, Martin-Nuñez G, Ribeiro ML. Molecular

study of congenital erythrocytosis in 70 unrelated patients revealed a

potential causal mutation in less than half of the cases (Where is/are the

missing gene(s)?). Eur J Haematol 2013;91:361-368.

Address for Correspondence/Yazışma Adresi: Ebru YILMAZ KESKİN, M.D.,

Süleyman Demirel University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Isparta, Turkey

Phone : +90 505 558 36 11

E-mail : ebruyilmaz81@hotmail.com ORCID-ID: orcid.org/0000-0002-1462-9876



DOI: 10.4274/tjh.2017.0213

373


A Case of Para-Bombay Phenotype Caused by Homozygous

Mutation of the FUT1 Gene

FUT1 Genindeki Homozigot Mutasyondan Kaynaklanan Bir Para-Bombay Fenotipi Olgusu

Jung-Kuang Yu 1 , Yi-Hong Liu 2 , Tze-Kiong Er 2,3

1

Asia University Hospital, Department of Orthopedics, Taichung, Taiwan

2

Asia University Hospital, Division of Laboratory Medicine, Taichung, Taiwan

3

Asia University Faculty of Medicine, Department of Health and Nutrition Biotechnology, Taichung, Taiwan

To the Editor,

A 79-year-old female patient presented at the hospital with

osteoarthritis. Examination of the patient revealed hemoglobin

level of 10.8 g/dL, RBC count of 3.45x10 6 /µL, WBC count of

10.1x103/µL, and platelet count of 122x10 3 /µL. Plasma levels of

blood urea nitrogen, creatinine, sodium, potassium, and alanine

aminotransferase were all within the normal ranges, while

aspartate aminotransferase was slightly higher than normal. A

blood sample obtained from the patient was submitted to our

division for blood typing and cross-matching, with a request

to receive 2 units of packed red blood cells. ABO typing was

performed using standard serological techniques after an

immediate spin. Testing the patient’s red blood cells revealed no

detectable ABO antigens upon forward/cell grouping (group O

blood type). On the other hand, reverse/serum grouping showed

the presence of A antibodies in the serum (group B blood type).

To resolve the discrepancy between cell and serum grouping we

performed an agglutination examination of anti-H serum; the

red blood cells from the sample did not exhibit an agglutination

reaction. Additionally, secretor status was determined in order

to assess the presence of soluble blood group substances.

Our results showed the presence of B and H antigens in the

saliva. Based on these results, the patient in the present case

was diagnosed as having a para-Bombay B phenotype (Table 1,

Figure 1).

Genotyping of the ABO and FUT1 genes was also performed.

Direct DNA sequencing of the patient’s ABO gene indicated

the B/O 1 genotype. To examine potential mutations in the

FUT1 gene, we amplified and sequenced the full coding region

of the gene. FUT1 gene sequence analysis revealed that the

patient harbored the homozygous mutation c.881_882delTT

(p.Phe294Cysfs*40). A heterozygous mutation in FUT1

(880delTT) has been previously reported as the cause of the

para-Bombay phenotype [1,2]. However, the homozygous

mutation c.881_882delTT (p.Phe294Cysfs*40) only rarely causes

the para-Bombay phenotype. Previously, a study indicated

that homozygous mutations are a cause of the para-Bombay

phenotype [3,4].

In patients with the para-Bombay blood group, ABH antigens

are present in saliva but not expressed in red blood cells. The

para-Bombay phenotype results either from an inactive FUT1

gene present together with a normal FUT2 gene or from a

mutated FUT1 gene present with or without an active FUT2 gene

[1]. H deficiency is slightly more common in Taiwan, affecting 1

of 8000 people [2]. More than 56 silencing or weakening FUT1

mutations have been reported in the dbRBC database (https://

www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.cgi?cmd=bgmut/

systems_info&system=hh).

Figure 1. Sequencing results confirm the wild-type (A) and the

presence of the FUT1 homozygous mutation c. 881_882delTT (p.

Phe294Cysfs*40) (B).

Table 1. Serologic and saliva test results of the patient: ABO group discrepancy.

Cell grouping Serum grouping Test for H antigen Saliva secretor status

Anti-A Anti-B Anti-D A 1

cells B cells O cells Anti-H A 1

cells B cells

0 0 4+ 4+ 0 0 0 2+ 0

0= No agglutination, 1+= multiple small agglutinates with hazy supernatant, 2+= multiple large agglutinates with clear supernatant, 3+= 2-3 large agglutinates with clear supernatant,

4+= single large agglutinates.

374



In conclusion, identification of this phenotype is very

important because this particular patient subgroup may be

clinically mislabeled as group O. If patients with anti-H in their

circulation receive transfusions of blood with the H antigen, it

may cause a transfusion reaction such as an acute hemolytic

reaction. Here we have reported a rare case of the para-Bombay

phenotype caused by the homozygous mutation c.881_882delTT

(p.Phe294Cysfs*40).

Acknowledgments

Our gratitude goes to Michael Burton, Asia University. This study

was supported by grants from Asia University and China Medical

University Hospital (ASIA-105-CMUH-07).

Keywords: Para-Bombay, Phenotype, FUT1 gene, Blood

transfusion

Anahtar Sözcükler: Para-Bombay, Fenotip, FUT1 geni, Kan

transfüzyonu




References

1. Cai XH, Jin X, Fan LF, Lu Q, Xiang D. Molecular genetic analysis for the para-

Bombay blood group revealing two novel alleles in the FUT1 gene. Blood

Transfus 2011;9:466-468.

2. Chen DP, Tseng CP, Wang WT, Peng CT, Tsao KC, Wu TL, Lin KT, Sun CF. Two

prevalent h allele in para-Bombay haplotypes among 250,000 Taiwanese.

Ann Clin Lab Sci 2004;34:314-318.

3. Zhang A, Chi Q, Ren B. Genomic analysis of para-Bombay individuals in

south-eastern China: the possibility of linkage and disequilibrium between

FUT1 and FUT2. Blood Tranfus 2015;13:472-477.

4. Lin-Chu M, Broadberry RE. Blood transfusion in the para-Bombay

phenotype. Br J Haematol 1990;75:568-572.

Address for Correspondence/Yazışma Adresi: Tze-Kiong ER, M.D.,

Asia University Hospital, Division of Laboratory Medicine, Taichung, Taiwan

E-mail : tzekiong92@gmail.com ORCID-ID: orcid.org/0000-0002-7068-1652



DOI: 10.4274/tjh.2017.0220

375


A Myopathy, Lactic Acidosis, Sideroblastic Anemia (MLASA) Case

Due to a Novel PUS1 Mutation

PUS1 Geninde Yeni Mutasyon Saptanan Miyopati, Laktik Asidoz, Sideroblastik Anemi (MLASA) Olgusu

Çiğdem Seher Kasapkara 1 , Leyla Tümer 1 , Nadia Zanetti 2 , Fatih Ezgü 1 , Eleonora Lamantea 2 , Massimo Zeviani 2,3

1

Gazi University Faculty of Medicine, Division of Metabolism, Ankara, Turkey

2

Fondazione IRCCS Carlo Besta, Molecular Neurogenetics Unit, Milan, Italy

3

Medical Research Council, Mitochondrial Biology Unit, Cambridge, United Kingdom

To the Editor,

The patient, the first child of Turkish first-cousins, was born at

term after an uncomplicated pregnancy. Birth parameters were

normal. The family history was negative for hematological or

neurological diseases. The newborn period was characterized by

hypoglycemia, lactic acidemia (6.1 mmol/L; normal values: up

to 1.9 mmol/L), and lactic, pyruvic, and dicarboxylic aciduria. At

10 months of age, hematological examination revealed marked

sideroblastic anemia. He started to receive transfusions every

3-4 weeks until 14 months of age, when the blood parameters

spontaneously normalized. He had exercise intolerance and

delayed motor milestones (walking at 3.5 years of age). At 14

years of age, pallor, progressive muscle weakness, and lethargy

occurred and sideroblastic anemia reappeared. The boy had

mild mental insufficiency, profound generalized hypotrophy

and weakness, and hyperlordosis of the trunk. He became

transfusion-dependent, requiring packed cell transfusions

every 2-3 weeks. The muscle biopsy showed subsarcolemmal

abnormal mitochondrial aggregates and diffuse negative

staining for cytochrome c oxidase. Due to the paucity of tissue,

the biochemical evaluation of respiratory chain complexes

was not performed. The clinical features oriented us towards a

mitochondrial pathology; CoQ10 was given (400 mg/day) and

dramatic improvement of muscle strength was observed with

reduction of the frequency of blood transfusions. Unfortunately,

the boy died when he was 18 years old due to severe respiratory

failure.

Myopathy, lactic acidosis, and sideroblastic anemia (MLASA)

is a rare mitochondrial disease [1]: MLASA1 (MIM #600462)

results from mutations in the PUS1 gene, encoding for

pseudouridylate synthase 1, and the enzyme is located in

both the nucleus and mitochondria, which is involved in posttranscriptional

modification of cytoplasmic and mitochondrial

tRNAs [2]; MLASA2 (MIM #613561) is caused by mutations in

the YARS2 gene that encodes for the mitochondrial tyrosyltRNA

synthetase [3]; and MLASA3 (MIM #500011) is caused

by heteroplasmic mutation m.8969G>A in the mitochondrial-

DNA-encoded ATP6 gene (MTATP6) [4].

Figure 1. Sequence analysis of the exon 2-intron 2 junction of the

PUS1 gene in a control (A), in the patient (B), and in the patient’s

mother (C). The yellow highlighted nucleotides belong to the

consensus sequence of the splice donor site. In the red circle the

mutated nucleotide is in homozygous (B) or heterozygous (C) form.

Our patient had typical features of MLASA, so we analyzed the

nuclear-encoded genes YARS2 and PUS1. YARS2 was normal,

but we identified the novel homozygous mutation c.302A>G in

exon 2 of the PUS1 gene, causing the substitution p.Gln101Arg

in the protein (Figure 1). This variant is reported as a singleton

in the ExAC browser, accounting for 0.001% of allele frequency,

and absent in dbSNP and EVS databases. The p.Gln101Arg

change was scored very high for the likelihood to be deleterious

by different bioinformatics tools for predicting pathogenic

variants, and furthermore the c.302A>G transition is predicted

to modify the consensus sequence of the splice donor site of

exon 2, probably affecting splicing (Table 1). The unavailability

of the patient’s cells did not allow us to confirm this hypothesis.

376



Table 1. Results of the in silico analysis obtained by different bioinformatics tools for the prediction of the impact of mutation

on mRNA and protein.

c.302A>G in splice donor site of exon 2 in the PUS1 gene

Tool Prediction Site

BDGP Donor site lost http://www.fruitfly.org/seq_tools/splice.html

Human SpliceFinder 3.0

Alteration of the WT donor site, most probably affecting

splicing

http://www.umd.be/HSF/

MutationTaster

Alteration within used splice site, likely to disturb normal

splicing; donor lost

http://www.mutationtaster.org/

MutPredSplice Splice affecting variant (0.78); loss of natural 5’ SS http://www.mutdb.org/mutpredsplice

p.Gln101Arg in pseudouridylate synthase 1

Tool Prediction Site

MutationTaster Disease (0.999) http://www.mutationtaster.org/

MutPred2 Probably pathogenic (0.897) http://mutpred2.mutdb.org/

Panther Probably damaging (0.95) http://www.pantherdb.org

Pmut (beta) Disease (0.82) http://mmb.pcb.ub.es/PMut/

PolyPhen-2 Probably damaging (0.931) http://genetics.bwh.harvard.edu/pph2/

SIFT Affected protein function http://siftdna.org/

BDGP: Berkeley Drosophila Genome Project, SIFT: Sorting Intolerant From Tolerant.

To date, eleven PUS1-mutated patients from six families have been

described [2,5,6,7,8] and five mutations are reported. Ours is the

first alteration allegedly causing a splicing aberration according

to prediction by in silico analysis. In our patient a high dose of

CoQ10 improved the clinical condition for a while, although it did

not reverse the course of the disease. To date, there is no effective

therapy for MLASA, although many studies are in progress to

address novel treatment options for mitochondrial diseases

[9]. Our report expands the genetic spectrum of the MLASA

syndrome, which must be considered in patients with congenital

sideroblastic anemia associated with myopathy.

Acknowledgments

This work was supported by Fondazione Pierfranco e Luisa Mariani

- CM23 (N.Z., E.L., M.Z.) and Institut de France - Grant NRJ (M.Z.).

Keywords: Myopathy, Lactic acidosis, Sideroblastic anemia

Anahtar Sözcükler: Miyopati, Laktik asidoz, Sideroblastik anemi




included.

References

1. Inbal A, Avissar N, Shaklai M, Kuritzky A, Schejter A, Ben-David E, Shanske

S, Garty BZ. Myopathy, lactic acidosis, and sideroblastic anemia: a new syndrome.

Am J Med Genet 1995;55:372-378.

2. Bykhovskaya Y, Casas K, Mengesha E, Inbal A, Fischel-Ghodsian N. Missense

mutation in pseudouridine synthase 1 (PUS1) causes mitochondrial myopathy

and sideroblastic anemia (MLASA). Am J Hum Genet 2004;74:1303-1308.

3. Riley LG, Cooper S, Hickey P, Rudinger-Thirion J, McKenzie M, Compton A,

Lim SC, Thorburn D, Ryan MT, Giege R, Bahlo M, Christodoulou J. Mutation

of the mitochondrial tyrosyl-tRNA synthetase gene, YARS2, causes myopathy,

lactic acidosis, and sideroblastic anemia-MLASA syndrome. Am J Hum Genet

2010;87:52-59.

4. Burrage LC, Tang S, Wang J, Donti TR, Walkiewicz M, Luchak JM, Chen LC, Schmitt

ES, Niu Z, Erana R, Hunter JV, Graham BH, Wong LJ, Scaglia F. Mitochondrial

myopathy, lactic acidosis, and sideroblastic anemia (MLASA) plus associated with

a novel de novo mutation (m.8969G>A) in the mitochondrial encoded ATP6

gene. Mol Genet Metab 2014;113:207-212.

5. Zeharia A, Fischel-Ghodsian N, Casas K, Bykhovskaya Y, Tamari H, Lev D, Mimouni

M, Lerman-Sagie T. Mitochondrial myopathy, sideroblastic anemia, and lactic

acidosis: an autosomal recessive syndrome in Persian Jews caused by a mutation

in the PUS1 gene. J Child Neurol 2005;20:449-452.

6. Fernandez-Vizarra E, Berardinelli A, Valente L, Tiranti V, Zeviani M. Nonsense

mutation in pseudouridylate synthase 1 (PUS1) in two brothers affected by

myopathy, lactic acidosis and sideroblastic anaemia (MLASA). J Med Genet

2007;44:173-180.

7. Metodiev MD, Assouline Z, Landrieu P, Chretien D, Bader-Meunier B, Guitton

C, Munnich A, Rötig A. Unusual clinical expression and long survival of a

pseudouridylate synthase (PUS1) mutation into adulthood. Eur J Hum Genet

2015;23:880-882.

8. Cao M, Donà M, Valentino ML, Semplicini C, Maresca A, Cassina M, Torraco A,

Galletta E, Manfioli V, Sorarù G, Carelli V, Stramare R, Bertini E, Carozzo R, Salviati

L, Pegoraro E. Clinical and molecular study in a long-surviving patient with

MLASA syndrome due to novel PUS1 mutations. Neurogenetics 2016;17:65-70.

9. Viscomi C, Bottani E, Zeviani M. Emerging concepts in the therapy of

mitochondrial disease. Biochim Biophys Acta 2015;1847:544-557

Address for Correspondence/Yazışma Adresi: Çiğdem Seher KASAPKARA, M.D.,

Gazi University Faculty of Medicine,

Division of Metabolism Diseases, Ankara, Turkey

E-mail : cskasapkara@gmail.com ORCID-ID: orcid.org/0000-0002-3569-276X



DOI: 10.4274/tjh.2017.0231

377


Frequency and Risk Factors for Secondary Malignancies in

Patients with Mycosis Fungoides

Mikozis Fungoidesli Hastalarda Sekonder Malignite Sıklığı ve Risk Faktörleri

Fatma Pelin Cengiz, Nazan Emiroğlu, Nahide Onsun

Bezmialem Vakıf University Faculty of Medicine, Department of Dermatovenereology, İstanbul, Turkey

To the Editor,

Mycosis fungoides (MF), the most common form of cutaneous

T-cell lymphoma (CTCL), has an incidence of 6.4 per million people

[1]. Patients with CTCL have an increased risk of the development

of secondary malignancies, particularly lymphomas [2,3]. We

conducted a 20-year population-based cohort study to assess the

risk factors of secondary cancers in MF patients from our center.

From 1998 to 2015, a total of 143 cases of CTCL were

documented in our database. In this same time period, 13 cases

(9.1%) of secondary malignancy excluding non-melanoma

skin cancer were diagnosed at least 3 months following the

diagnosis of CTCL (Table 1). MF patients were grouped by their

tumor stage from I to IV. Statistical analysis was performed with

SPSS 15. Odds ratios (ORs) and 95% confidence intervals (CIs)

were calculated.

Risk factors significantly associated with secondary cancers in

univariate analyses were entered into a multivariate logistic

regression model. Significance was set at p<0.05.

The vast majority of patients had early-stage disease: 64

(45.35%) stage IA, 30 (20.97%) stage IB, 24 (16.78%) stage IIA,

13 (9.09%) stage IIB, 4 (2.79%) stage IIIA, 3 (2.09%) stage IIIB, 4

(2.79%) stage IVA, and 1 (1.43%) stage IVB.

Table 1. Clinical features of mycosis fungoides patients with secondary malignancies.

Sex

Age at

diagnosis of

MF (years)

Age at diagnosis

of malignancy

(years)

Stage

of MF

Type of

malignancy

Presence of

lymphomatoid

papulosis

Systemic treatment

for MF

Patient 1 Male 8 15 IVA Non-Hodgkin No Interferon,

acitretin

Patient 2 Male 63 65 IB Adult T-cell

leukemia

Patient 3 Male 62 63 IIA Lung cancer No None

No

Interferon,

acitretin

Patient 4 Male 59 59 IIIB Nasopharynx Yes Acitretin

Patient 5 Male 69 68 IIA Lung cancer

+ adult T-cell

leukemia

Patient 6 Female 56 59 IB Renal cell

carcinoma

Patient 7 Male 60 60 IB Lung cancer No None

Patient 8 Female 18 30 IB Hodgkin Yes Interferon,

acitretin

Patient 9 Male 46 50 IIB Bladder cancer No Acitretin

Patient 10 Female 48 51 IIB Superficial

spreading

malignant

melanoma

Patient 11 Female 22 23 IIA Hodgkin No None

Patient 12 Female 36 36 IB Hodgkin Yes None

Patient 13 Female 33 35 IIA Non-Hodgkin

lymphoma

MF: Mycosis fungoides.

Yes

Yes

No

No

None

None

None

Interferon,

UVA1

378



Stage IV disease, the presence of lymphomatoid papulosis, and

duration of disease (more than 10 years) were shown to be the

factors that increased the risk of developing secondary solid

tumors (OR: 21.958, 95% CI: 2.039-839.657; OR: 19.926, 95% CI:

2.387-166.362; OR: 0.635, 95% CI: 0.420-0.959, respectively).

In the vast majority of the patients, secondary malignancies

occurred during the first year of diagnosis of MF (60%).

Our study supports previous findings about an increased risk

of developing a second primary malignancy, especially Hodgkin

lymphoma, chronic leukemia, and lung cancer, in patients with

MF. In previous epidemiological studies, patients with MF had

an elevated risk of secondary neoplasms (mean relative risk:

1.73, range: 1.32-2.4) [2,3]. Some authors have suggested that

anti-lymphoma drugs [4] and particularly alkylating agents may

lead to leukemia [5]. MF and hematological malignancies may

have the same genetic origin, carcinogens, or viruses that affect

lymphocyte precursors, and additionally the production of

cytokines by the first neoplasm may induce the development of

the secondary neoplasm [5]. It was shown that MF is a T helper

cell 2 (Th2) mediated disease and is associated with human

leukocyte antigen 2 alleles. The antigens causing inappropriate

antigens presenting to T lymphocytes are still unknown. Viruses

(Epstein-Barr virus, herpes simplex virus), deficiency of vitamin

D, and medications are possible causative agents. In addition to

these factors, increased levels of transforming growth factor-β,

interleukin-10, and Th2 cytokines and the activation of STAT-3

oncogenes make the host immunosuppressed. We found that

older age, stage of MF, and the presence of lymphomatoid

papulosis increased the risk of coexistence of two other

malignancies besides MF. Therefore, extensive evaluation

for secondary malignancies in the adult population would

be warranted, particularly if the patient has lymphomatoid

papulosis.

Keywords: T-cell neoplasms, Non-Hodgkin lymphoma,

Oncogenes, T-cell mediated immunity

Anahtar Sözcükler: T hücreli neoplazmlar, Hodgkin dışı lenfoma,

Onkogenler, T hücre aracılı immünite

References

1. Bradford PT, Devesa SS, Anderson WF, Toro JR. Cutaneous lymphoma

incidence patterns in the United States: a population-based study of 3884

cases. Blood 2009;113:5064-5073.

2. Vakeva L, Pukkala E, Ranki A. Increased risk of secondary cancers in patients

with primary cutaneous T cell lymphoma. J Invest Dermatol 2000;115:62-

65.

3. Scarisbrick JJ, Child FJ, Evans AV, Fraser-Andrews EA, Spittle M, Russell-

Jones R. Secondary malignant neoplasms in 71 patients with Sezary

syndrome. Arch Dermatol 1999;135:1381-1385.

4. Au WY, Ma SK, Chung LP, Chim CS, Kwong YL. Two cases of therapy-related

acute promyelocytic leukemia (t-APL) after mantle cell lymphoma and

gestational trophoblastic disease. Ann Hematol 2002;81:659-661.

5. Green MH, Young RC, Merrill JM, De Vita VT. Evidence of a treatment dose

response in acute nonlymphocytic leukemias which occur after therapy of

non-Hodgkin’s lymphoma. Cancer Res 1983;43;1891-1898.

Address for Correspondence/Yazışma Adresi: Fatma Pelin CENGİZ, M.D.,

Bezmialem Vakıf University Faculty of Medicine, Department of Dermatovenereology, İstanbul, Turkey

Phone : +90 506 701 54 06

E-mail : fpelinozgen@hotmail.com ORCID-ID: orcid.org/0000-0003-0669-6232



DOI: 10.4274/tjh.2017.0234

379


Leishmaniasis: Bone Marrow Aspirate Smear and Rapid Antibody

Test

Layşmanyazis: Kemik İliği Aspirasyon Yayması ve Hızlı Antikor Testi

Beuy Joob 1 , Viroj Wiwanitkit 2

1

Sanitation 1 Medical Academic Center, Bangkok, Thailand

2

DY Patil University Faculty of Medicine, Pune, India

To the Editor,

The publication by Dorji et al. [1], “Microscopic Image of

Leishman-Donovan Bodies in Bone Marrow Aspirate Smear of

Patient Suffering from Unexplained Intermittent Low-Grade

Fever and Cough”, is very interesting. Indeed, the clinical

presentation of leishmaniasis is usually nonspecific and it is hard

to discriminate it from other tropical infections [2]. However,

the common presentation that might be useful for the inclusion

of leishmaniasis in differential diagnosis is splenomegaly [2].

In the present report, an important observation is a negative

antibody test for Leishmania pathogens. The gold standard for

the diagnosis of leishmaniasis is usually the examination of the

blood or marrow and identification of the parasite. However,

it is considered harmful. A new rapid test might be a solution

and a less invasive technique. Nevertheless, the problem of the

diagnostic properties of the available rapid test is frequently

reported [3]. There are many possible explanations for false

negative results. One important explanation is the prozone

phenomenon [4] due to heavy infection. In the present case,

there might be parasites and excessive antigens that could

induce false negative results due to the immunological testing

and the dilution of the blood sample before the test could be

a simple method to reduce the problem of the prozone effect.

Focusing on the examination of bone marrow a spirates, a

similar problem with diagnostic false negatives can be expected

[5]. At present, the test with the best diagnostic properties is the

polymerase chain reaction-based test [5].

Keywords: Leishmaniasis, Bone marrow, Aspirate, Smear,

Antibody

Anahtar Sözcükler: Layşmanyazis, Kemik iliği, Aspirasyon,

Yayma, Antikor




included.

References

1. Dorji K, Tobgay T, Jamtsho R, Samal PD, Rai P. Microscopic image of

Leishman-Donovan bodies in bone marrow aspirate smear of patient

suffering from unexplained intermittent low-grade fever and cough. Turk J

Hematol 2017;34:266-267.

2. Wiwanitkit V. Bone marrow leishmaniasis: a review of situation in Thailand.

Asian Pac J Trop Med 2011;4:757-759.

3. Khan MG, Alam MS, Bhuiyan AT, Jamil MA, Saha B, Islam M, Haque R,

Hossain M, Jamil KM. Short communication: evaluation of a new rapid

diagnostic test for quality assurance by kala azar elimination programme in

Bangladesh. J Parasitol Res 2011;2011:862475.

4. Itoh Y, Yamaguchi T. Factors that affect analytical results in an enzyme

immunoassay. Nihon Rinsho 1995;53:2143-2148.

5. Piarroux R, Gambarelli F, Dumon H, Fontes M, Dunan S, Mary C, Toga B,

Quilici M. Comparison of PCR with direct examination of bone marrow

aspiration, myeloculture, and serology for diagnosis of visceral leishmaniasis

in immunocompromised patients. J Clin Microbiol 1994;32:746-749.

Address for Correspondence/Yazışma Adresi: Beuy JOOB, M.D.,

Sanitation 1 Medical Academic Center, Bangkok, Thailand

E-mail : beuyjoob@hotmail.com ORCID-ID: orcid.org/0000-0002-5281-0369



DOI: 10.4274/tjh.2017.0291

380



Receiver Operating Characteristic Curve Analysis May be Helpful

to Study the Prognostic Value of miR-155 in B-Cell Non-Hodgkin

Lymphoma

İşlem Karakteristik Eğrisi Analizi B-Hücre Non-Hodgkin Lenfomada miR-155’in Prognostik

Değerini Çalışmada Yardımcı Olabilir

Long Su

The First Hospital of Jilin University, Department of Hematology, Changchun, China

To the Editor,

Aberrant expression of microRNA (miR-155) has been reported

previously in several hematological malignancies [1,2,3,4].

Recently, Bedewy et al. [5] published their excellent findings

in this journal. They reported that miR-155 expression was

significantly upregulated in patients with B-cell non-Hodgkin

lymphoma (NHL) compared with normal controls. In patients

with B-cell NHL, a high level of miR-155 expression was

associated with the presence of B symptoms, involvement of

extranodal sites, and high Eastern Cooperative Oncology Group

score. In patients with diffuse large B-cell lymphoma (DLBCL),

high miR-155 levels were related to non-germinal B-cell-like

type and higher International Prognostic Index scores. High

miR-155 expression was also associated with inferior event-free

survival. Accordingly, the authors concluded that miRNA-155

might be a potential biomarker of prognosis and monitoring in

B-cell NHL, and especially that of the DLBCL type [5].

Receiver operating characteristic (ROC) curve analysis is a

graphical plot that illustrates the diagnostic ability of a binary

classifier system as its discrimination threshold is varied.

The best cutoff value can be calculated by ROC analysis for

continuous variables to predict dichotomous variables with the

best sensitivity and specificity. In Bedewy et al.’s [5] study, the

patients were divided into high-expression and low-expression

groups based on the median level of miR-155 relative expression

units. I wonder if the median value of miR-155 expression level

in this study was the cutoff value with the best sensitivity

and specificity between patients and normal controls by ROC

analysis. Similarly, in patients with B-cell NHL, whether there

is another cutoff value that is associated with clinical stage,

treatment response, long-term outcomes, and so on remains to

be determined.

Keywords: Non-Hodgkin lymphoma, miR-155, Prognostic value

Anahtar Sözcükler: Non-Hodgkin lenfoma, miR-155, Prognostik

değer




included.

References

1. Jones K, Nourse JP, Keane C, Bhatnagar A, Gandhi MK. Plasma microRNA are

disease response biomarkers in classical Hodgkin lymphoma. Clin Cancer Res

2014;20:253-264.

2. Wallace JA, Kagele DA, Eiring AM, Kim CN, Hu R, Runtsch MC, Alexander

M, Huffaker TB, Lee SH, Patel AB, Mosbruger TL, Voth WP, Rao DS, Miles

RR, Round JL, Deininger MW, O’Connell RM. miR-155 promotes FLT3-ITDinduced

myeloproliferative disease through inhibition of the interferon

response. Blood 2017;129:3074-3086.

3. Alizadeh AA, Eisen MB, Davis RE, Ma C, Lossos IS, Rosenwald A, Boldrick JC,

Sabet H, Tran T, Yu X, Powell JI, Yang L, Marti GE, Moore T, Hudson J Jr, Lu

L, Lewis DB, Tibshirani R, Sherlock G, Chan WC, Greiner TC, Weisenburger

DD, Armitage JO, Warnke R, Levy R, Wilson W, Grever MR, Byrd JC, Botstein

D, Brown PO, Staudt LM. Distinct types of diffuse large B-cell lymphoma

identified by gene expression profiling. Nature 2000;403:503-511.

4. Marcucci G, Maharry KS, Metzeler KH, Volinia S, Wu YZ, Mrózek K, Nicolet

D, Kohlschmidt J, Whitman SP, Mendler JH, Schwind S, Becker H, Eisfeld

AK, Carroll AJ, Powell BL, Kolitz JE, Garzon R, Caligiuri MA, Stone RM,

Bloomfield CD. Clinical role of microRNAs in cytogenetically normal acute

myeloid leukemia: miR-155 upregulation independently identifies high-risk

patients. J Clin Oncol 2013;31:2086-2093.

5. Bedewy AML, Elmaghraby SM, Shehata AA, Kandil NS. Prognostic value of

miRNA-155 expression in B-cell non-Hodgkin lymphoma. Turk J Hematol

2017;34:207-212.

Address for Correspondence/Yazışma Adresi: Long SU, M.D.,

The First Hospital of Jilin University, Department of Hematology, Changchun, China

Phone : +86 0431 887 821 57

E-mail : sulongjdyy@163.com ORCID-ID: orcid.org/0000-0002-5360-468X



DOI: 10.4274/tjh.2017.0297

381

AUTHOR INDEX 2017

34 th Volume Index / 34. Cilt Dizini

AUTHOR INDEX - YAZAR DİZİNİ 2017

A. Emre Eşkazan................................. 233, 291

Abdulkadir Ünsal......................................... 116

Abdurrahman Kara........................................ 40

Abraham Pouliakis......................................... 52

Ageliki Tirogala............................................... 52

Ahmad Antar................................................358

Ahmed A. Shehata.......................................207

Ahmed M. L. Bedewy..................................207

Ahmet Emin Kürekçi..................................... 64

Ahmet Koluman............................................. 93

Ahmet Muzaffer Demir............................... 89

Ahmet Pekoğlu............................................... 64

Ahmet Uygun................................................359

Ahu Senem Demiröz......................................81

Aida Berenjian.............................................. 118

Aidin Meshksar.............................................250

Ajlan Tükün...................................................151

Aleksandar Jankovic...................................268

Alessandro Allegra.......................................184

Alexandra Tsoka............................................. 52

Ali Eser............................................................ 118

Ali Fettah.......................................40, 183, 372

Ali Şahin Küçükaslan..................................174

Ali Ünal............................................................. 27

Alişan Yıldıran...............................................345

Ana Bulatovic...............................................268

Ana Catarina Oliveira.................................372

Ana K. Nuñez-Cortes..................................239

Anastasia Livada............................................ 52

Andaç Salman............................................... 118

Andreia Lopes...............................................372

Andrés A. Leon-Peña..................................239

Anna J. Kopinska..........................................186

Anna Koclega................................................186

Anurag Gupta...............................................370

Argyrios Tsantes............................................. 52

Arie C. Van Der Spek...................................124

Arzu Akyay..................................................... 114

Arzu Ekici.......................................................264

Ashutosh Kumar..........................................369

Aslı Akın Belli...................................... 122, 192

Aslı Korur....................................196, 280, 324

Aspasia Argyrou............................................. 52

Asude Kara........................................... 122, 192

Aşkın Şen........................................................ 114

Ayfer Gedük..................................................190

Ayla Eker Sarıboyacı...................................213

Aylin Okçu Heper.........................................194

Aynur Küçükçongar Yavaş........................264

Aysel Pekel....................................................... 64

Ayşe Salihoğlu.................................... 233, 291

Ayşegül Üner................................................... 34

Aytekin Ünlü................................................... 64

Azhar Sattar..................................................278

Bahattin Tunç........................................40, 183

Baohua Huang..............................................126

Baoshan Liu...................................................200

Barış Malbora................................................179

Behnaz Valibeigi..........................................300

Bengi Öztürk................................................... 16

Bengü Nisa Akay..........................................194

Berna Atabay................................................179

Betül Orhaner...............................................264

Betül Tavil........................................................ 40

Beuy Joob..............................................101, 380

Bin Wang........................................................200

Buket Kosova................................................174

Burcu Belen...................................................179

Burcu Yazıcıoğlu..........................................143

Burçin Tezcanlı Kaymaz.............................174

Can Boğa...........................196, 270, 280, 324

Carl Hsieh.......................................................328

Caterina Musolino.......................................184

Celalettin Üstün............................................. 16

Celeste Bento................................................372

Cengiz Bayram................................................ 40

Cengiz Beyan................................................359

Cengiz Ceylan...............................................197

Cengizhan Açıkel........................................... 64

Chen Fangping................................................10

Cheng-Wei Chou.........................................159

Chieh-lin Jerry Teng...................................159

Chingiz Asadov.............................................258

Chunhua Song..............................................366

Çağatay Taşkapan........................................ 114

Çağdaş Aktan................................................174

Çağla Sarıtürk...............................................324

Çetin Demir..................................................... 34

Çiğdem Gereklioğlu.................196, 280, 324

Çiğdem Seher Kasapkara...........................376

Demet Aydın.................................................167

Demet Çekdemir............................................ 27

Deniz Kızmazoğlu.......................................... 46

Deniz Yılmaz Karapınar..............................254

Deniz Yücelten.............................................. 118

Devrim Dündar.............................................340

Dildar Bahar Genç.......................................109

Dilek Gürlek Gökçebay...............................183

Dimitra Agoritsa............................................. 52

Dimitrios Maltezos........................................ 52

Ebru Yılmaz Keskin......................................372

Eda Ataseven................................................... 46

Eda Güngörürler..........................................213

Ekaterini Kontopanou.................................. 52

Ekaterini Manaka........................................... 52

Eldar Abdulalimov.......................................258

Eleonora Lamantea.....................................376

Elias Kyriakou.................................................. 52

Elias Markopoulos......................................... 52

Elif Azarsız.....................................................254

Elif Gülsüm Ümit........................................... 89

Elisavet Grouzi................................................ 52

Elise Schapkaitz.............................................. 59

Emel Özyürek................................................345

Emin Kaya........................................................ 27

Emin Sami Arısoy.........................................340

Emine Begüm Gençer.................................314

Emine Dağıstan............................................120

Emine Zengin................................................340

Emmanouil Lysikatos.................................... 52

Emre Ali Acar................................................181

Erdal Karaöz..................................................213

Erdal Kurtoğlu.................................... 122, 192

Erden Atilla........................................1, 16, 244

Erkan Maytalman........................................280

Esra T. Demirsoy...........................................190

Fadıl Vardar...................................................254

Fahri Şahin..............................................27, 174

Fatih Demircioğlu........................................120

Fatih Ezgü......................................................376

Fatih Kandemir.............................................280

Fatma Demir Yenigürbüz............................ 46

Fatma Demirel................................................ 40

Fatma Dilek Kaymakçı................................ 118

Fehmi Hindilerden......................................... 72

Ferda Perçin...................................................143

Ferit Avcu......................................................... 64

Fevzi Fırat Yalnız..........................................233

Feyza Bayrakdar Çağlayan........................ 116

Figen Kuloğlu.................................................. 89

Filiz Vural.......................................................... 27

Funda Ceran..................................................226

Funda Şimşek................................................167

Füsun Özdemirkıran...................................... 27

George Karam...............................................358

Gökhan Güngör............................................356

Gökhan Özgür...............................................359

Gönül Oğur....................................................345

Grzegorz Helbig...........................................186

Guifang Ouyang...........................................362

Guillermo J. Ruiz-Argüelles......................239

Guillermo J. Ruiz-Delgado........................239

Gunay Aliyeva...............................................258

Gül Nihal Özdemir......................................... 99

Gülay Erman..................................................213

Güldane Seval...............................................137

Gülsüm Çağlıyan............................................ 27

Gülsüm Özet..................................................226

Gülşen Bolat.................................................... 16

Günhan Gürman............................................ 16

Güray Saydam.......................................27, 174

Gürcan Dikme................................................. 99

Gürsel Güneş.................................................103

Habip Gedik...................................................167

Hakan Erdoğan.............................................264

Hakan Göker.................................................103

Hakan Hanımoğlu........................................ 113

Hakan Özdoğu.................196, 270, 280, 324

Haldun Öniz...................................................179

Hale Ören......................................................... 46

Halil Gürhan Karabulut..............................151

AUTHOR INDEX 2017

Haluk Demiroğlu..........................................103

Hamdi Akan..................................................... 16

Hande Kızılocak.............................................. 99

Hasan Özdil.................................................... 107

Hatice Ilgın Ruhi..........................................151

Hatice Şanlı...................................................194

Hayri Özsan...................................................282

Hayriye Mine Miskioğlu.............................181

He Qun...............................................................10

Hilal Aydın.....................................................120

Hong Qian......................................................334

Hsin-Chen Lin...............................................159

Hu Sun............................................................200

Hüseyin Önay................................................120

Hüseyin Tokgöz............................................360

Ioannis Tsolakis............................................... 52

Işık Atagündüz.....................................118, 352

İbrahim C. Haznedaroğlu............27, 93, 103

İbrahim Eker.................................................... 64

İbrahim Kulaç.................................................. 34

İdil Yenicesu..................................................143

Ilana Levy.......................................................289

İlknur Kozanoğlu......................196, 280, 324

İpek Yönal Hindilerden................................. 72

İsmail Yaşar Avcı............................................ 64

İsmet Aydoğdu.............................................181

Jan E. Dyr........................................................111

Jaroslav Cermak............................................111

Jasmita Dass..................................................188

Jelena Tosic Dragovic.................................268

Jia Meng.........................................................334

Jiajia Jin..........................................................334

Jian Tong........................................................334

Jianning Zhao...............................................334

Jie Zhang........................................................200

Jing Huang........................................................10

Juan Carlos Olivares-Gazca......................239

Jung-Kuang Yu.............................................374

Junqing Xu.....................................................126

Kanay Yararbaş.............................................105

Katarina Markovic.......................................268

Kazuo Wakayama........................................354

Klara Dalva.....................................................314

Klara Pecankova............................................111

Konstantinos Malekas.................................. 52

Konstantinos Stamoulis............................... 52

Kuang-Hsi Chang........................................159

Kuenzang Dorji.............................................266

Lale Olcay.......................................................151

Leontini Fountoulaki-Paparisos................ 52

Levent Karapınar..........................................179

Leyan Wang......................................................10

Leyla Cinel...................................................... 118

Leyla Tümer...................................................376

Liming Chen..................................................126

Long Su...........................................................381

Lu Chen..............................................................10

Madhu Mati Goel........................................369

Mahmut Yeral..................196, 270, 280, 324

Mani Ramzi....................................................300

Manu Goyal...................................................370

Maria Baka....................................................... 52

Maria Gavalaki............................................... 52

Maria Mouratidou......................................... 52

Mariana A. Mendez Huerta......................239

Marianna Politou........................................... 52

Marije M. Van Santen.................................124

Mario Soliman..............................................366

Massimo Zeviani..........................................376

Maurice Kfoury............................................358

Mehdi Khansalar..........................................300

Mehmet Can Uğur.......................................197

Mehmet Gündüz............................................ 16

Mehmet Halil Çeliksoy...............................345

Mehmet Özen................................................. 16

Mehmet Yılmaz.............................................. 27

Mehran Karimi..............................................250

Melda Cömert........................................27, 174

Melek Erdem................................................... 46

Meliha Nalçacı.......................................72, 131

Melikşah Uzakgider.....................................179

Meltem Aylı...................................................137

Meral Beksaç..........................................16, 314

Meral Türker..................................................179

Meriban Karadoğan....................................340

Meriç Kaymak Cihan...................................151

Mesude Falay................................................226

Michael Halefom Mezgebe......................... 59

Milan Pantelic...............................................268

Mili Jain..........................................................369

Mine Hekimgil..............................................122

Mohammad Mostafa Safarpour.............250

Mónica Leon-Gonzalez..............................239

Muhammad Ikram Ullah...........................278

Muhit Özcan................................................... 16

Muhlis Cem Ar.................................... 233, 291

Murat Akyıldız..............................................356

Murat Büyükdoğan.....................................360

Murat Dayangaç..........................................356

Murat Elli.......................................................345

Murat Sarı......................................................131

Murat Seval...................................................137

Murat Sünbül................................................ 107

Murat Yıldırım..............................................359

Musa Barış Aykan........................................359

Mustafa Erkoçoğlu......................................120

Mustafa Nuri Yenerel................................... 72

Mutlu Arat..............................................16, 350

Mürüvvet Seda Aydın.................................103

Naciye Demirel.............................................167

Nada Dimkovic.............................................268

Nadia Zanetti................................................376

Nadim El-Majzoub......................................358

Nafiye Urgancı..............................................109

Namık Özbek.................................................183

Nannan Li.......................................................126

Narges Rezaei...............................................300

Nargess Arandi.............................................300

Nazan Dalgıç.................................................109

Nazan Sarper................................................340

Nazif Zeybek................................................... 64

Nejat Akar...............................................93, 105

Neşe Yaralı..............................................40, 183

Neval Özkaya...................................................81

Nevin Kılıç......................................................264

Nihal Karadaş Özdemir..............................254

Nihan Akkoç..................................................233

Niki Vgontza.................................................... 52

Nilgün Sayınalp............................................103

Ning Xu...........................................................362

Nirong Bao.....................................................334

Nishant Verma..............................................369

Nitin Sood......................................................272

Noha S. Kandil..............................................207

Nur Efe İris........................................... 167, 350

Nur Soyer................................................27, 174

Nuray Başsüllü.................................................81

Nurhilal Büyükkurt.....................................324

Nuri Barış Hasbal......................................... 116

Nüket Yürür Kutlay.....................................151

Nükhet Tüzüner.....................................81, 291

Oktay Bilgir...................................................... 27

Orhan Gürsel................................................... 64

Osman İlhami Özcebe.................................103

Osman İlhan.............................................16, 27

Osman Kara...................................................352

Osman Yokuş.................................................167

Önder Arslan..........................................16, 244

Önder Şirikçi..................................................352

Özcan Çeneli.................................................364

Özge Özer Kaya............................................197

Özlem Çakıcı..................................................179

Özlem Özdemir.............................................264

Özlen Bektaş..................................................364

Paraskevi Papadopoulou.............................. 52

Pavel Majek....................................................111

Pelin Aytan.......................................... 196, 280

Pervin Topçuoğlu........................16, 194, 244

Petar Djuric....................................................268

Pınar Çetinalp Demircan...........................213

Pınar Genç.....................................................264

Pınar Yurdakul..............................................314

Pratap Rai......................................................266

Puja Devi Samal...........................................266

Qi Han.............................................................366

Recep Sancak................................................345

Reyhan Diz-Küçükkaya.............72, 350, 356

Rıza Aytaç Çetinkaya.................................... 64

Ritesh Sachdev.............................................272

Rixin Jamtsho...............................................266

Roger K. Schindhelm..................................124

Rongdi Yan....................................................200

Rosalba Morreale.........................................184

Ruchika K Goel.............................................272

Sabina Langer...............................................188

Sabina Russo.................................................184

Salih Aksu......................................................103

Sanjay Mishra...............................................369

Saqib Mahmood...........................................278

Sebahattin Yılmaz......................................... 64

Seçil Saral.......................................................194

Seda Yılmaz...................................................364

Selami Koçak Toprak..............................1, 244

Selim Öncel....................................................340

Selim Sayın....................................................359

Selin Berk.......................................................291

Selma Sarı......................................................131

Sema Aylan Gelen.......................................340

Sema Vural.....................................................109

Semra Şen......................................................254

Sena Sert........................................................ 107

Serena Valsami............................................... 52

AUTHOR INDEX 2017

Serkan Güvenç.............................................360

Serkan İşgören..............................................190

Sezaneh Haghpanah......................... 250, 300

Sezen Güntekin Ergün...............................143

Sezgin Etgül..................................................103

Shabbir Hussain...........................................278

Shahida Mohsin...........................................278

Shalini Goel...................................................272

Shao-Min Han..............................................159

Shereen M. Elmaghraby.............................207

Shinya Kimura..............................................354

Shyam Aggarwal..........................................188

Sıdıka Fındık..................................................364

Simten Dağdaş.............................................226

Sinan Demircioğlu.......................................364

Sinan Özkavukçu.........................................137

Sinem Civriz Bozdağ...................................244

Slavisa Sekulic..............................................268

Slawomira Kyrcz Krzemien.......................186

Smeeta Gajendra............................... 100, 272

Soner Solmaz................................................324

Soner Yılmaz................................................... 64

Songül Tepebaşı...........................................324

Sora Yasri........................................................102

Sousanna Pefani............................................ 52

Sreeja Thandilath Thekkelakayil..............370

Stavros Nousis................................................ 52

Stavroula Tsitlakidou.................................... 52

Stephan Borte...............................................345

Suar Çakı Kılıç...............................................340

Suchi Mittal...................................................188

Surmaya Gafarova.......................................258

Süheyl Asma...............................196, 280, 324

Süheyla U. Bozkurt............................ 190, 352

Sülen Sarıoğlu..............................................282

Süreyya Bozkurt...........................................103

Şadiye Mehtat Ünlü....................................282

Şebnem Yılmaz Bengoa............................... 46

Şencan Acar..................................................356

Şeniz Öngören.................................... 233, 291

Şöhret Aydemir............................................254

Şule Toprak....................................................372

Şükrü Kasap...................................................122

Şükrü Nail Güner.........................................345

Tahira Mammadova....................................258

Tamar Tadmor...............................................289

Taner Baştürk.....................................1, 16, 116

Taner Yıldırmak............................................167

Tao Yuan.........................................................334

Tashi Tobgay..................................................266

Tayfur Toptaş.................................................352

Teoman Soysal.................................... 233, 291

Tezer Kutluk..................................................... 34

Tiraje Celkan.................................................... 99

Tuğçe Kurtaraner.........................................109

Tuğrul Elverdi................................................291

Tuncay Aslan.................................................103

Tuncay Sahutoğlu........................................ 116

Tunç Fışgın.....................................................345

Tülin Ergun.................................................... 118

Tülin Fıratlı Tuğlular....................................352

Türkiz Gürsel..................................................143

Tze-Kiong Er..................................................374

Ufuk Demirci.................................................181

Uğur Demirsoy..............................................340

Uğur Muşabak................................................ 64

Ülker Koçak...................................................143

Ümit Yavuz Malkan.....................................103

Ümran Çalışkan............................................360

Ünsal Özgen.................................................. 114

Vanessa Innao...............................................184

Vani Rajashekharaiah.................................328

Vassiliki Pournara........................................... 52

Vassiliki Rekari................................................ 52

Veysel Sabri Hançer....................................360

Vildan Çulha..................................................183

Viroj Wiwanitkit........................ 101, 102, 380

Volkan Karakuş................................... 122, 192

Wen-Li Hwang..............................................159

Wen-shuang Sun.........................................334

Wen-xiang Chen..........................................334

Xiao-Ping Huang.........................................362

Xiaoqian Liu...................................................126

Xiaoxia Chu...................................................126

Xilian Zhou....................................................366

Xing Cui..........................................................200

Xu Yajing...........................................................10

Yahveth Cantero-Fortiz.............................239

Yahya Büyükaşık...................................34, 103

Yaman Tokat..................................................356

Yan Gu.............................................................366

Yanyan Luan..................................................126

Yasemin Ardıçoğlu.......................................105

Yasushi Kubota.............................................354

Yaşar Bekir Kutbay......................................197

Yegana Guliyeva..........................................258

Yelda Dere............................................ 122, 192

Yener Koç....................................................... 116

Yıldız Aydın.......................................... 233, 291

Yi-Hong Liu...................................................374

Yinghui Liu.....................................................126

Yuanfeng Zhang...........................................126

Yuka Hirakawa..............................................354

Yüksel Pabuşçu.............................................181

Zafer Başlar..........................................113, 291

Zafer Gülbaş..................................................190

Zahide Eriş.....................................................350

Zehra Işık Haşıloğlu....................................... 99

Zerrin Ertaş...................................................... 64

Zeynep Kayra Tanrıverdi............................ 114

Zhao Xielan.......................................................10

Zheng Ge........................................................366

Zhi-Yuan Shi.................................................159

Zhuan-Bo Luo...............................................362

Zoran Milojevic............................................268

Zühre Kaya.....................................................143

Zümrüt Şahbudak Bal................................254

SUBJECT INDEX 2017

34 th Volume Index / 34. Cilt Dizini

SUBJECT INDEX - KONU DİZİNİ 2017

Acute Leukemia

Relapsed/refractory leukemia / Nüks/refrakter lösemi, 46

FLAG regimen / FLAG tedavisi, 46

Chemotherapy / Kemoterapi, 46

Childhood / Çocukluk çağı, 46

Hypogammaglobulinemia / Hipogamaglobulinemi, 89

Leukemia / Lösemi, 89, 109, 126, 186, 190

Lymphoma / Lenfoma, 89

Myeloma / Miyelom, 89, 181, 182

Vancomycin-resistant Enterococcus / Vankomisin dirençli entekok, 89

Wernicke’s encephalopathy / Wernicke ensefalopatisi, 99

Thiamine deficiency / Tiamin eksikliği, 99

Pediatric leukemia / Pediatrik lösemi, 99, 340

Trisomy 6 / Trizomi 6, 103

Acute myeloid leukemia / Akut miyeloid lösemi, 103, 192, 340, 300, 307

Breast cancer / Meme kanseri, 103

Granulocytic sarcoma / Granülositik sarkom, 122

Acute monoblastic leukemia / Akut monoblastik lösemi, 122

CD34 / CD34, 122

Myeloperoxidase / Miyeloperoksidaz, 122

Monosomal karyotype / Monozomal karyotip, 126

Prognosis / Prognoz, 126, 143, 207

Methotrexate / Metotreksat, 143

Toxicity / Toksisite, 143

Folate-related gene polymorphisms / Folat-ilişkili gen

polimorfizmleri, 143

Acute lymphoblastic leukemia / Akut lenfoblastik lösemi, 40, 151, 366

Glucocorticoid receptor gene / Glukokortikoid reseptör geni, 151

BclI and N363S polymorphisms / BclI ve N363S polimorfizmler, 151

TET2 / TET2, 174

ASXL1 / ASXL1, 174

IDH1 / IDH1, 174

IDH2 / IDH2, 174

Single nucleotide polymorphisms / Tek nükleoitid polimorfizm, 174

Ph-negative myeloproliferative neoplasms / Ph-negatif

Miyeloproliferatif neoplaziler, 174


Lymphadenopathy / Lenfadenopati, 190

Azacitidine / Azasitidin, 192

Leukemia cutis / Lösemi kutis, 192

Febrile neutropenia / Netropenik Ateş, 109, 167, 254, 340

Infection / Enfeksiyon, 340

Acute myelogenous leukemia / Akut myeloid lösemi, 350

Bullous pyoderma gangrenosum / Büllöz piyoderma gangrenosum, 350

Pathergy / Paterji, 350

EZH2 / EZH2, 366

Adult / Erişkin, 366

T-cell / T-hücre, 366

Gene mutation / Gen mutasyonu, 300

FLT3 / FLT3, 300

NPM1 / NPM1, 300

Autoantibody / Otoantikor, 307

Cancer / Kanser, 307

Carbonic anhydrase / Karbonik anhidraz, 307

Anemia

Schistocyte / Şistosit, 59

Thrombotic microangiopathy / Trombotik mikroanjiopati, 59

Microscopy / Mikroskop, 59

ADVIA 2120 / ADVIA 2120, 59

Standardization / Standardizasyon, 59

Refractory anemia with ring sideroblasts / Halka sideroblastlı

refrakter anemi, 100

RARS with thrombocytosis / Trombositoz ile birlikte RARS, 100

Myelodysplastic syndrome/myeloproliferative neoplasm with

ring sideroblasts and thrombocytosis / Halka sideroblast ve trombositoz

ile birlikte miyelodisplastik sendrom/miyeloproliferatif neoplaz, 100

Abnormal hemoglobins / Anormal hemoglobinler, 114

Hb Kansas / Hb Kansas 114

Children / Çocuk, 120

Griscelli syndrome / Griscelli sendromu, 120

Status epilepticus / Status epileptikus, 120

Hemophagocytic lymphohistiocytosis / Hemofagositik

lenfohistiyositoz, 120

Adult / Erişkin, 196, 366

Sickle cell anemia / Orak hücreli anemi, 196, 358

Genetic counseling / Genetik danışmanlık, 196

Transplantation / Transplantasyon, 102, 196, 345

Blood components / Kan içeriği, 244

Blood processing / Kan ürünlerini işleme, 244

Donors / Verici, 244

Transfusion strategy / Transfüzyon stratejisi, 244

Thalassemia / Talasemi, 258

Sickle/β-thalassemia / Orak/β talasemi, 258

Codon / Kodon, 258

Genotype / Genotip, 258

Phenotype / Fenotip, 258

Osteomyelitis / Osteomiyelit, 358

Salmonella typhi / Salmonella typhi, 358

Myopathy / Miyopati, 376

Lactic acidosis / Laktik asidoz, 376

Sideroblastic anemia / Sideroblastik anemi, 376

Hidden blood loss / Gizli kan kaybı, 334

Antioxidants / Antioksidanlar, 334

Proanthocyanidin / Proantosiyanidin, 334

Hydrogen water / Hidrojenli su, 334

Bleeding Disorders

Hemophilia A / Hemofili A, 113

Intradiploic hematoma / İntradiploik hematom, 113

Coagulopathy / Koagülopati, 113

Intraosseous / İntraosseoz, 113



SUBJECT INDEX 2017




Afibrinogenemia / Afibrinojenemi, 183

Bone cysts / Kemik kistleri, 183

Child / Çocuk, 183

Rare / Nadir, 183

Acquired hemophilia / Edinsel hemofili, 184

Multiple myeloma / Multipl miyelom, 131, 184, 226, 233, 282

Factor VIII / Faktör VIII, 184


Coagulation disorder / Koagülasyon bozukluğu, 184

Congenital / Konjenital, 250

Factor V deficiency / Faktör V eksikliği, 250


Rare bleeding disorders / Nadir kanama bozuklukları, 250

Psychogenic purpura / Psikojenik purpura, 274

Gardner-Diamond syndrome / Gardner-Diamond sendromu, 274

Antidepressant / Antidepresan, 274

Adolescent / Adölesan, 274


FVIII gene / FVIII geni, 278

Intron 1 / İntron 1, 278

Polymerase chain reaction / Polimeraz zincirleme reaksiyonu, 278

Pakistan / Pakistan, 278

Dysfibrinogenemia / Disfibrinojenemi, 356

Liver transplantation / Karaciğer nakli, 356

Subdural hematoma / Subdural hematom, 356

Chronic Leukemia

Imatinib / İmatinib, 10

Sokal score / Sokal skoru, 10

Hasford score / Hasford skoru, 10

European Treatment Outcome Study score / Avrupa Tedavi

İzlem Çalışması skoru, 10

Chronic myeloid leukemia / Kronik miyeloid lösemi, 16, 137, 197, 362

Allogeneic transplantation / Allojenik transplantasyon, 16

Tyrosine kinase inhibitors / Tirozin kinaz inhibitörleri, 16

Hematologic remission / Hematolojik remisyon, 16

TP53 / TP53, 34

Immunohistochemistry / İmmünohistokimya, 34

Chronic lymphocytic lymphoma / Kronik lenfositik lenfoma, 34

Proliferation centers / Proliferasyon merkezleri, 34

Nilotinib / Nilotinib, 137, 197, 362

Fertility / Doğurganlık, 137

Mouse / Fare, 137

Gonads / Gonad, 137

Richter’s syndrome / Richter sendromu, 188

Flow cytometry / Akım sitometri, 188

Chronic lymphocytic leukemia / Kronik lenfositik lösemi, 188

CD5-positive / CD5- pozitif, 188

Diffuse large B-cell lymphoma / Diffüz büyük B-hücreli lenfoma, 188

Cytogenetic / Sitogenetik, 197

Molecular hematology / Moleküler hematoloji, 197

Life-quality / Yaşam kalitesi, 197

Dasatinib / Dasatinib, 197

Hairy cell leukemia / Saçlı hücreli lösemi, 270, 289, 291

Splenectomy / Splenektomi, 270, 291

Sweet’s syndrome / Sweet’s sendromu, 270

Systemic mastocytosis / Sistemik mastositoz, 276

Systemic mastocytosis with an associated hematological neoplasm /

Hematolojik malignite ile seyreden sistemik mastositozis, 276


KIT D816V / KIT D816V, 276

Purine Analogs / Pürin Analogları, 289

Cladribine / Kladribin, 289, 291

Pentostatin /Pentostatin, 289

Imatinib mesylate / İmatinib mesilat, 362

Interstitial pneumonitis / İnterstisyel pnömonitis, 362

Tuberculosis / Tüberküloz, 362

Interferon / İnterferon, 291

Coagulation

Ankaferd Blood Stopper / Ankaferd Blood Stopper, 93

Shiga-toxigenic Escherichia coli / Shigatoksijenik Escherichia coli, 93

Salmonella / Salmonella, 93

Campylobacter / Campylobacter, 93

Listeria monocytogenes / Listeria monocytogenes, 93

Monitoring / İzlem, 101

International normalized ratio / Uluslararası normalleştirilmiş oran, 101

Hemostasis / Hemostaz, 101

Acute myocardial infarction / Akut miyokard infarktüsü, 107

Immune thrombocytopenic purpura / İmmün trombositopenik

purpura, 107

Eltrombopag / Eltrombopag, 107







Child / Çocuk, 183

Rare / Nadir, 183






Sticky platelet syndrome / Yapışkan trombosit sendromu, 239

Thrombophilia / Trombofili, 239

Miscarriages / Düşükler, 239

Congenital / Konjenital, 250

Factor V deficiency / Faktör V eksikliği, 250


Rare bleeding disorders / Nadir kanama bozuklukları, 250









Hematological Malignancies

Imatinib / İmatinib, 10

Sokal score / Sokal skoru, 10

Hasford score / Hasford skoru, 10

European Treatment Outcome Study score / Avrupa Tedavi

SUBJECT INDEX 2017

İzlem Çalışması skoru, 10

Chronic myeloid leukemia / Kronik miyeloid lösemi, 16, 137, 197, 362




Chronic myeloproliferative neoplasms / Kronik miyeloproliferatif

neoplaziler, 27

Treatment / Tedavi, 27

Survival / Sağkalım, 27

JAK2 mutation / JAK2 mutasyonu, 27

Relapsed/refractory leukemia / Nüks/refrakter lösemi, 46

FLAG regimen / FLAG tedavisi, 46

Chemotherapy / Kemoterapi, 46

Childhood / Çocukluk çağı, 46


Leukemia / Lösemi, 89, 109, 126, 186, 190


Myeloma / Miyelom, 89, 181, 182, 352











CD34 / CD34, 122





Prognostic factors / Prognostik faktörler, 131

Serum immunoglobulins / Serum immunoglobulin, 131

Nephelometric measurement / Nefelometrik ölçüm, 131

M-protein / M-protein, 131



Mouse / Fare, 137

Gonads / Gonad, 137







BclI and N363S polymorphisms / BclI ve N363S polimorfizmler, 151

TET2 / TET2, 174

ASXL1 / ASXL1, 174

IDH1 / IDH1, 174

IDH2 / IDH2, 174


Ph-negative myeloproliferative neoplasms /

Ph-negatif Miyeloproliferatif neoplaziler, 174

Myeloma / Miyelom, 89, 181, 182

Infection / Enfeksiyon, 182, 340

Atlas / Atlas, 182

Fracture / Kırık, 182



Autoantibody / Otoantikor, 184, 307


Bing-Neel syndrome / Bing-Neel sendromu, 186

Waldenström’s macroglobulinemia / Waldenström makroglobulinemisi,

186

Central nervous system / Merkezi sinir sistemi, 186

MYD88 L265P mutation / MYD88 L265P mutasyonu, 186

Cerebrospinal fluid / Beyin-omurilik sıvısı, 186

Leukemia / Lösemi, 89, 109, 126, 186, 190









Azacitidine / Azasitidin, 192

Leukemia cutis / Lösemi kutis, 192

Mycosis fungoides / Mikozis fungoides, 194

Ichthyosiform / İktiyotik, 194

Sezary syndrome / Sezary sendromu, 194

Anaplastic / Anaplastik, 194

CD3/TCR / CD3/TCR, 194





Myelodysplastic syndromes / Miyelodisplastik sendromlar, 200

Telomere-binding proteins / Telomer-bağlayıcı proteinler, 200

Reverse transcription-polymerase chain reaction /

Ters transkripsiyon-polimeraz zincir reaksiyonu, 200

International Prognostic Scoring System /

Uluslararası Prognoz Skorlama Sistemi, 200

World Health Organization Prognostic Scoring System /

Dünya Sağlık Örgütü Prognoz Skorlama Sistemi, 200

MicroRNA-155 / MikroRNA-155, 207

Non-Hodgkin lymphoma / Hodgkin dışı lenfoma, 207, 378, 381

CD56 / CD56, 226

CD117 / CD117, 226


Multiple myeloma / Multipl miyeloma, 131, 184, 226, 233, 282

Relapse refractory / Relaps refrakter, 233

Bendamustine / Bendamustin, 233

Distal arteriovenous fistula / Distal arteriyovenöz fistül, 268

Multislice computer tomography/ Çok kesitli bilgisayarlı tomografi, 268

Hemodialysis / Hemodiyaliz, 268




Hodgkin’s lymphoma / Hodgkin lenfoma, 272

Metastatic adenocarcinoma / Metastatik adenokarsinom, 272

Reed-Sternberg cells / Reed-Sternberg hücreleri, 272

PET/CT / PET/BT, 272

Systemic mastocytosis / Sistemik mastositoz, 276

Systemic mastocytosis with an associated hematological neoplasm /

Hematolojik malignite ile seyreden sistemik mastositozis, 276

SUBJECT INDEX 2017


KIT D816V / KIT D816V, 276

Pediatric leukemia / Çocukluk çağı lösemileri, 340

Febrile neutropenia / Nötropenik Ateş, 109, 167, 254, 340

Purine analogs / Pürin analogları, 289

Cladribine / Kladribin, 289, 291

Pentostatin / Pentostatin, 289

Acute myelogenous leukemia / Akut myeloid lösemi, 350



Plasmacytoma / Plazmasitom, 352

Intracranial / İntrakraniyal, 352

Essential thrombocythemia / Esansiyel trombositemi, 360

Primary myelofibrosis / Primer miyelofibroz, 124, 360

Calreticulin / Kalretikulin, 360




Follicular lymphoma / Foliküler lenfoma, 364

Extranodal / Ekstranodal, 364

Prostatic involvement / Prostat Tutulumu, 364

EZH2 / EZH2, 366




Hemoglobin San Diego / Hemoglobin San Diego, 372

Hemoglobin variant / Hemoglobin varyantı, 372

T-cell neoplasms / T hücreli neoplazmlar, 378

Oncogenes / Onkogenler, 378

T-cell mediated immunity / T hücre aracılı immünite, 378

miR-155 / miR-155, 381

Prognostic value / Prognostik değer, 381

Interferon / İnterferon, 291


FLT3 / FLT3, 300

NPM1 / NPM1, 300



Monoclonal gammopathy of renal significance / Renal öneme sahip

monoklonal gammopati, 282

Plasma cell disorders / Plazma hücre hastalıkları, 282

Renal involvement / Böbrek tutulumu, 282

Kidney / Böbrek, 282

Cast nephropathy / Kast nefropati, 282

Immunohematology

Leukemia / Lösemi, 89, 109, 126, 186, 190

Febrile neutropenia / Febril nötropeni, 109, 167, 254, 340

Candida / Candida, 109

Immune response inflammatory syndrome / İmmün yanıt

enflamatuvar sendromu, 109



Serum immunoglobulins / Serum immunoglobulin, 131



Neutrophil / Nötrofil, 167

CD64 / CD64, 167

Bacteremia / Bakteriyemi, 167, 254

Diagnostic parameter / Diyagnostik parametre, 167

Acute compartment syndrome / Akut kompartman sendromu, 179

Inflammation / Enflamasyon, 179

Immune deficiency / İmmün yetmezlik, 179




CD5-positive / CD5-pozitif, 188


Immunoregulatory effect / Bağışıklık baskılayıcı etki, 213

Co-culture / Ko-kültür, 213

Mesenchymal stem cells / Mezenkimal kök hücre, 213

T cells / T hücre, 213

CD56 / CD56, 226

CD117 / CD117, 226


IL-6 / IL-6, 254

IL-8 / IL-8, 254

IL-10 / IL-10, 254

Hematopoitic stem cell / Hematopoetik kök hücre, 345

Transplantation /Transplantasyon, 102, 196, 345


Immunodeficiency / İmmün yetmezlik, 345





Cord blood / Göbek kordon kanı, 314

Aldehyde dehydrogenase / Aldehit dehidrogenaz, 314

Colony-forming unit-granulocyte/macrophage / Koloni

oluşturan birim granülositer/makrofaj, 314

L-Carnitine / L-Karnitin, 328

Plasma / Plazma, 328

Antioxidant enzymes / Antioksidan enzimler, 328

Lipid peroxidation / Lipid peroksidasyonu, 328

Protein oxidation / Protein oksidasyonu, 328





Iron Disorder

Iron overload / Demir yüklenmesi, 1

Hematopoietic stem cell transplantation / Hematopoietik kök

hücre nakli, 1

Ferritin / Ferritin, 1

Iron chelation / Demir şelasyonu, 1

Iron / Demir, 102

Overload / Aşırı yüklenme, 102

Hematopoietic stem cell / Hematopoietik kök hücre, 102


Infection Disorders


Leukemia / Lösemi, 89, 109, 126, 186, 190


Myeloma / Miyelom, 89, 181, 182, 352




SUBJECT INDEX 2017




Febrile neutropenia / Febril nötropeni, 109, 167, 254, 340

Candida / Candida, 109

Immune response inflammatory syndrome / İmmün yanıt

enflamatuvar sendromu, 109

Allogeneic hematopoietic stem cell transplantation /

Allojeneik hematopoietik kök hücre transplantasyonu, 159

Cytomegalovirus / Sitomegalovirüs, 159

Graft-versus-host disease / Graft versus host hastalığı, 159

Taiwan / Tayvan, 159

Neutrophil / Nötrofil, 167

CD64 / CD64, 167

Bacteremia / Bakteriyemi, 167, 254

Diagnostic parameter / Diyagnostik parametre, 167




IL-6 / IL-6, 254

IL-8 / IL-8, 254

IL-10 / IL-10, 254

Leishman-Donovan body / Leishman-Donovan cismi, 266

Bone marrow aspirate smear / Kemik iliği aspirasyonu yayması, 266

Microscopic image / Mikroskobik görüntü, 266


Pediatric leukemia / Çocuk çağı lösemileri, 340


Leukoagglutination / Lökoaglütinasyon, 354

Cold agglutinin / Soğuk aglütinin, 354

Mycoplasma pneumoniae / Mycoplasma pneumoniae, 354

Eosinophilia / Eozinofili, 354

Pseudoleukopenia / Psödolökopeni, 354


Osteomyelitis / Osteomiyelit, 358

Salmonella typhi / Salmonella typhi, 358

Cholangitis / Kolanjit, 359

Döhle bodies / Döhle cisimciği, 359

May-Hegglin anomaly / May-Hegglin anomalisi, 359



Chronic myeloid leukemia / Kronik miyeloid lösemi, 362



Leishmaniasis / Layşmanyazis, 380

Bone marrow / Kemik iliği, 380

Aspirate / Aspirasyon, 380

Smear / Yayma, 380

Antibody / Antikor, 380

Lymphoma

Diagnosis of lymphoma / Lenfoma tanısı, 81

World Health Organization lymphoma classification / Dünya Sağlık

Örgütü lenfoma sınıflaması, 81

Discrepancies in diagnosis / Tanı tutarsızlıkları, 81

Hematopathology / Hematopatoloji, 81


Waldenström’s macroglobulinemia /

Waldenström makroglobulinemisi, 186




Leukemia / Lösemi, 89, 109, 126, 186, 190
























miR-155 / miR-155, 381


Molecular Hematology





Variant / Varyant, 105


Prenatal diagnosis / Prenatal tanı, 105

Beta globin gene / Beta globin geni, 105

Turkish / Türk, 105



Leukemia / Lösemi, 89, 109, 126, 186, 190

Chronic myeloid leukemia / Kronik miyeloid lösemi, 16, 137, 197



Mouse / Fare, 137

Gonads / Gonad, 137







BclI and N363S polymorphisms / BclI ve N363S polimorfizmleri, 151

TET2 / TET2, 174

ASXL1 / ASXL1, 174

IDH1 / IDH1, 174

IDH2 / IDH2, 174

SUBJECT INDEX 2017





Waldenström’s macroglobulinemia / Waldenström

makroglobulinemisi, 186




Leukemia / Lösemi, 89, 109, 126, 186, 190

Adult / Erişkin, 196, 366



Transplantation / Transplantasyon, 102, 196



Reverse transcription-polymerase chain reaction /

Ters transkripsiyon-polimeraz zincir reaksiyonu, 200




Dünya Sağlık Örgütü Prognoz Skorlama Sistemi, 200








Sickle/β-thalassemia / Orak/β talasemi, 258

Codon / Kodon, 258

Genotype / Genotip, 258

Phenotype / Fenotip, 258, 374

Wolman disease / Wolman hastalığı, 264

Hemophagocytic lymphohistiocytosis /

Hemafagositik lenfohistiyositozis, 264

Hemophagocytosis / Hemofagositoz, 264









EZH2 / EZH2, 366



Hemoglobin San Diego / Hemoglobin San Diego, 372

Hemoglobin variant / Hemoglobin varyantı, 372

Para-Bombay / Para-Bombay, 374

FUT1 gene / FUT1 geni, 374

Blood transfusion / Kan transfüzyonu, 374




miR-155 / miR-155, 381



FLT3 / FLT3, 300

NPM1 / NPM1, 300

Multiple Myeloma



Serum immunoglobulins / Serum immünglobulin, 131



Myeloma / Miyelom, 89, 181, 182, 352


Atlas / Atlas, 181






CD56 / CD56, 226

CD117 / CD117, 226





Multislice computer tomography / Çok kesitli bilgisayarlı tomografi, 268


Plasmacytoma / Plazmasitom, 352

Intracranial / İntrakraniyal, 352

Monoclonal gammopathy of renal significance / Renal öneme

sahip monoklonal gammopati, 282





Myelodysplastic Syndromes

Refractory anemia with ring sideroblasts / Halka sideroblastlı refrakter

anemi, 100


Myelodysplastic syndrome/myeloproliferative neoplasm with ring

sideroblasts and thrombocytosis / Halka sideroblast ve trombositoz ile

birlikte miyelodisplastik sendrom/miyeloproliferatif neoplazi, 100



Reverse transcription-polymerase chain reaction / Ters

transkripsiyon-polimeraz zincir reaksiyonu, 200




Dünya Sağlık Örgütü Prognoz Skorlama

Sistemi, 200

Myeloproliferative Disorders

Chronic myeloproliferative neoplasms / Kronik

miyeloproliferatif neoplaziler, 27

Treatment / Tedavi, 27

Survival / Sağkalım, 27

JAK2 mutation / JAK2 mutasyonu, 27


Internuclear bridging / Nükleuslar arası köprüleşme, 124

Erythrocytes / Eritrositler, 124

SUBJECT INDEX 2017

TET2 / TET2, 174

ASXL1 / ASXL1, 174

IDH1 / IDH1, 174

IDH2 / IDH2, 174






Stem Cell Transplantation

Iron overload / Demir yüklenmesi, 1

Hematopoietic stem cell transplantation / Hematopoietik kök

hücre nakli, 1

Ferritin / Ferritin, 1

Iron chelation / Demir şelasyonu, 1





Iron / Demir, 102

Overload / Aşırı yüklenme, 102

Hematopoietic stem cell / Hematopoietik kök hücre, 102, 345, 321


Allogeneic hematopoietic stem cell transplantation /

Allojeneik hematopoietik kök hücre transplantasyonu, 159

Cytomegalovirus / Sitomegalovirüs, 159

Graft-versus-host disease / Graft versus host hastalığı, 159

Taiwan / Tayvan, 159


Waldenström’s macroglobulinemia / Waldenström

makroglobulinemisi, 186




Leukemia / Lösemi, 89, 109, 126, 186, 190













Hematopoietic stem and progenitor cells / Hematopoietik

stem ve progenitor hücreler, 280

Stem cell mobilization / Stem hücre mobilizasyonu, 280

Apheresis / Aferez, 280

ISBT 128 / ISBT 128, 280

JACIE / JACIE, 280

Labeling / Etiketleme, 280

Traceability / Takip edilebilirlik, 280


Immunodeficiency / İmmün yetmezlik, 345

Cord blood / Göbek kordon kanı, 314

Aldehyde dehydrogenase / Aldehit dehidrogenaz, 314

Colony-forming unit-granulocyte/macrophage / Koloni

oluşturan birim granülositer/makrofaj, 314

Donor / Donör, 321

Informed consent / Bilgilendirilmiş onam, 321

Audiovisual method / Audovizuel yöntem, 321

Bone marrow transplantation / Kemik iliği nakli, 321

L-Carnitine / L-Karnitin, 328

Plasma / Plazma, 328

Antioxidant enzymes / Antioksidan enzimler, 328

Lipid peroxidation / Lipid peroksidasyonu, 328

Protein oxidation / Protein oksidasyonu, 328

Thalassemia


Variant / Varyant, 105


Prenatal diagnosis / Prenatal tanı, 105

Beta globin gene / Beta globin geni, 105

Turkish / Türk, 105

Thrombosis

Monitoring / İzlem, 101

International normalized ratio / Uluslararası normalleştirilmiş oran, 101

Hemostasis / Hemostaz, 101



purpura, 107


Vasculitis / Vaskülit, 116

Anticoagulants / Antikoagülan, 116

Rivaroxaban / Rivaroksaban, 116




Thrombocytopenia

Platelet / Trombosit, 64

Cryopreservation / Kriyoprezervasyon, 64

Microparticle generation / Mikropartikül, 64

Hemostatic activity / Hemostatik aktivite, 64

Immune thrombocytopenia / İmmün trombositopeni, 72

Rituximab / Rituksimab, 72

Early response / Erken yanıt, 72

Late response / Geç yanıt, 72

Sustained response / Sürekli yanıt, 72



purpura, 107











Thrombocytopenia / Trombositopeni, 370

SUBJECT INDEX 2017

Megakaryocytic emperipolesis / Megakaryositik emperipolez, 370

GATA1 / GATA1, 370

Other


Endocrine / Endokrin, 40

Late effects / Geç yan etkiler, 40







Wernicke’s encephalopathy / Wernicke ensefalopatisi, 99

Thiamine deficiency / Tiamin eksikliği, 99

Pediatric leukemia / Pediatrik lösemi, 99





Bullous / Büllöz, 118

Figurate erythema / Figüre eritem, 118

Myelodysplastic syndrome / Miyelodisplastik sendrom, 118

Sweet’s syndrome / Sweet sendromu, 118


BclI and N363S polymorphisms / BclI ve N363S polimorfizmleri, 151




Myeloma / Miyelom, 89, 181, 182, 352


Atlas / Atlas, 181




Child / Çocuk, 183

Rare / Nadir, 183

















Wolman disease / Wolman hastalığı, 264

Hemophagocytic lymphohistiocytosis /

Hemafagositik lenfohistiyositozis, 264

Hemophagocytosis / Hemofagositoz, 264








Hematopoietic stem and progenitor cells / Hematopoietik stem

ve progenitor hücreler, 280

Stem cell mobilization / Stem hücre mobilizasyonu, 280

Apheresis / Aferez, 280

ISBT 128 / ISBT 128, 280

JACIE / JACIE, 280

Labeling / Etiketleme, 280

Traceability / Takip edilebilirlik, 280

Leukoagglutination / Lökoaglütinasyon, 354

Cold agglutinin / Soğuk aglütinin, 354

Mycoplasma pneumoniae / Mycoplasma pneumoniae, 354

Eosinophilia / Eozinofili, 354

Pseudoleukopenia / Psödolökopeni, 354




Neuroblastoma / Nöroblastoma, 369

Circulating tumor cells / Dolaşan tümör hücreleri, 369

Metastasis / Metastaz, 369








Hematopoietic stem cell / Hematopoietik kök hücre, 321

Donor / Donör, 321

Informed consent / Bilgilendirilmiş onam, 321

Audiovisual method / Audovizuel yöntem, 321

Bone marrow transplantation / Kemik iliği nakli, 321





Monoclonal gammopathy of renal significance / Renal öneme

sahip monoklonal gammopati, 282





Pathology

TP53 / TP53, 34

Immunohistochemistry / İmmünohistokimya, 34

Chronic lymphocytic lymphoma / Kronik lenfosittik lenfoma, 34

Proliferation centers / Proliferasyon merkezleri, 34

Schistocyte / Şistosit,

thrombotic microangiopathy / trombotik mikroanjiopati, 59

Microscopy / Mikroskop, 59

ADVIA 2120 / ADVIA 2120, 59

Standardization / Standardizasyon, 59

Diagnosis of lymphoma / Lenfoma tanısı, 81

World Health Organization lymphoma classification / Dünya

Sağlık Örgütü lenfoma sınıflaması, 81

SUBJECT INDEX 2017

Discrepancies in diagnosis / Tanı tutarsızlıkları, 81

Hematopathology / Hematopatoloji, 81

Refractory anemia with ring sideroblasts / Halka sideroblastlı

refrakter anemi, 100


Myelodysplastic syndrome/myeloproliferative neoplasm with

ring sideroblasts and thrombocytosis / Halka sideroblast ve

trombositoz ile birlikte miyelodisplastik sendrom/

miyeloproliferatif neoplazi, 100

Vasculitis / Vaskülit, 116

Anticoagulants / Antikoagülan, 116

Rivaroxaban / Rivaroksaban, 116

Bullous / Büllöz, 118

Figurate erythema / Figüre eritem, 118

Myelodysplastic syndrome / Miyelodisplastik sendrom, 118

Sweet’s syndrome / Sweet sendromu, 118



CD34 / CD34, 122



Internuclear bridging / Nükleuslar arası köprüleşme, 124

Erythrocytes / Eritrositler, 124







Leukemia / Lösemi, 89, 109, 126, 186, 190


Leishman-Donovan body / Leishman-Donovan cismi, 266

Bone marrow aspirate smear / Kemik iliği aspirasyonu yayması, 266

Microscopic image / Mikroskobik görüntü, 266


Multislice computer tomography / Çok kesitli bilgisayarlı tomografi, 268









Acute myelogenous leukemia / Akut miyeloid lösemi, 350



Cholangitis / Kolanjit, 359

Döhle bodies / Döhle cisimciği, 359

May-Hegglin anomaly / May-Hegglin anomalisi, 359




Neuroblastoma / Nöroblastoma, 369

Circulating tumor cells / Dolaşan tümör hücreleri, 369

Metastasis / Metastaz, 369

Thrombocytopenia / Trombositopeni, 370

Megakaryocytic emperipolesis / Megakaryositik emperipolez, 370

GATA1 / GATA1, 370

Leishmaniasis / Layşmanyazisis, 380

Bone marrow / Kemik iliği, 380

Aspirate / Aspirasyon, 380

Smear / Yayma, 380

Antibody / Antikor, 380

Autoimmune Disorders

Immune thrombocytopenia / İmmün trombositopeni, 72

Rituximab / Rituksimab, 72

Early response / Erken yanıt, 72

Late response / Geç yanıt, 72

Sustained response / Sürekli yanıt, 72



purpura, 107


Transfusion

Red blood cell / Kırmızı kan hücresi, 52

Transfusion practice / Transfüzyon uygulamaları, 52

Blood storage age / Kan depolama yaşı, 52

Platelet / Trombosit, 64

Cryopreservation / Kriyoprezervasyon, 64

Microparticle generation / Mikropartikül, 64

Hemostatic activity / Hemostatik aktivite, 64

Blood components / Kan içeriği, 244

Blood processing / Kan ürünlerini işleme, 244

Donors / Verici, 244

Transfusion strategy / Transfüzyon Stratejisi, 244

Para-Bombay / Para-Bombay, 374


FUT1 gene / FUT1 geni, 374

Blood transfusion / Kan transfüzyonu, 374

2017-4

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