v33i1

Volume 33 Issue 1 March 2016 80 TL

ISSN 1300-7777

Review Article

The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome

Doruk Erkan, Jane E. Salmon; New York, United States

Research Articles

Deregulated Levels of the NF-κB1, NF-κB2, and Rel Genes in Ukrainian Patients with Leukemia and

Lymphoma in the Post-Chernobyl Period

Hakan Savlı, et al.; Kocaeli, Turkey, Kyiv, Ukraine

Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin Expression

Tze Yan Lee, et al.; Serdang, Malaysia

The Feasibility of Magnetic Resonance Imaging for Quantification of Liver, Pancreas, Spleen, Vertebral

Bone Marrow, and Renal Cortex R2* and Proton Density Fat Fraction in Transfusion-Related Iron Overload

İlkay S. İdilman, et al.; Ankara, Turkey

Freezing of Apheresis Platelet Concentrates in 6% Dimethyl Sulfoxide: The First Preliminary

Study in Turkey

Soner Yılmaz, et al.; Ankara, Turkey

Is It Possible to Predict Pulmonary Complications and Mortality in Hematopoietic Stem Cell

Transplantation Recipients from Pre-Transplantation Exhaled Nitric Oxide Levels?

Nurdan Köktürk, et al.; Ankara, Turkey

A Quasi-Experimental Study Analyzing the Effectiveness of Portable High-Efficiency Particulate

Absorption Filters in Preventing Infections in Hematology Patients during Construction

Mehmet Özen, et al.; Ankara, Turkey

Comparison of the Efficiencies of Buffers Containing Ankaferd and Chitosan on Hemostasis in an

Experimental Rat Model with Femoral Artery Bleeding

Serkan Abacıoğlu, et al.; Osmaniye, Ankara, Zonguldak, Turkey

Cover Picture:

Işıl Erdoğan Özünal

Ağva, İstanbul

1

Editor-in-Chief

Reyhan Küçükkaya

İstanbul Bilim University, İstanbul, Turkey

Associate Editors

Ayşegül Ünüvar

İstanbul University, İstanbul, Turkey

Cengiz Beyan

Gülhane Military Medical Academy,

Ankara, Turkey

Hale Ören

Dokuz Eylül University, İzmir, Turkey

İbrahim C. Haznedaroğlu

Hacettepe University, Ankara, Turkey

M. Cem Ar

İstanbul University Cerrahpaşa Faculty of

Medicine, İstanbul, Turkey

Selami Koçak Toprak

Ankara University, Ankara, Turkey

Semra Paydaş

Çukurova University, Adana, Turkey

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

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

Osmangazi University, Eskişehir, Turkey

Şule Ünal


Veysel Sabri Hançer

İstanbul Bilim 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

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, 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

Turkish Society of Hematology Editorial Board

Ahmet Muzaffer Demir, President

Güner Hayri Özsan, General Secretary

T. Tiraje Celkan, Vice President

M. Cem Ar, Research Secretary

E. Naci Tiftik, Treasurer

Meltem Yüksel, Member

İlknur Kozanoğlu, Member

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

Ahmet Muzaffer Demir

Üç ayda bir yayımlanan İngilizce süreli yayındır.

International scientific journal published quarterly.

Publishing Manager

Sorumlu Yazı İşleri Müdürü

Güner Hayri Özsan

Management Address

Yayın İdare Adresi

Türk Hematoloji Derneği

İlkbahar Mahallesi, Turan Güneş Bulvarı 613. Sk. No:8 06550

Çankaya, Ankara / Turkey

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: Senk Ajans Reklam Matbaacılık San. ve Tic.Ltd.Şti.

Sanayi Mah. Sultan Selim Cad. Aybike Sk.No:22/-3, Kağıthane,

İstanbul, Türkiye Tel: +90 212 264 38 77

Printing Date / Basım Tarihi

15.02.2016

Cover Picture

Işıl Erdoğan Özünal was born in 1982, Turkey. She is currently working at

İstanbul University Cerrahpaşa Faculty of Medicine, Department of Internal

Medicine, Division of Hematology, İstanbul, Turkey.

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 Englishlanguage

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: 0.360

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 Diz 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 Yayinevi

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

Telephone : 0212 621 99 25

Fax : 0212 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.

A-III

TURKISH JOURNAL OF HEMATOLOGY

INSTRUCTIONS TO 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 kind of reviewing system.

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, 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 not exceed 2500 words. The word count for an abstract

should be 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/coi_disclose.pdf.

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, perspectives, 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 what

ever 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 (http://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

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

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

A-IV

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

parentheses. 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

and 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 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 posthepatitis

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 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

A-V

therel evant 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

Article length: Not exceed 200 words.

Authors can submit for consideration an illustration and photos that

is 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 figure or

table. No abstract, discussion or conclusion are 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 on 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. High-resolution 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 1 author.

Contributor’s Statement

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

manuscript should contain substantial contributions to idea and

design, acquisition of data, or 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 experimentation. Approval of

research protocols by the relevant ethics committee, in accordance

with international agreements (Helsinki Declaration of 1975, revised

2002 available at http://www.wma.net/e/policy/b3.htm, “Guide for

the Care and use of Laboratory Animals” www.nap.edu/catalog/5140.

html/), 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-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 re-publishing a manuscript in different languages.

Salamisation: To create more than one publication by dividing the

results of a study preternaturally.

We disapprove of such unethical practices as plagiarism, fabrication,

duplication, and salamisation, as well as efforts to influence the review

process with such practices as gifting authorship, inappropriate

acknowledgements, and references. Additionally, authors must respect

participant 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 those that are presented in an electronic environment are not

accepted pre-published work. Authors in such 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)

A-VI

We use iThenticate to screen all submissions for plagiarism before

publication.

Turkish Journal of Hematology uses plagiarism screening service to

verify the originality of content submitted 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, 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

copyright system. Corresponding authors can log in to the submission

system at any time to check the status of any co-author’s copyright

form. All accepted manuscripts become the permanent property of The

Turkish Journal of Hematology and may not be published elsewhere-in

whole or in part-without written permission.

Note: We cannot accept any copyright that has been altered, revised,

amended, or otherwise changed. Our original copyright form must

be used as is.

Units of Measurement

Measurements should be reported using the metric system, according

to the International System of Units (SI). Consult the SI Unit

Conversion Guide, New England Journal of Medicine Books, 1992.

An extensive list of conversion factors can be found at http://www.

unc.edu/~rowlett/units/scales/clinical_data.html. For more details, see

http://www.amamanualofstyle.com/oso/public/jama/si_conversion_

table.html. Example for CBC.

Hematology component SI units

RBC 6.7-11 x 10 12 /L

WBC 5.5-19.5 x10 9 /L

Hemoglobin 116-168 g/L

PCV 0.31-0.46 L/L

MCV 39-53 fL

MCHC 300-360 g/L

MCH 19.5-25 pg

Platelets 300-700 x 10 9 /L

Source: http://www.vetstream.com/felis/Corporate/993fhtm/ha-mat.htm

Abbreviations and Symbols

Use only standard abbreviations. Avoid abbreviations in the title and

abstract. The full term for an abbreviation should precede its first

use in the text, unless it is a standard abbreviation. All acronyms

used in the text should be expanded at first mention, followed by

the abbreviation in parentheses; thereafter the acronym only should

appear in the text. Acronyms may be used in the abstract if they occur

3 or more times therein, but must be reintroduced in the body of the

text. Generally, abbreviations should be limited to those defined in the

AMA Manual of Style, current edition. A list of each abbreviation (and

the corresponding full term) used in the manuscript must be provided

on the title page.

Online Manuscript Submission Process

The Turkish Journal of Hematology uses submission software powered

by ScholarOne Manuscripts. The website for submissions to The Turkish

Journal of Hematology is http://mc.manuscriptcentral.com/tjh. This

system is quick and convenient, both for authors and reviewers.

Setting up an account

New users to the submission site will need to register and enter their

account details before they can submit a manuscript. Log in, or click

the “Create Account” button if you are a first-time user. To create a

new account: After clicking the “Create Account” button, enter your

name and e-mail address, and then click the “Next” button. Your

e-mail address is very important. Enter your institution and address

information, as appropriate, and then click the “Next” Button. Enter

a user ID and password of your choice, select your area of expertise,

and then click the “Finish” button.

If you have an account, but have forgotten your log-in details, go to

“Password Help” on the journal’s online submission system and enter

A-VII

your e-mail address. The system will send you an automatic user ID

and a new temporary password.

Full instructions and support are available on the site, and a user ID

and password can be obtained during your first visit. Full support

for authors is provided. Each page has a “Get Help Now” icon that

connects directly to the online support system. Contact the journal

administrator with any questions about submitting your manuscript

to the journal (info@tjh.com.tr). For ScholarOne Manuscripts

customer support, click on the “Get Help Now” link on the top right

hand corner of every page on the site.

The Electronic Submission Process

Log in to your author center. Once you have logged in, click the

“Submit a Manuscript” link in the menu bar. Enter the appropriate

data and answer the questions. You may copy and paste directly from

your manuscript. Click the “Next” button on each screen to save your

work and advance to the next screen.

Upload Files

Click on the “Browse” button and locate the file on your computer.

Select the appropriate designation for each file in the drop-down

menu next to the “Browse” button. When you have selected all the

files you want to upload, click the “Upload Files” button. Review

your submission before sending to the journal. Click the “Submit”

button when you are finished reviewing. You can use ScholarOne

Manuscripts at any time to check the status of your submission. The

journal’s editorial office will inform you by e-mail once a decision has

been made. After your manuscript has been submitted, a checklist will

then be completed by the Editorial Assistant. The Editorial Assistant

will check that the manuscript contains all required components

and adheres to the author guidelines. Once the Editorial Assistant is

satisfied with the manuscript it will be forwarded to the Senior Editor,

who will assign an editor and reviewers.

The Review Process

Each manuscript submitted to The Turkish Journal of Hematology is

subject to an initial review by the editorial office in order to determine

if it is aligned with the journal’s aims and scope, and complies with

essential requirements. Manuscripts sent for peer review will be

assigned to one of the journal’s associate editors that has expertise

relevant to the manuscript’s content. All manuscripts are single-blind

peer reviewed. All accepted manuscripts are sent to a statistical and

English language editor before publishing. Once papers have been

reviewed, the reviewers’ comments are sent to the Editor, who will

then make a preliminary decision on the paper. At this stage, based on

the feedback from reviewers, manuscripts can be accepted, rejected, or

revisions can be recommended. Following initial peer-review, articles

judged worthy of further consideration often require revision. Revised

manuscripts generally must be received within 3 months of the date of

the initial decision. Extensions must be requested from the Associate

Editor at least 2 weeks before the 3-month revision deadline expires;

The Turkish Journal of Hematology will reject manuscripts that are

not received within the 3-month revision deadline. Manuscripts with

extensive revision recommendations will be sent for further review

(usually by the same reviewers) upon their re-submission. When a

manuscript is finally accepted for publication, the Technical Editor

undertakes a final edit and a marked-up copy will be e-mailed to the

corresponding author for review and to make any final adjustments.

Submission of Revised Papers

When revising a manuscript based on the reviewers’ and Editor’s

feedback, please insert all changed text in red. Please do not use

track changes, as this feature can make reading difficult. To submit

revised manuscripts, please log into your author center at ScholarOne

Manuscripts. Your manuscript will be stored under “Manuscripts with

Decisions”. Please click on the “Create a Revision” link located to the right

of the manuscript title. A revised manuscript number will be created for

you; you will then need to click on the “Continue Submission” button.

You will then be guided through a submission process very similar to

that for new manuscripts. You will be able to amend any details you

wish. At stage 6 (“File Upload”), please delete the file for your original

manuscript and upload the revised version. Additionally, please upload

an anonymous cover letter, preferably in table format, including a

point-by-point response to the reviews’ revision recommendations. You

will then need to review your paper as a PDF and click the “Submit”

button. Your revised manuscript will have the same ID number as the

original version, but with the addition of an R and a number at the end,

for example, TJH-2011-0001 for an original and TJH-2011-0001.R1,

indicating a first revision; subsequent revisions will end with R2, R3,

and so on. Please do not submit a revised manuscript as a new paper, as

revised manuscripts are processed differently. If you click on the “Create

a Revision” button and receive a message stating that the revision option

has expired, please contact the Editorial Assistant at info@tjh.com.tr to

reactivate the option.

English Language Editing

All manuscripts are professionally edited by an English language

editor prior to publication.

Online Early

The Turkish Journal of Hematology publishes abstracts of accepted

manuscripts online in advance of their publication in print. Once an

accepted manuscript has been edited, the authors have submitted

any final corrections, and all changes have been incorporated, the

manuscript will be published online. At that time the manuscript

will receive a Digital Object Identifier (DOI) number. Both forms

can be found at www.tjh.com.tr. Authors of accepted manuscripts

will receive electronic page proofs directly from the printer, and are

responsible for proofreading and checking the entire manuscript,

including tables, figures, and references. Page proofs must be returned

within 48 hours to avoid delays in publication.

A-VIII

CONTENTS

Review Article

1 The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome

Doruk Erkan, Jane E. Salmon

Research Articles

8 Deregulated Levels of the NF-κB1, NF-κB2, and Rel Genes in Ukrainian Patients with Leukemia and Lymphoma in the

Post-Chernobyl Period

Hakan Savlı, Ramis Ufuk Akkoyunlu, Naci Çine, Daniil F. Gluzman, Michael P. Zavelevich, Lilia M. Sklyarenko,

Stella V. Koval, Deniz Sünnetçi

15 Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin Expression

Tze Yan Lee, Logeswaran Muniandy, Lai Kuan Teh, Maha Abdullah, Elizabeth George, Jameela Sathar, Mei I Lai

21 The Feasibility of Magnetic Resonance Imaging for Quantification of Liver, Pancreas, Spleen, Vertebral Bone Marrow, and Renal

Cortex R2* and Proton Density Fat Fraction in Transfusion-Related Iron Overload

İlkay S. İdilman, Fatma Gümrük, Mithat Haliloğlu, Muşturay Karçaaltıncaba

28 Freezing of Apheresis Platelet Concentrates in 6% Dimethyl Sulfoxide: The First Preliminary Study in Turkey

Soner Yılmaz, Rıza Aytaç Çetinkaya, İbrahim Eker, Aytekin Ünlü, Metin Uyanık, Serkan Tapan, Ahmet Pekoğlu, Aysel Pekel,

Birgül Erkmen, Uğur Muşabak, Sebahattin Yılmaz, İsmail Yaşar Avcı, Ferit Avcu, Emin Kürekçi, Can Polat Eyigün

34 Is It Possible to Predict Pulmonary Complications and Mortality in Hematopoietic Stem Cell Transplantation Recipients from

Pre-Transplantation Exhaled Nitric Oxide Levels?

Nurdan Köktürk, Fatma Yıldırım, Müge Aydoğdu, Şahika Zeynep Akı, Zeynep Arzu Yeğin, Zübeyde Nur Özkurt, Elif Suyanı,

İpek Kıvılcım Oğuzülgen, Gülsan Türköz Sucak

41 A Quasi-Experimental Study Analyzing the Effectiveness of Portable High-Efficiency Particulate Absorption Filters in

Preventing Infections in Hematology Patients during Construction

Mehmet Özen, Gülden Yılmaz, Belgin Coşkun, Pervin Topçuoğlu, Bengi Öztürk, Mehmet Gündüz, Erden Atilla, Önder Arslan,

Muhit Özcan, Taner Demirer, Osman İlhan, Nahide Konuk, İsmail Balık, Günhan Gürman, Hamdi Akan

48 Comparison of the Efficiencies of Buffers Containing Ankaferd and Chitosan on Hemostasis in an Experimental Rat Model with

Femoral Artery Bleeding

Serkan Abacıoğlu, Kemal Aydın, Fatih Büyükçam, Ural Kaya, Bahattin Işık, Muhammed Evvah Karakılıç

Brief Reports

53 Antifungal Treatment in Stem Cell Transplantation Centers in Turkey

Hamdi Akan, Erden Atilla

56 Hemoglobin H Disease in Turkey: Experience from Eight Centers

Selma Ünal, Gönül Oktay, Can Acıpayam, Gül İlhan, Edip Gali, Tiraje Celkan, Ali Bay, Barış Malbora, Nejat Akar,

Yeşim Oymak, Tayfur Toptaş

A-IX

60 The Impact of Variant Philadelphia Chromosome Translocations on the Clinical Course of Chronic Myeloid Leukemia

Damla Eyüpoğlu, Süreyya Bozkurt, İbrahim Haznedaroğlu, Yahya Büyükaşık, Deniz Güven

Case Report

66 Secondary Neoplasms in Children with Hodgkin’s Lymphoma Receiving C-MOPP and Radiotherapy: Presentation of Four Cases

Sevgi Gözdaşoğlu, Ali Pamir, Emel Ünal, İsmail Haluk Gökçora, Ömer Uluoğlu, Koray Ceyhan, Haluk Deda, Erdoğan Işıkman,

Gülsan Yavuz, Nurdan Taçyıldız, Ayhan Çavdar

Letters to the Editor

71 First Observation of Hemoglobin G-Waimanalo and Hemoglobin Fontainebleau Cases in the Turkish Population

Duran Canatan, Türker Bilgen, Vildan Çiftçi, Gülsüm Yazıcı, Serpil Delibaş, İbrahim Keser

72 Serum Lipids in Turkish Patients with β-Thalassemia Major and β-Thalassemia Minor

Yasemin Işık Balcı, Şule Ünal, Fatma Gümrük

74 Extranodal Natural Killer/T-Cell Lymphoma: A Rare Nasal-Type Case

Esra Sarıbacak Can, Harika Okutan, Murat Albayrak, Ünsal Han

75 T-Cell Lymphoma Presenting with Auricular and Parotid Gland Involvement

Birgül Öneç, Alper Koç, Elif Nisa Ünlü, İlhan Ünlü, Hüseyin Yaman, Durdu Mehmet Köş

77 Immune Thrombocytopenia Resolved by Eltrombopag in a Carrier of Glucose-6-Phosphate Dehydrogenase Deficiency

Laura Scaramucci, Pasquale Niscola, Massimiliano Palombi, Andrea Tendas, Marco Giovannini, Paolo de Fabritiis

78 Wernicke’s Encephalopathy in an Acute Myeloid Leukemia Patient: A Case Study

Muhammet Maden, Gülsüm Pamuk, Yahya Çelik, Ercüment Ünlü

79 Kaleidoscopic Views in the Bone Marrow: Oxalate Crystals in a Patient Presenting with Bicytopenia

Yelda Dere, Simge Erbil, Murat Sezak, Başak Doğanavşargil, Mümtaz Yılmaz, Nazan Özsan, Mine Hekimgil

81 An Unexpected Innocent Complication Associated with Azacitidine Treatment of Myelodysplastic Syndrome: Erythema

Annulare Centrifugum

Esra Turan Erkek, Sevgi Kalayoğlu Beşışık

83 Thrombotic Microangiopathic Hemolytic Anemia without Evidence of Hemolytic Uremic Syndrome

Şinasi Özsoylu

Images in Hematology

84 Radiologic Image of a Child with Leukemia Who Developed Sepsis and Fulminant Thrombosis during Induction Therapy

Eda Ataseven, Ömer Özden, Şebnem Yılmaz Bengoa, Handan Güleryüz, Murat Duman, Hale Ören

86 Hereditary Elliptocytosis with Pyropoikilocytosis

Turan Bayhan, Şule Ünal, Fatma Gümrük

A-X

Advisory Board of This Issue (Mart 2016)

Ahmet Emre Eşkazan, Turkey

Ahmet Kürekçi, Turkey

Ali Bay, Turkey

Ali Ghasemi, Iran

Amir Steinberg, USA

Aytemiz Gürgey, Turkey

Balint Nagy, Hungary

Canan Vergin, Turkey

Cengiz Beyan, Turkey

Christopher Dandoy, USA

Emin Kaya, Turkey

Erol Atalay, Turkey

Fevzi Altuntaş, Turkey

Francesca Malvestiti, Italy

Füsun Özdemirkıran, Turkey

Gamal Abdul Hamid, Yemen

Gülden Yılmaz, Turkey

Gülsün Karasu, Turkey

İbrahim Haznedaroğlu, Turkey

İdil Yenicesu, Turkey

İlknur Kozanoğlu, Turkey

İnci Alacacıoğlu, Turkey

Keith R. McCrae, USA

Luke Curtis, USA

Mehmet Doğu, Turkey

Mehmet Gündüz, Turkey

Mehmet Kantar, Turkey

Mesut Ayer, Turkey

Muhit Özcan, Turkey

Murat Akova, Turkey

Murat İnanç, Turkey

Mustafa Pehlivan, Turkey

Mustafa Sünbül, Turkey

Müge Sayitoğlu, Turkey

Namık Özbek, Turkey

Nazan Sarper, Turkey

Nazlı Başak, Turkey

Nil Güler, Turkey

Rejin Kebudi, Turkey

Selami Koçak Toprak, Turkey

Selma Ünal, Turkey

Şule Ünal, Turkey

Sümeyye Ekmekçi, Turkey

Tahir Ali, Pakistan

Tezer Kutluk, Turkey

Türkan Patıroğlu, Turkey

Vahid Afshar-Kharghan, USA

Yusuf Baran, Turkey

Zeynep Karakaş, Turkey

REVIEW

DOI: 10.4274/tjh.2015.0197

Turk J Hematol 2016;33:1-7

The Role of Complement Inhibition in Thrombotic Angiopathies

and Antiphospholipid Syndrome

Trombotik Anjiyopati ve Antifosfolipid Sendromunda Kompleman İnhibisyonunun Rolü

Doruk Erkan, Jane E. Salmon

Hospital for Special Surgery, Weill Cornell Medicine, New York, United States

Abstract

Antiphospholipid syndrome (APS) is characterized by thrombosis

(arterial, venous, small vessel) and/or pregnancy morbidity occurring

in patients with persistently positive antiphospholipid antibodies

(aPL). Catastrophic APS is the most severe form of the disease,

characterized by multiple organ thromboses occurring in a short

period and commonly associated with thrombotic microangiopathy

(TMA). Similar to patients with complement regulatory gene mutations

developing TMA, increased complement activation on endothelial

cells plays a role in hypercoagulability in aPL-positive patients. In

mouse models of APS, activation of the complement is required and

interaction of complement (C) 5a with its receptor C5aR leads to

aPL-induced inflammation, placental insufficiency, and thrombosis.

Anti-C5 antibody and C5aR antagonist peptides prevent aPL-mediated

pregnancy loss and thrombosis in these experimental models. Clinical

studies of anti-C5 monoclonal antibody in aPL-positive patients are

limited to a small number of case reports. Ongoing and future clinical

studies of complement inhibitors will help determine the role of

complement inhibition in the management of aPL-positive patients.

Keywords: Antiphospholipid syndrome, Complement inhibition,

Eculizumab, Thrombotic angiopathy

Öz

Antifosfolipid sendromu (APS), ısrarcı antifosfolipid antikor (aPL)

pozitifliği olan hastalarda görülen tromboz (arteriyel, venöz, küçük

damar) ve/veya gebelik ile ilişkili morbidite ile karakterizedir.

Hastalığın en şiddetli formu olan katastrofik APS, kısa süre içerisinde

gelişen çoklu organ trombozları ile karakterizedir ve sıklıkla trombotik

mikroanjiyopati (TMA) ile ilişkilidir. TMA geliştiren kompleman

düzenleyici gen mutasyonları bulunan hastalarla benzer olarak, aPLpozitif

hastalardaki hiperkoagülopatide, endotel hücrelerinde artmış

kompleman aktivasyonunun rolü vardır. APS’nin fare modellerinde,

kompleman aktivasyonunun olması zorunludur ve kompleman (C) 5a

ile reseptörü C5aR’nin etkileşmesi aPL-ile uyarılmış yangı, plasenta

yetmezliği ve tromboza neden olur. Anti-C5 antikoru ve C5aR

antagonisti olan peptidler bu deneysel modellerde, aPL-aracılı gebelik

kaybı ve trombozu önler. aPL-pozitif hastalarda anti-C5 monoklonal

antikorun klinik kullanımı az sayıdaki olgu sunumları ile sınırlıdır.

Halen devam etmekte olan ve gelecekte yapılacak klinik çalışmalar,

aPL-pozitif hastaların yönetiminde kompleman inhibitörlerinin rolünü

belirlemede yardımcı olacaktır.

Anahtar Sözcükler: Antifosfolipid sendromu, Komplaman inhibisyonu,

Eculizumab, Trombotik anjiyopati

Introduction

Antiphospholipid syndrome (APS) is characterized by thrombosis

(arterial, venous, small vessel) and/or pregnancy morbidity occurring

in patients with persistently positive antiphospholipid antibodies (aPL)

[1]. The current treatment in APS focuses on final thrombosis rather

than the initial aPL-induced prothrombotic and proinflammatory

phenotypes. In parallel to our increased understanding of the mediators

and mechanisms of the aPL-induced clinical events, the blockade of

early pathogenic effects of aPL on target cells (monocytes, endothelial

cells, or platelets) has been increasingly investigated.

The proposed mechanism of aPL-mediated thrombosis is

the binding of aPL to endothelial cells [via β 2 -glycoprotein-I

(β 2 GPI)] inducing a procoagulant state through different

mechanisms including the expression of adhesion molecules

and tissue factor (a physiologic initiator of coagulation and

thrombin formation), and complement activation. In addition,

products of complement activation, complement 3 (C3), C5a,

and membrane attack complex (MAC), are potent mediators of

platelet and endothelial cell activation; thus, the complement

system is likely a critical step in the pathogenesis of APS [2].

Eculizumab, a humanized monoclonal antibody directed against

C5, is approved for paroxysmal nocturnal hemoglobinuria

(PNH) and atypical hemolytic uremic syndrome (aHUS) [3,4].

Given several recent case reports describing positive outcomes

of severely ill aPL-positive patients treated with eculizumab,

Address for Correspondence/Yazışma Adresi: Doruk ERKAN, M.D.,

Hospital for Special Surgery, Weill Cornell Medicine, New York, United States

Phone : +90 212 774 22 91

E-mail : erkand@hss.edu

Received/Geliş tarihi: May 13, 2015

Accepted/Kabul tarihi: September 28, 2015

1

Erkan D and Salmon JE: The Role of Complement Inhibition in Thrombotic Angiopathies and Antiphospholipid Syndrome


the purpose of this review is to discuss the importance of

the complement system in the pathogenesis of APS, and the

potential role of complement inhibition to prevent organ

damage in aPL-positive patients.

Complement System

The complement system, composed of 30 proteins, protects

the host against infections and initiates inflammation to kill

microbes, remove dying cells, and dispose of immune complexes.

The system is activated in a rapid fashion to opsonize or lyse

a bacterium, while simultaneously triggering the release of

proinflammatory and chemotactic peptides. The complement

cascade can be triggered through 3 pathways: 1) the classical

pathway, initiated by multivalent binding of the Fc fragments

of antibody binding to the C1 complex; 2) the lectin pathway,

binding specific sugars on a microbe to mannose binding

lectin-associated proteases; and 3) the alternative pathway,

spontaneous low-grade cleavage of C3 in plasma (Figure 1) [5,6].

These 3 pathways converge to generate C3 convertases, which

cleave C3 into C3a and C3b. C3a is an anaphylatoxin that

recruits and activates leukocyte effectors; C3b tags pathogens

and immune complexes for opsonization. C3b leads to the

assembly of C5 convertase and subsequent cleavage of C5

into C5a and C5b. C5a is a potent chemotactic molecule

that recruits and stimulates leukocytes and endothelial cells,

triggering release of cytokines/chemokines and the expression

of adhesion molecules. Binding of C5b to cell surface assembles

C5b-9 MAC, which inserts itself into membranes, damages cells,

and activates proinflammatory pathways [5,6]. Furthermore,

complement activation products contribute to thrombosis by

augmenting the inflammatory responses of leukocytes and the

endothelium, which in turn potentiate coagulation [6].

Factor B, factor D, and properdin contribute to the generation

of C3b directly through the alternative pathway or through the

amplification loop where C3b is formed. The production of C3b,

triggered from engagement of the classical or lectin pathways,

is augmented through the alternative pathway amplification [7].

Because of its potency, complement activation is regulated

at each step. The major regulators of the alternate pathway

amplification loop are plasma proteins complement factor H

(CFH) and complement factor I (CFI), and a membrane cofactor

protein (MCP) (CD46) [6].

Thrombotic Microangiopathies and Paroxysmal

Nocturnal Hemoglobinuria

Definitions

Thrombotic microangiopathy (TMA) is defined as thrombosis in

arterioles and capillaries, which is commonly associated with

thrombocytopenia, microangiopathic hemolytic anemia, and/

or kidney failure [8]. Diseases associated with TMA are either

hereditary or acquired; selected TMA syndromes are described

in Table 1 [9]. Antiphospholipid antibody-positive patients,

especially those with catastrophic APS [10], can develop TMA

with or without medium-to-large vessel thrombosis (further

discussed below).

Figure 1. Human complement system. Three pathways are activated

by immune complexes and apoptotic cells (classical); by microbes

and stressors (lectin); and spontaneously (alternative). The effect of

complement: clearance of apoptotic cells, opsonization of pathogens

and immune complexes for phagocytosis, release of anaphylatoxins

and lysis (shown in italics), and activation of effector cells that express

receptors for C5a and/or C3a (neutrophils, monocytes, and platelets)

are shown on the left. Complement inhibitors are indicated in red.

Soluble inhibitors are factor I (FI), C4b-binding protein (C4BP), factor

H (FH), and FH-like protein (FHL-1). Membrane-bound inhibitors

include MCP (CD46), DAF (CD55), and CD59. Reprinted from Placenta

2010;31:561-567, Lynch AM, Salmon JE, Dysregulated complement

activation as a common pathway of injury in preeclampsia and

other pregnancy complications. Copyright (2015) with permission

from Elsevier.

Table 1. Hereditary and acquired thrombotic

microangiopathies (adapted from George and Nester [9]).

Hereditary:

Thrombotic thrombocytopenic purpura (TTP) (ADAMTS13

deficiency-mediated TMA)

Complement-mediated TMA

Acquired (Primary):

Thrombotic thrombocytopenic purpura (TTP) (ADAMTS13

deficiency-mediated TMA)

Complement-mediated TMA

Shiga-toxin-mediated TMA (hemolytic uremic syndrome)

Acquired (Secondary):

Infection

Cancer

Preeclampsia

HELLP (hemolysis, elevated liver enzymes, and low platelets)

Syndrome

Autoimmune Disorders (e.g., Lupus, Systemic Sclerosis,

Antiphospholipid Syndrome)

Hematopoietic Stem Cell or Organ Transplantation

2



Thrombotic thrombocytopenic purpura (TTP), which is

hereditary (ADAMTS13 mutations) or acquired (antibodies

against ADAMTS13), can present with a wide spectrum of

manifestations including microangiopathic hemolytic anemia,

thrombocytopenia, neurologic manifestations, gastrointestinal

symptoms, purpura, and/or renal disease [9].

Hereditary [regulatory (CFH, CFI, or CD46) or effector

(complement factor B or C3) gene mutations] or primary

acquired (antibodies against factor H) complement-mediated

TMA is due to the uncontrolled activation of the alternative

pathway of the complement resulting in acute kidney injury

and hypertension. These patients were previously classified as

having “aHUS”, clinically defined as thrombocytopenia and

microangiopathic hemolysis (with ADAMTS13 activity of >5%

and a negative stool test for Shiga-toxin-producing infection)

and one of the following: neurological symptoms, renal

impairment, or gastrointestinal symptoms [9].

Hemolytic uremic syndrome (HUS) is a TMA featuring the

triad of hemolytic anemia, thrombocytopenia, and acute renal

impairment, mainly caused by Shiga-toxin-producing Escherichia

coli. It is often preceded by bloody diarrhea (although one-third

of the patients do not have bloody diarrhea), accounts for 90%

of HUS cases in childhood, and does not relapse, and renal

function recovers completely in >90% of cases. Traditionally,

aHUS has been distinguished from HUS by the absence of

diarrhea secondary to an Escherichia coli infection and a more

severe clinical course [6]; however, rarely aHUS patients can

present with gastrointestinal symptoms.

Several other conditions can be associated with secondary TMA

(Table 1). Disseminated intravascular coagulation is associated

with intravascular activation and consumption of the different

coagulation system components depending on the underlying

cause. Preeclampsia is the onset of hypertension and proteinuria

after 20 weeks of gestation; disease manifestations range from

mild blood pressure elevations to severe hypertension, the HELLP

syndrome (hemolysis, elevated liver enzymes, and low platelets),

and eclampsia (seizures).

PNH is a disease of hematopoietic stem cells resulting in

intravascular hemolysis, hemoglobinuria, and thromboembolism

due to the deficiency of two proteins (CD55-decay accelerating

factor; CD59-membrane inhibitor of reactive lysis) that inhibit

the activation and cytolytic functions of the complement

system [11].

Complement System in Thrombotic Microangiopathies and

Paroxysmal Nocturnal Hemoglobinuria

Mouse studies demonstrate that the absence of complement

regulatory proteins is associated with TMA and pregnancy

loss [12]. As discussed above, mutations in complement

regulatory proteins result in aHUS and PNH, both associated

with microvascular endothelial cell activation, cell injury, and

thrombosis [13].

Furthermore, a relationship exists between activation of the

complement system and development of an imbalance in

angiogenic factors. Mouse models show that C5a induces release

of antiangiogenic factors [14] and complement deletion prevents

placental insufficiency in mouse models of preeclampsia [15].

Excess production of an antiangiogenic factor by the placenta

and inflammatory cells leads to impaired placental development

and placental dysfunction [5,14,16]. In normal pregnancies,

excessive complement activation is prevented by complement

regulatory proteins that are highly expressed on trophoblast

membranes (CD55 and CD59) and circulating complement

regulatory proteins (CFH, CFI, and C4 binding protein).

In preeclampsia, complement activation products (C4d and

C5b-9) are detected on trophoblasts, and in vivo hypoxia

enhances MAC deposition on villous trophoblasts [17].

Complement Inhibition in Thrombotic Microangiopathies and

Paroxysmal Nocturnal Hemoglobinuria

Eculizumab is a recombinant humanized monoclonal antibody

that binds to the terminal complement protein C5, inhibits its

cleavage into C5a and C5b, and prevents the generation of

MAC. In PNH patients, eculizumab reduces the frequency of

hemolysis, hemoglobinuria, transfusion, and thrombosis [18].

In aHUS patients, eculizumab inhibits complement-mediated

TMA (resolving thrombocytopenia and TMA) and improves

renal transplantation outcomes by allowing plasma exchangedependent

patients to stop this treatment [4]. Eculizumab

has been also used off-label in TTP patients refractory to

plasmapheresis [19].

Antiphospholipid Syndrome

Definitions

Antiphospholipid antibodie are a heterogeneous family of

autoantibodies directed against phospholipid-binding plasma

proteins, most commonly β 2 GPI. The predominant hypothesis

regarding the origin of aPL states that an incidental exposure to

environmental agents with β 2 GPI-like peptides induces aPL in

susceptible individuals (molecular mimicry) [20].

A positive aPL test is not always “clinically significant”; transient

aPL positivity is not uncommon, especially during infections

[21]. Documentation of aPL persistence is therefore important.

High titers of anticardiolipin antibodies (aCL) and aβ 2 GPI as well

as IgG/M isotypes are more concerning than lower titers and

IgA isotypes. Whereas a positive lupus anticoagulant (LA) test

is a better predictor of thrombosis than aCL and aβ 2 GPI, falsepositive

and false-negative LA tests can occur for patients on

anticoagulation [22]. Documentation of a positive LA test requires

4 criteria according to International Society of Thrombosis and

3



Haemostasis guidelines [23]: 1) demonstration of a prolonged

phospholipid-dependent coagulation screening test, such as

activated partial thromboplastin time or dilute Russell viper

venom time; 2) failure to correct the prolonged screening test by

mixing the patient’s plasma with normal platelet-poor plasma,

demonstrating the presence of an inhibitor; 3) shortening or

correction of the prolonged screening test by the addition of

excess phospholipid, demonstrating phospholipid dependency;

and 4) exclusion of other inhibitors.

Our definition of a “clinically significant” aPL profile is: 1) LA

test positivity in accordance with the above guidelines [23]; 2)

aCL IgG/M of ≥40 U [1]; and/or 3) aβ 2 GPI IgG/M of ≥40 U tested

twice at least 12 weeks apart. Clinical judgment is required to

determine thrombosis risk when aPL results are equivocal, e.g.,

LA test not measurable because the patient is anticoagulated,

aCL or aβ 2 GPI IgG/M titers are between 20 and 39 U, and/or aCL

or aβ 2 GPI IgA is the only positive aPL test.

In patients with clinically significant aPL profiles, clinical

manifestations may be varied: 1) asymptomatic aPL positivity

(no history of thrombosis or pregnancy morbidity); 2)

non-criteria manifestations of aPL, e.g., livedo reticularis,

thrombocytopenia, hemolytic anemia, cardiac valve disease, aPLassociated

nephropathy, or cognitive dysfunction; 3) pregnancy

morbidity (recurrent embryonic or fetal loss, preeclampsia,

placental insufficiency, and fetal growth restriction); 4) venous,

arterial, or small vessel thrombosis (stroke is the most common

presentation of arterial thrombosis; deep vein thrombosis is the

most common venous manifestation); and 5) catastrophic APS

(multiple organ thrombosis developing in a short period), which

is usually associated with TMA [1,10].

Mechanisms of Antiphospholipid Antibody-Mediated Injury

Antiphospholipid antibodies induce thrombosis and placental

injury through multiple mechanisms. The process begins

with activation or apoptosis of platelets, endothelial cells, or

trophoblasts, during which phosphatidylserine (a negatively

charged phospholipid) migrates from the inner to the normally

electrically neutral outer cell membrane. β 2 GPI, which potentially

exists in the circulation in a circular form [24], then binds to PS

independently of aβ 2 GPI (via β 2 GPI surface receptors such as

apoER2′, annexin A2, or a toll-like receptor). After this binding,

the circular β 2 GPI opens up to expose domain I and aPL binds

to β 2 GPI, directly stimulating cells through surface receptors.

Antiphospholipid antibodies can also stimulate cells indirectly

by activating the classic complement pathway. The generation

of C5a induces expression of adhesion molecules and tissue

factor and activation of monocytes, polymorphonuclear cells,

and platelets, and it triggers the release of proinflammatory

mediators (e.g., tumor necrosis factor, vascular endothelial

growth factor receptor-1) and initiation of the proadhesive

and prothrombotic state. Thus, multiple pathways are engaged

by aPL binding to the surface. Crosslinking apoER2′ receptors

antagonize endothelial nitric oxide synthase, blocking its

phosphorylation and leading to increased leukocyte adhesion

and thrombosis. Both nuclear factor κB and p38 mitogenactivated

protein kinase play a role in the intracellular signaling

cascade. Antiphospholipid antibodies also downregulate the

expression of trophoblast signal transducer and activator of

transcription 5 (STAT5), reducing the endometrial stromal cell

production of prolactin (PRL) and insulin growth factor binding

protein-1 (IGFBP-1) [25].

Patients with aPL may have thrombocytopenia, and its

mechanisms include: 1) promoting platelet activation and

aggregation; 2) antiplatelet antibodies directed against platelet

membrane glycoproteins such as aGPIIb/IIIa [26]; and 3) platelet

destruction as seen in TMA, including catastrophic APS.

Thrombotic microangiopathy, which is common in catastrophic

APS and/or transplant rejection, is the most characteristic lesion

of APS nephropathy; the pathologic changes may be similar to

other TMAs, e.g., HUS, TTP, and preeclampsia. In addition, aPLnephropathy

patients can develop chronic cortical ischemia/

infarction (arteriosclerosis, arteriolosclerosis, arterial fibrous

intimal hyperplasia, glomerular ischemia, interstitial fibrosis,

tubular thyroidization, tubular atrophy, and/or organized

thrombi with/without recanalization) [1,27,28]. Recently, it has

been shown that in APS patients these vascular renal lesions

are associated with the activation of the mammalian target of

rapamycin pathway [29]. Given the tissue damage prominent in

renal TMA, it is likely that inflammatory damage by recruited

leukocytes and vascular cell activation are amplified by

complement activation products generated as a consequence of

the alternative pathway.

Complement System in Antiphospholipid Antibody-Mediated

Injury

Passive transfer of human aPL results in endothelial cell activation

and pregnancy loss in animal models [30,31]. Endothelial cell

activation correlates with a prothrombotic phenotype in vitro

and enhances thrombus formation in vivo [30,32]. Complement

activation, specifically C5, is a necessary intermediary event in

both thrombosis and pregnancy complications associated with

aPL in rodent models [33].

Complement activation initiates and amplifies the cellular

features characteristic of APS: endothelial cell activation,

monocyte tissue factor expression, and platelet aggregation.

Generation of C5a contributes to vascular inflammation

[34,35]. Complement 5a interacts with its receptor, C5aR, to

promote recruitment and activation of neutrophils (PMNs) and

monocytes, and activation of EC [29]. C5a-C5aR ligation also:

1) upregulates neutrophil-derived TF expression, thought to be

one mechanism of aPL-mediated coagulation and disseminated

4



thrombosis [36]; 2) leads to trophoblast injury and angiogenic

factor imbalance in aPL-induced fetal injury [37]; and 3)

produces lesions such as those seen in TMA in mouse models.

Mice deficient in complement components C3, C5, C6, or C5a

receptors are resistant to aPL-induced enhanced thrombophilia

and endothelial cell activation [38]. Treatment with anti-C5

monoclonal antibody or C5aR antagonist peptides attenuates

thrombosis in mouse models of APS [31]. In mouse models of

surgically induced thrombus formation, complement activation

plays an important role in the increased thrombosis and

adhesion of leukocytes to endothelial cells caused by treatment

with aPL. Heparin has anticomplement effects, as well as acting

as an anticoagulant, which may explain some of its efficacy in

APS [39].

In addition, mouse models of aPL-induced pregnancy loss and

growth restriction show that C4, factor B, C3, C5, and C5aR are

required for placental injury [30]. Complement deposition is

present in human placenta from patients with APS [40]. Patients

with aPL, with or without clinical manifestation of APS, show

elevated circulating levels of Bb and C3a fragments [41]; the

fact that the complement contributes to placental injury is

suggested by the evidence for C4d on trophoblasts in patients

with APS [42]. In a recent study, APS patients were found to

have elevated C3a levels in plasma, but there was no correlation

with the development of thrombosis [43].

Complement Inhibition in Antiphospholipid Antibody-Positive

Patients

Clinical studies of anti-C5 monoclonal antibody (eculizumab)

in aPL-positive patients are limited to a small number of case

reports.

The first report was published in 2010 by Lonze et al. describing

improvement of TMA after kidney transplant in an eculizumabtreated

patient with a history of catastrophic aPL syndrome [44].

Of note, the patient also received systemic anticoagulation and

standard immunosuppression. Velik-Salchner et al. questioned

the effectiveness of eculizumab in this patient by drawing

attention to the ability of heparin to inhibit complement in

APS mouse models and the possibility of TMA in aPL-positive

patients that does not involve complement activation [45].

In 2011, Hadaya et al. reported an aPL-positive systemic lupus

erythematosus patient who underwent a living-related kidney

transplantation, which was complicated by recurrent thrombotic

microangiopathy [46]. Despite the standard posttransplantation

regimen and daily plasma exchange, renal function did not

improve. The patient received 5 weekly infusions of eculizumab,

and the renal function normalized after 6 months. Darnige et

al. studied aPL titers in 20 PNH patients receiving eculizumab

[47]. Only 3 patients had preinfusion low-titer aCL or aβ 2 GPI

(negative lupus anticoagulant); there was no significant change

in the titers after 11 weeks of treatment.

In 2012, Shapira et al. reported a catastrophic APS

patient resistant to anticoagulation, immunosuppression,

plasmapheresis, and rituximab; eculizumab successfully blocked

complement activity, aborted progressive thrombosis, and

reversed thrombocytopenia [48].

In 2013, Canaud et al. reported 3 aPL-nephropathy patients treated

with eculizumab following TMA after kidney transplantation

due to aPL-nephropathy recurrence [49]. Based on pre- and

posttransplantation biopsies, the investigators showed that:

1) eculizumab results in remission in plasmapheresis-resistant

thrombotic angiopathy related to aPL-nephropathy recurrence;

2) persistent C5b-9 deposition is commonly found in allografts

developing posttransplant thrombotic microangiopathy; and 3)

chronic vascular changes related to aPL may not be related to

complement activation. In the same year, another case report

[50] and personal communications during the 14th International

Congress on aPL described catastrophic APS patients who failed

to respond to eculizumab [51].

In 2014, several case reports or series described the outcomes

of eculizumab-treated aPL-positive patients: 1) Bakhtar et al.

described a lupus and APS patient who developed biopsy-proven

TMA, thrombocytopenia, and hemolysis 3 years after livingrelated

kidney transplantation; after 7 months of eculizumab,

there was no evidence of TMA on biopsy and both hemoglobin

and platelets were normal [52]; 2) Lonze et al. reported 3 APS

patients (2 with catastrophic APS, and including the follow-up

information of the first eculizumab-receiving patient reported

in 2010) who were treated with anticoagulation and eculizumab

prior to and following live donor renal transplantation (2 also

received plasmapheresis); after a follow-up ranging from 4

months to 4 years, all patients had functioning renal allografts

[53]; 3) Strakhan et al. reported another catastrophic APS patient

(multiple strokes, non-ST elevation myocardial infarction, endstage

renal disease due to TMA, intraretinal hemorrhage, and

thrombocytopenia) who had no response to corticosteroids

and plasma exchange (no heparin during the acute period); the

patient’s condition stabilized after eculizumab [54]; 4) Zapantis

et al. reported 3 APS patients with recurrent thrombosis and

thrombocytopenia unresponsive to conventional therapy with

significant improvement of thrombocytopenia after eculizumab

administration (personal communication) [55].

Given the above reports, complement inhibition may have a role

as an adjuvant or main therapy for APS patients refractory to

anticoagulation; however, publication bias is a concern as well as

the lack of systematic clinical studies. Thus, more mechanistic and

clinical studies are needed before eculizumab can be recommended

[50]. Clinicians should keep in mind that the infection risk of

eculizumab is mainly with encapsulated organisms, specifically

5



meningococcal. Patients must be immunized against Neisseria

meningitidis before treatment with eculizumab.

Ongoing Observational and Interventional Complement-Related

Clinical Studies in Antiphospholipid Antibody-Positive Patients

Potential novel approaches to target terminal complement

activation include C5aR antagonists (antibodies or peptides)

and soluble and targeted complement regulatory proteins.

The PROMISE Study (Predictors of pRegnancy Outcome:

bioMarkers In APS and Systemic lupus Erythematosus)

(clinicaltrials.gov#: NCT00198068), a prospective, multicenter

observational study, aims to translate findings in mice to

humans and evaluate the role of complement in lupus and

aPL-associated pregnancy complications. The study, which has

recruited over 700 patients as of December 2014, is ongoing

and will test the hypotheses that classical, alternative, and

terminal complement pathway activation and dysregulation

of angiogenic factors will be detected in the circulation of

patients destined for pregnancy complications. The PROMISE

Study has the potential to identify new biomarkers for adverse

pregnancy outcomes that in addition to being good predictors

of these outcomes are also part of the mechanistic process of

these pregnancy complications. In the future, it may be possible

to identify those patients in whom complement inhibitors are

likely to prevent or modify the inflammatory-related sequelae

associated with adverse pregnancy outcomes.

One open-label interventional phase II prevention trial

(clinicaltrials.gov#: NCT01029587) is investigating whether

blocking the complement cascade with eculizumab in 10

patients with a prior history of catastrophic APS who are

undergoing kidney transplant will lead to increased transplant

success. Three patients included in the protocol have already

been reported [44,51] and the estimated completion date was

August 2015.

Another open-label multicenter international phase IIa

treatment trial (clinicaltrials.gov#: NCT02128269) is evaluating

the safety and tolerability of an intravenous C5a inhibitor in

persistently aPL-positive patients with at least one of the

following non-criteria manifestations of APS: aPL-nephropathy,

skin ulcers, and/or thrombocytopenia.

Conclusion

Animal and human studies have confirmed the relevance

of complement inhibition in many inflammatory and

microthrombotic diseases including APS. Thus, complement

inhibition may have a role for APS patients refractory to

anticoagulation; however, more clinical data are needed.

Future mechanistic and clinical studies of eculizumab and

other complement inhibitors will be necessary to individualize

treatment. We hope that the results from the ongoing studies

will be available for further discussion at the 15th International

Congress on aPL (İstanbul, Turkey) (www.apsistanbul2016.org).

Authorship Contributions

Concept: Doruk Erkan, Jane E. Salmon, Design: Doruk Erkan, Jane

E. Salmon, Data Collection or Processing: Doruk Erkan, Jane E.

Salmon, Analysis or Interpretation: Doruk Erkan, Jane E. Salmon,

Literature Search: Doruk Erkan, Jane E. Salmon, Writing: Doruk

Erkan, Jane E. Salmon.

Conflict of Interest: Doruk Erkan and Jane Salmon: Alexion

(Clinical Trial Investigator, Advisory Board)

References

1. Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, Derksen

RH, DE Groot PG, Koike T, Meroni PL, Reber G, Shoenfeld Y, Tincani A,

Vlachoyiannopoulos PG, Krilis SA. International consensus statement on an

update of the classification criteria for definite antiphospholipid syndrome

(APS). J Thromb Haemost 2006;4:295-306.

2. Girardi G, Redecha P, Salmon JE. Heparin prevents antiphospholipid

antibody-induced fetal loss by inhibiting complement activation. Nat Med

2004;10:1222-1226.

3. Hillmen P, Young NS, Schubert J, Brodsky RA, Socié G, Muus P, Röth A, Szer

J, Elebute MO, Nakamura R, Browne P, Risitano AM, Hill A, Schrezenmeier

H, Fu CL, Maciejewski J, Rollins SA, Mojcik CF, Rother RP, Luzzatto L. The

complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria.

N Engl J Med 2006;355:1233-1243.

4. Legendre CM, Licht C, Muus P, Greenbaum LA, Babu S, Bedrosian C,

Bingham C, Cohen DJ, Delmas Y, Douglas K, Eitner F, Feldkamp T, Fouque D,

Furman RR, Gaber O, Herthelius M, Hourmant M, Karpman D, Lebranchu Y,

Mariat C, Menne J, Moulin B, Nürnberger J, Ogawa M, Remuzzi G, Richard

T, Sberro-Soussan R, Severino B, Sheerin NS, Trivelli A, Zimmerhackl LB,

Goodship T, Loirat C. Terminal complement inhibitor eculizumab in atypical

hemolytic-uremic syndrome. N Engl J Med 2013;368:2169-2181.

5. Lynch AM, Salmon JE. Dysregulated complement activation as a common

pathway of injury in preeclampsia and other pregnancy complications.

Placenta 2010;31:561-567.

6. Java A, Atkinson J, Salmon J. Defective complement inhibitory function

predisposes to renal disease. Annu Rev Med 2013;64:307-324.

7. Holers VM. The spectrum of complement alternative pathway-mediated

diseases. Immunol Rev 2008;223:300-316.

8. Benz K, Amann K. Thrombotic microangiopathy: new insights. Curr Opin

Nephrol Hypertens 2010;19:242-247.

9. George JN, Nester CM. Syndromes of thrombotic microangiopathy. N Engl

J Med 2014;371:1847-1848.

10. Aguiar CL, Erkan D. Catastrophic antiphospholipid syndrome: how to

diagnose a rare but highly fatal disease. Ther Adv Musculoskeletal Dis

2013;5:305-314.

11. Parker JC. Paroxysmal nocturnal hemoglobinuria. Curr Opin Hematol

2012;19:141-148.

12. Xu C, Mao D, Holers VM, Palanca B, Cheng AM, Molina H. A critical role

for murine complement regulator crry in fetomaternal tolerance. Science

2000;287:498-501.

13. Hillmen P, Young NS, Schubert J, Brodsky RA, Socié G, Muus P, Röth A, Szer

J, Elebute MO, Nakamura R, Browne P, Risitano AM, Hill A, Schrezenmeier

H, Fu CL, Maciejewski J, Rollins SA, Mojcik CF, Rother RP, Luzzatto L. The

complement inhibitor eculizumab in paroxysmal nocturnal hemoglobinuria.

N Engl J Med 2006;355:1233-1243.

14. Girardi G, Yarilin D, Thurman JM, Holers VM, Salmon JE. Complement

activation induces dysregulation of angiogenic factors and causes fetal

rejection and growth restriction. J Exp Med 2006;203:2165-2175.

15. Gelber SE, Brent E, Redecha P, Perino G, Tomlinson S, Davisson RL, Salmon

JE. Prevention of defective placentation and pregnancy loss by blocking

innate immune pathways in a syngeneic model of placental insufficiency. J

Immunol 2015;195:1129-1138.

6



16. Lynch AM, Murphy JR, Byers T, Gibbs RS, Neville MC, Giclas PC, Salmon

JE, Holers VM. Alternative complement pathway activation fragment

Bb in early pregnancy as a predictor of preeclampsia. Am J Obstet Gynecol

2008;198:385.1-9.

17. Rampersad R, Barton A, Sadovsky Y, Nelson DM. The C5b-9 membrane attack

complex of complement activation localizes to villous trophoblast injury in vivo

and modulates human trophoblast function in vitro. Placenta 2008;29:855-861.

18. Hillmen P, Hall C, Marsh JC, Elebute M, Bombara MP, Petro BE, Cullen MJ, Richards

SJ, Rollins SA, Mojcik CF, Rother RP. Effect of eculizumab on hemolysis and

transfusion requirements in patients with paroxysmal nocturnal hemoglobinuria.

N Engl J Med 2004;350:552-559.

19. Tsai E, Chapin J, Laurence JC, Tsai HM. Use of eculizumab in the treatment of a

case of refractory, ADAMTS13-deficient thrombotic thrombocytopenic purpura:

additional data and clinical follow-up. Br J Haematol 2013;162:558-559.

20. Shoenfeld Y. Etiology and pathogenetic mechanisms of the antiphospholipid

syndrome unraveled. Trends Immunol 2003;24:2-4.

21. Avcin T, Toplak N. Antiphospholipid antibodies in response to infection. Curr

Rheumatol Rep 2007;9:212-218.

22. Galli M, Luciani D, Bertolini G, Barbui T. Lupus anticoagulants are stronger risk

factors for thrombosis than anticardiolipin antibodies in the antiphospholipid

syndrome: a systematic review of the literature. Blood 2003;101:1827-1832.

23. Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, De Groot PG; Subcommittee

on Lupus Anticoagulant/Antiphospholipid Antibody of the Scientific and

Standardisation Committee of the International Society on Thrombosis and

Haemostasis. Update of the guidelines for lupus anticoagulant detection.

Subcommittee on Lupus Anticoagulant/Antiphospholipid Antibody of the

Scientific and Standardisation Committee of the International Society on

Thrombosis and Haemostasis. J Thromb Haemost 2009;7:1737-1740.

24. Giannakopoulos B, Krilis SA. The pathogenesis of the antiphospholipid syndrome.

N Engl J Med 2013;368:1033-1044.

25. Erkan D, Salmon J, Lockshin MD. Antiphospholipid syndrome. In: Firestein GS, Budd

RC, Gabriel SE, McInnes IB, Odell JR (eds). Kelley’s Textbook of Rheumatology, 9th

ed. Philadelphia, Elsevier Saunders, 2013.

26. Alpert D, Mandl LA, Erkan D, Yin W, Peersche EI, Salmon JE. Anti-heparin platelet

factor 4 antibodies in systemic lupus erythematosus are associated with IgM

antiphospholipid antibodies and the antiphospholipid syndrome. Ann Rheum Dis

2008;67:395-401.

27. D’Cruz D. Renal manifestations of the antiphospholipid syndrome. Curr Rheumatol

Rep 2009;11:52-60.

28. Gigante A, Gasperini ML, Cianci R, Barbano B, Giannakakis K, Di Donato D, Fuiano

G, Amoroso A. Antiphospholipid antibodies and renal involvement. Am J Nephrol

2009;30:405-412.

29. Canaud G, Bienaimé F, Tabarin F, Bataillon G, Seilhean D, Noël LH, Dragon-Durey

MA, Snanoudj R, Friedlander G, Halbwachs-Mecarelli L, Legendre C, Terzi F.

Inhibition of the mTORC pathway in the antiphospholipid syndrome. N Engl J Med

2014;371:303-312.

30. Girardi G, Berman J, Redecha P, Spruce L, Thurman JM, Kraus D, Hollmann TJ,

Casali P, Caroll MC, Wetsel RA, Lambris JD, Holers VM, Salmon JE. Complement C5a

receptors and neutrophils mediate fetal injury in the antiphospholipid syndrome. J

Clin Invest 2003;112:1644-1654.

31. Pierangeli SS, Colden-Stanfield M, Liu X, Barker JH, Anderson GL, Harris EN.

Antiphospholipid antibodies from antiphospholipid syndrome patients activate

endothelial cells in vitro and in vivo. Circulation 1999;99:1997-2002.

32. Simantov R, LaSala JM, Lo SK, Gharavi AE, Sammaritano LR, Salmon JE, Silverstein

RL. Activation of cultured vascular endothelial cells by antiphospholipid antibodies.

J Clin Invest 1995;96:2211-2219.

33. Bulla R, Bossi F, Fischetti F, De Seta F, Tedesco F. The complement system at the

fetomaternal interface. Chem Immunol Allergy 2005;89:149-157.

34. Giannakopoulos B, Passam F, Rahgozar S, Krilis SA. Current concepts on the

pathogenesis of the antiphospholipid syndrome. Blood 2007;109:422-430.

35. Peerschke EI, Yin W, Ghebrehiwet B. Complement activation on platelets:

implications for vascular inflammation and thrombosis. Mol Immunol

2010;47:2170-2175.

36. Ritis K, Doumas M, Mastellos D, Micheli A, Giaglis S, Magotti P, Rafail S, Kartalis G,

Sideras P, Lambris JD. A novel C5a receptor-tissue factor crosstalk in neutrophils

links innate immunity to coagulation pathways. J Immunol 2006;177:4794-4802.

37. Redecha P, Tilley R, Tencati M, Salmon JE, Kirchhofer D, Mackman N, Girardi G.

Tissue factor: a link between C5a and neutrophil activation in antiphospholipid

antibody induced fetal injury. Blood 2007;110:2423-2431.

38. Pierangeli SS, Girardi G, Vega-Ostertag M, Liu X, Espinola RG, Salmon J. Requirement

of activation of complement C3 and C5 for antiphospholipid antibody-mediated

thrombophilia. Arthritis Rheum 2005;52:2120-2124.

39. Licht C, Fremeaux-Bacchi V. Hereditary and acquired complement dysregulation in

membranoproliferative glomerulonephritis. Thromb Haemost 2009:101:271-278.

40. Shamonki JM, Salmon JE, Hyjek E, Baergen RN. Excessive complement activation is

associated with placental injury in patients with antiphospholipid antibodies. Am

J Obstet Gynecol 2007;196:167.1-5.

41. Oku K, Amengual O, Atsumi T. Pathophysiology of thrombosis and pregnancy

morbidity in the antiphospholipid syndrome. Eur J Clin Invest 2012;42:1126-1135.

42. Viall CA, Chamley LW. Histopathology in the placentae of women with

antiphospholipid antibodies: a systematic review of the literature. Autoimmun Rev

2014;14:446-471.

43. Devreese K, Hoylaerts MF. Is there an association between complement

activation and antiphospholipid antibody related thrombosis? Thromb Haemost

2010;104:1279-1281.

44. Lonze BE, Singer AL, Montgomery RA. Eculizumab and renal transplantation in a

patient with CAPS. N Engl J Med 2010; 362:1744-1745.

45. Velik-Salchner C, Lederer W, Wiedermann F. Eculizumab and renal transplantation

in a patient with catastrophic antiphospholipid syndrome: effect of heparin on

complement activation. Lupus 2011;20:772.

46. Hadaya K, Ferrari-Lacraz S, Fumeaux D, Boehlen F, Toso C, Moll S, Martin PY, Villard

J. Eculizumab in acute recurrence of thrombotic microangiopathy after renal

transplantation. Am J Transplant 2011;11:2523-2527.

47. Darnige L, Peffault de Latour R, Zemori L, Socié G, Fischer AM, Helley D; French

Society of Haematology. Antiphospholipid antibodies in patients with paroxysmal

nocturnal haemoglobinuria receiving eculizumab. Br J Haematol 2011;153:789-

791.

48. Shapira I, Andrade D, Allen SL, Salmon JE. Brief report: induction of sustained

remission in recurrent catastrophic antiphospholipid syndrome via inhibition of

terminal complement with eculizumab. Arthritis Rheum 2012;64:2719-2723.

49. Canaud G, Kamar N, Anglicheau D, Esposito L, Rabant M, Noël LH, Guilbeau-

Frugier C, Sberro-Soussan R, Del Bello A, Martinez F, Zuber J, Rostaing L, Legendre

C. Eculizumab improves posttransplant thrombotic microangiopathy due to

antiphospholipid syndrome recurrence but fails to prevent chronic vascular

changes. Am J Transplant 2013;13:2179-2185.

50. Mushin SA, Khianey R, Erkan D. Discordant aPTT and anti-FXa values in a catastrophic

antiphospholipid syndrome patient receiving intravenous unfractionated heparin.

In: 14th International Congress on Antiphospholipid Antibodies Abstract Book,

2013;1:14-76 (abstract).

51. Erkan D, Aguiar CL, Andrade D, Cohen H, Cuadrado MJ, Danowski A, Levy RA,

Ortel TL, Rahman A, Salmon JE, Tektonidou MG, Willis R1, Lockshin MD. 14th

International Congress on Antiphospholipid Antibodies: Task force report on

antiphospholipid syndrome treatment trends. Autoimmun Rev 2014;13:685-696.

52. Bakhtar O, Thajudeen B, Braunhut BL, Yost SE, Bracamonte ER, Sussman AN,

Kaplan B. A case of thrombotic microangiopathy associated with antiphospholipid

antibody syndrome successfully treated with eculizumab. Transplantation

2014;98:17-18.

53. Lonze BE, Zachary AA, Magro CM, Desai NM, Orandi BJ, Dagher NN, Singer

AL, Carter-Monroe N, Nazarian SM, Segev DL, Streiff MB, Montgomery RA.

Eculizumab prevents recurrent antiphospholipid antibody syndrome and enables

successful renal transplantation. Am J Transplant 2014;14:459-465.

54. Strakhan M, Hurtado-Sbordoni M, Galeas N, Bakirhan K, Alexis K, Elrafei T. 36-yearold

female with catastrophic antiphospholipid syndrome treated with eculizumab:

a case report and review of literature. Case Rep Hematol 2014;2014:704371.

55. Zapantis E, Furie R, Horowitz D. Eculizumab in antiphospholipid antibody

syndrome. Arthritis Rheum 2014;66(Suppl):6(abstract).

7

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0190


Deregulated Levels of the NF-κB1, NF-κB2, and Rel Genes in

Ukrainian Patients with Leukemia and Lymphoma in the

Post-Chernobyl Period

Çernobil Sonrası Ukraynalı Lösemi ve Lenfoma Hastalarında Değişken NF-κB1, NF-κB2 ve

Rel Gen Düzeyleri

Hakan Savlı 1 , Ramis Ufuk Akkoyunlu 1 , Naci Çine 1 , Daniil F. Gluzman 2 , Michael P. Zavelevich 2 , Lilia M. Sklyarenko 2 , Stella V. Koval 2 ,

Deniz Sünnetçi 1

1Kocaeli University Faculty of Medicine, Department of Medical Genetics, Kocaeli, Turkey

2National Academy of Sciences of Ukraine, R.E. Kavetsky Institute of Experimental Pathology, Oncology, and Radiobiology, Kyiv, Ukraine

Abstract

Objective: Nuclear factor kappa B (NF-κB) is an important

transcription factor in cancer and NF-κB activation has been seen

in angiogenesis, tumor progression, and metastasis. Relationships

between specific NF-κB gene networks, leukemogenesis, and radiation

exposure are still unknown. Our aim was to study the expression levels

of the NF-κB1, NF-κB2, and Rel genes in hematological malignancies

in the post-Chernobyl period.

Materials and Methods: We analyzed gene expression levels of NFκB1,

NF-κB2, and Rel in 49 B-cell chronic lymphocytic leukemia, 8

B-cell non-Hodgkin’s lymphoma, 3 acute myeloid leukemia, 3 chronic

myeloid leukemia, 2 hairy cell leukemia, 2 myelodysplastic syndrome,

and 2 T-cell large granular lymphocytic leukemia patients using realtime

polymerase chain reaction.

Results: Expression levels of NF-κB1, NF-κB2, and Rel genes were

found to be deregulated.

Conclusion: These results could be accepted as specific gene traces

to radiation-induced leukemia or as potential candidates for new

diagnostic biomarker studies. Larger experiments and non-exposed

control malignant cell populations are needed to clarify these

suggestions.

Keywords: Chronic lymphocytic leukemia, Non-Hodgkin’s lymphoma,

B-cell neoplasms, Cancer, Thrombosis, T-cell neoplasms, B-cell

neoplasms, Acute leukemia, Myelodysplastic syndromes, Chronic

leukemia

Öz

Amaç: Nükleer faktör kappa B (NF-κB), kanserde önemli bir

transkripsiyon faktörü olup aktivasyonu anjiyogenez, tümör gelişimi

ve metastazın birçok basamağında görülmektedir. Spesifik NF-κB gen

ağları, lökomogenez ve radyasyon maruziyeti arasındaki ilişki halen

belirsizdir. Çalışmamızda Çernobil sonrası hematolojik kanserlerde

NF-κB1, NF-κB2 ve Rel genlerinin ekspresyon düzeylerini incelemeyi

amaçladık.

Gereç ve Yöntemler: Gerçek zamanlı polimeraz zincir reaksiyonu ile 49

B-hücreli kronik lenfositik lösemi, 8 B-hücreli non-Hodgkin lenfoma,

3 akut myeloid lösemi, 3 kronik myeloid lösemi, 2 tüylü hücre lösemi,

2 miyelodisplastik sendrom ve 2 T-hücreli büyük granüler lenfositik

lösemi hastasında NF-κB1, NF-κB2 ve Rel gen ekspresyon düzeylerini

analiz ettik.

Bulgular: NF-κB1, NF-κB2 ve Rel genlerine ait ekspresyon düzeyleri

değişmiş olarak saptandı.

Sonuç: Bu sonuçlar, radyasyonla indüklenmiş lösemilerdeki spesifik

gen izleri veya yeni tanısal biyobelirteç çalışmalarına muhtemel aday

önerileri olarak da kabul edilebilir. Bu düşünceleri açıklığa kavuşturmak

için daha geniş deneyler ve radyasyon maruziyeti olmayan kontrol

malign hücre popülasyonlarına ihtiyaç vardır.

Anahtar Sözcükler: Kronik lenfositik lösemi, Non-Hodgkin lenfoma,

B-hücreli neoplazmalar, Kanser, Tromboz, T-hücreli neoplaziler,

B-hücreli neoplaziler, Akut lösemi, Myelodisplastik sendromlar, Kronik

lösemi

Address for Correspondence/Yazışma Adresi: Deniz SÜNNETÇİ, PhD.,

Kocaeli University Faculty of Medicine,

Department of Medical Genetics, Kocaeli, Turkey

E-mail : sun_deniz@hotmail.com


Accepted/Kabul tarihi: October 13, 2014

8


Savlı H, et al: Deregulated Levels of NF-κB1, NF-κB2, and Rel Genes in Leukemia and Lymphoma

Introduction

The derivations of signatures using proteomics and genomics

are increasingly integrated in the design of prognostic and

predictive markers in oncology. Some of these markers are

also well-known targets for therapeutic approaches, such

as bortezomib, a nuclear factor kappa B (NF-κB) inhibitor

possessing clinical activity in mantle cell lymphoma patients

[1]. NF-κB is an important transcription factor in immunity, cell

proliferation, cell survival, and cancer [2,3,4,5]. NF-κB activation

has been demonstrated in angiogenesis, tumor progression, and

metastasis [6,7].

Relationships between gene networks, leukemogenesis, and

radiation exposure are still unknown. Our aim was to study

expression levels of the NF-κB1 gene family in Ukrainian B-cell

chronic lymphocytic leukemia (B-CLL), B-cell non-Hodgkin’s

lymphoma (NHL), acute myeloid leukemia (AML), myelodysplastic

syndrome (MDS), chronic myeloid leukemia (CML), hairy cell

leukemia (HCL), and T-cell large granular lymphocytic leukemia

(T-cell LGLL) patients in the post-Chernobyl period.

Materials and Methods

Samplesof theperipheral bloodand bonemarrow of49 B-CLL,8

B-cell NHL,3 AML,3 CML,2 HCL,2 MDS,and 2T-cell LGLL patients

were obtainedfrom the R.E. Kavetsky Institute of Experimental

Pathology, Oncology, and Radiobiology of the National Academy

of Sciences of Ukraine in 2008 and 2009. The mean age ofthe

B-CLL groupwas 58.7 years and the median was 60 years

(minimum 36 yrs, maximum 87 yrs). In theB-cell NHL group, the

mean age was 57.3 years and the median was 60 years (minimum

43 yrs, maximum 69 yrs). Patients were analyzed morphologically

and immunocytochemically according to the new World Health

Organization classification, asshown in Table 1 along with

demographical data. Thecontrol group comprised the peripheral

blood samples of 8 healthy donors from Ukraine. Themean age

of the secontrol subjects was 45.9 years and the median was 42.5

years (minimum 27 yrs, maximum 78 yrs). All B-CLL cases under

study were of the typical B-CLL immunophenotype without

adverse prognostic markers such as CD38+. Total ribonucleic

acid (RNA) was isolated from leukocytes using the QIAamp

RNA Blood Mini Kit (QIAGEN, Valencia, CA, USA) and treated

with DNase I according to the manufacturer’s instructions.

Quantity and purity were checked using a NanoDrop 2000

UV-Vis Spectrophotometer (Thermo Scientific, Wilmington,

DE, USA). Complementary deoxyribonucleic acid (cDNA) was

synthesized using a RevertAid First Strand cDNA Synthesis Kit

(Fermentas Inc., Hanover, MD, USA) from 100 ng/µL total RNA

as starting material. Gene expression levels were determined by

quantitative reverse transcription-polymerase chain reaction

as described previously [8,9]. Standard curves were obtained

using serial dilutions of the beta-globulin gene (DNA Control

Kit, Roche, Penzberg, Germany). Gene-specific primers (Table 2)

were obtained from Integrated DNA Technologies (Coralville, IA,

USA). Obtained gene expression values were normalized using

a housekeeping gene of beta-2 microglobulin. Gene expression

ratios were compared in patient and control groups using the

Relative Expression Software Tool (REST).

Statistical Analysis

Statistical analysis was performed using independent sample

t-tests to analyze the statistical significance of our results by

comparing controls with B-CLL, B-cell NHL, AML, MDS, CML,

HCL, and T-cell LGLL patients. The p-values are shown in Table 3.

Results

The NF-κB1, NF-κB2, and Rel genes were found to be upregulated

in 49 B-CLL, 8 B-cell NHL, 3 AML, and 2 HCL patients in the

post-Chernobyl period (Table 3). NF-κB1 was decreased 1.301-

fold in B-CLL, 1.473-fold in B-cell NHL, 1.534-fold in AML, and

1.862-fold in HCL cases. NF-κB2 was upregulated 1.720-fold in

B-CLL, 8.545-fold in B-cell NHL, 16.257-fold in AML, and 1.676-

fold in HCL cases. We found Rel expression upregulated 2.736-

fold in B-CLL, 4.039-fold in B-cell NHL, 65.526-fold in AML, and

6.912-fold in HCL cases.

In the MDS group, NF-κB2 was found to be significantly

upregulated (50.563-fold). Rel was 2.272-fold upregulated

whereas NF-κB1 was 1.100-fold downregulated in the same

group.

In the CML group, NF-κB2 was 2.110-fold upregulated while

NF-κB1 and Rel were downregulated 1.056-fold and 1.239-fold,

respectively.

We found downregulation of the NF-κB1, NF-κB2, and Rel genes

in T-cell LGLL cases at 4.557-fold, 3.771-fold, and 2.632-fold,

respectively.

Discussion

We had already found deregulated levels of NF-κB in our

genomic experiments on prostate cancer [10], papillary thyroid

cancer [11], and leukemia [12,13] in our previous studies.

Recently, our proteomic results confirmed the upregulation of

NF-κB in microarray screening in a breast cancer population

[14]. This is our first observation of NF-κB deregulations in

hematopoietic malignancies.

Transcription of proteins that promote cell survival, stimulate

growth, induce angiogenesis, and reduce susceptibility to

apoptosis are upregulated by NF-κB. The NF-κB signaling

pathway was found activated in MDS, AML, acute lymphoblastic

leukemia (ALL), CML, CLL, multiple myeloma, and lymphoma

cases before. These 3 genes were defined as deregulated before

9



Table 1. Patient data, clinical features, and individual gene expression ratios.

Leukemia

Type

Patient ID Sex, Age WBC Clinical Data NF-κB1 Gene

Expression Fold

Change

NF-κB2 Gene

Expression Fold

Change

Rel Gene

Expression Fold

Change

B-CLL U 4 B F/73 Le-132x109/L

Ly-85%

B-CLL U 11 B F/71 Le-19.2x109/L

Ly-57%

B-CLL U 20 B F/46 Le-23.2x109/L

Ly-64%

B-CLL U 23 B M/55 Le-15.0x10 9 /L

Ly-65%

B-CLL U 27 B M/49 Le-43.3x10 9 /L

Ly-91%

Stage 3

Lymph nodes +,

lien +, hepar +

Stage 2

Lymph nodes +

Stage 2

Lien +

Stage 2

Lymph nodes +, lien +,

hepar +

Stage 2

Lymph nodes +

1.528/U 2.676/U 3.444/U

1.397/U 1.366/D 5.220/U

1.818/U 15.562/U 6.653/U

1.233/U 1.214/D 3.350/U

2.953/U 3.434/U 8.598/U

B-CLL U 28 B M/62 Le-105x109/L Stage 1-2 1.916/D 17.630/D 1.773/D

Ly-72%

B-CLL U 29 B F/72 B-CLL 34.824/U n/a 2.740/U

B-CLL U 30 B M/69 B-CLL 22.816/U n/a 1.489/U

B-CLL U 31 B M/36 B-CLL 2.661/U 2.497/D 1.120/U

B-CLL U 32 B F/59 B-CLL 1.134/U 7.362/D 1.598/D

B-CLL U 33 B F B-CLL 1.402/D 1.693/U 2.880/D

B-CLL U 34 B M/67 B-CLL 3.463/U 1.240/D 2.020/U

B-CLL U 35 B F/66 B-CLL 12.658/U 2.908/D 2.288/U

B-CLL U 36 B F/87 B-CLL 1.656/D 2.990/D 1.450/D

B-CLL U 37 B F/63 B-CLL 2.238/U 1.310/U 2.272/U

B-CLL U 43 B F/50 B-CLL 1.216/U 1.257/U 1.924/U

B-CLL U 44 B M/74 B-CLL 1.022/U 1.079/D 5.007/U

B-CLL U 45 B F/57 B-CLL 2.141/D 1.569/D 1.820/U


B-CLL U 51 B M/62 B-CLL 16.772/D n/a 2.556/U


B-CLL U 54 B F/52 B-CLL 1.327/D 3.117/D 4.737/U

B-CLL U 1 P M/70 B-CLL 2.518/U 2.657/U 2.452/U

B-CLL U 12 P M/57 B-CLL 3.245/D 1.729/U 1.252/U

B-CLL U 24 P F/53 B-CLL 2.102/U 5.657/D 1.060/U

B-CLL U 33 P F/36 B-CLL 1.282/D 1.301/U 4.482/U

B-CLL U 34 P M/85 B-CLL 1.027/D 2.549/U 2.504/U

B-CLL U 40 P M/62 B-CLL 1.309/D 2.868/D 1.722/U

B-CLL U 42 P M/56 B-CLL 1.393/D 8.574/D 1.607/U

B-CLL U 47 P F/71 B-CLL 1.482/D 8.938/U 6.426/U

B-CLL U 51 P F/70 B-CLL 1.116/D 4.959/U 8.779/U

10



Table 1. Continuation.

B-CLL U 69 P M/41 Preliminary diagnosis: B-CLL

NHL of spleen could not be

excluded

1.155/D 5.776/U 1.584/U

B-CLL U 76 P M/70 B-CLL 1.282/D 3.074/D 6.746/U

B-CLL U 77 P F/69 B-CLL 3.254/U 1.301/U 11.035/U

B-CLL U 80 P M/51 B-CLL 1.462/D 7.945/U 6.122/U

B-CLL U 81 P M/70 B-CLL 2.025/D 18.252/U 2.521/U

B-CLL U 82 P M/60 B-CLL 1.889/D 3.945/U 4.608/U

B-CLL U 83 P F/48 B-CLL 1.051/U n/a 1.686/U

B-CLL U 85 P F/57 B-CLL 1.668/D 14.420/U 5.184/U

B-CLL U 87 P F B-CLL 2.691/D 9.781/U 2.320/U

B-CLL U 88 P M/51 B-CLL 1.436/U 3.811/D 7.537/U

B-CLL U 208 P F/62 B-CLL 1.259/U 20.821/U 1.128/U

B-CLL U 211 P F/55 B-CLL 1.213/D 17.630/U 1.399/U

B-CLL U 7 B F/78 Le-149.9x109/L

Ly-71%

Stage 3

Lymph nodes +, lien +,

hepar +

B-CLL/PLL (NHL)

B-CLL U 15 B F/67 Stage 1-2

Lymph nodes +

B-CLL/PLL (NHL)

B-CLL U 41 B M/53 Chronic lymphoproliferative

disease, Waldenström

macroglobulinemia

B-CLL U 55 B F/59 Chronic lymphoproliferative

disease

1.571/U 2.848/U 14.065/U

1.524/D 2.014/D 1.978/U

1.507/U n/a 1.428/U

1.116/D 2.567/D 2.797/U

B-CLL U 57 B M/78 B-CLL 1.147/D 1.064/D 3.396/U

B-CLL U 58 B F/57 B-CLL 2.415/U 1.454/U 5.293/U

B-cell NHL U 8 B F/43 Le-20.5x109/L

Ly-76%

B-cell NHL U 14 B M/53 Le-15.1x10 9 /L

Ly-49%

B-cell NHL U 19 B M/69 Le-10x10 9 /L

Ly-68%

Lien +, hepar + 1.336/D 1.310/U 3.565/U

Lymph nodes +,

lien +

Lymph nodes +,

hepar +

Large B-cell lymphoma

1.196/D 1.094/D 1.082/U

1.015/U n/a n/a

B-cell NHL U 48 B M/45 B-cell NHL 4.061/U n/a 2.006/U

B-cell NHL U 13 P M/60 Diffuse large B-cell lymphoma 2.717/U n/a n/a

plasmablastic variant

B-cell NHL U 209 P M/62 B-cell NHL 1.207/U 37.531/U 1.552/U

B-cell NHL U 15 P F/60 Lymphoma of marginal zone 3.660/U n/a 7.230/U

of spleen, leukemic transition

11



Table 1. Continuation.

B-cell NHL U 56 B M/66 Splenic lymphoma 1.402/D 4.925/D 1.951/U

AML U 5 P F/51 AML M5 12.484/U 1341.843/U 29,004.719/U

AML U 17 B M/68 Le-33.7x10 9 /L AML M4 n/a 4.000/U n/a

Lien +, hepar +

Blast cells-82%

AML U 46 B M/35 AML M3 3.686/U 1.248/D 2.8967/U

CML U 9 B M/20 Lymph nodes +,

lien +

Acceleration phase

CML U 26 B F/59 Lien +

Le-47.8x109/L

Blast cells-19%

Acceleration phase

CML 1.935/U 10.703/U 2.402/U

CML 1.611/D 1.670/U 3.195/D

CML U 47 B M/20 CML 1.412/D 1.905/D 1.430/D

HCL U 24 B M/49 Le-3.0x10 9 /L

Ly-70%

Thrombocytopenia

HCL 1.229/D 1.803/D 1.595/U

HCL U 50 B F/36 HCL 4.263/U n/a 29.940/U

MDS U 3 P M/74 MDS RAEB 1.282/D 15.348/U n/a

MDS U 207 P M/57 MDS RA n/a 166.572/U 2.646/U

T-cell LGLL U 7 P M/70 T-cell LGLL 11.778/D 2.204/D 2.323/D

T-cell LGLL U 42 B F/78 1.763/D n/a 2.981/D

NF-κB: Nuclear factor kappa B, B-CLL: B-cell chronic lymphocytic leukemia, AML: acute myeloid leukemia, MDS: myelodysplastic syndrome, CML: chronic myeloid leukemia,

HCL: hairy cell leukemia, T-cell LGLL: T-cell large granular lymphocytic leukemia, NHL: non-Hodgkin’s lymphoma, WBC: white blood cell, D: downregulated, U: upregulated, F: female,

M: male, n/a: not applicable.

Table 2. Primer sequences of the studied genes.

Genes

Primer Sequences

Beta-2 microglobulin (F) 5’ TGA CTT TGT CAC AGC CCA AGA TA 3’

(R) 5’ AAT CCA AAT GCG GCA TCT TC 3’

NF-κB1 (F) 5’ AGC ACG AAT GAC AGA GGC GTG TA 3’

(R) 5’ TTC TGC TTG CAA ATA GGC AAG GT 3’

NF-κB2 (F) 5’ AGA CGA GTG TGG TGA GCT TTCT 3’

(R) 5’ AGT CAG GCA TAT GCA ACA 3’

Rel (F) 5’ TGC CGA TGA CAT AGT CGG AAT 3’

(R) 5’ GGA CAT CTG ATG GAG CTG TCT 3’

NF-κB: Nuclear factor kappa B.

in hematological malignancies [15]. Here we have observed

deregulated levels in radiation-induced leukemia populations.

Results supported that radiation-exposed and non-exposed

hematological malignancies use the same gene pathways and

are shaped around the NF-κB gene network.

It was shown that losses in the 13q chromosomal region are

also associated with B-CLL and these losses deregulate the

NF-κB pathway [16]. Deregulation of the NF-κB pathway by

gains at chromosomal loci including the NF-κB1, NF-κB2, and

Rel genes was reported previously. A gain at the (2)(p16.1p14)

region including the Rel gene, an oncogene, was reported in

17p-deleted CLL with poor prognosis [17]. Rearrangements such

as translocations and deletions occurred in 10q24 affecting

the NF-κB2 gene, a protooncogene. These rearrangements are

known to lead to deletion of 3’ sequences of the NF-κB2 gene

and cause production of carboxy-truncated constitutively

nuclear proteins that may have a role in the tumorigenesis

of B-CLL and B-cell NHL at high levels [18]. Unlike its relative

NF-κB2, NF-κB1 has few rearrangements reported in leukemias

and lymphomas. There is evidence in the literature that

NF-κB2 is involved in oncogenesis in T-cell ALL as a result of

LYL1 translocation [19]. Further studies are needed to assess the

NF-κB1 rearrangements leading to B-CLL and B-cell NHLs. These

observations give us new clues about relationships between

NF-κB deregulation in leukemias and chromosomal regions.

We are planning to continue our further studies by array

comparative genomic hybridization analysis to focus on fine

mapping of 13q and 2p in particular.

Over the last decade, the problem of association between B-CLL

and ionizing radiation has become a matter of considerable

scientific interest [20]. Nevertheless, the experimental studies on

the relationship between ionizing radiation and CLL are limited.

Lyng et al. indicated that activation of the NF-κB pathway

12



Table 3. Gene expression levels in groups of studied patients.

Number of NF-κB1 Gene Expression Change NF-κB2 Gene Expression Change REL Gene Expression Change

Patients

B-CLL 49 1.301 U

p=0.569

B-cell NHL 8 1.473 U

p=0.039

AML 3 1.534 U

p=0.001

CML 3 1.056 D

p=0.758

1.720 U

p=0.335

8.545 U

p=0.391

16.257 U

p=0.698

2.110 U

p=0.99

2.736 U

p=0.018

4.039 U

p=0.018

65.526 U

p=0.786

1.239 D

p=0.127

HCL 2 1.862 U

p=0.272

1.676 U 6.912 U

p=0.519

MDS 2 1.100 D 50.563 U

p=0.72

2.272 U

T-cell LGLL 2 4.557 D

p=0.236

3.771 D 2.632 D

p=0.623

NF-κB: Nuclear factor kappa B, B-CLL: B-cell chronic lymphocytic leukemia, AML: acute myeloid leukemia, MDS: myelodysplastic syndrome, CML: chronic myeloid leukemia,

HCL: hairy cell leukemia, T-cell LGLL: T-cell large granular lymphocytic leukemia, NHL: non-Hodgkin’s lymphoma, D: downregulated, U: upregulated.

may suppress the apoptotic response in U698 cells, a malignant

B-lymphocyte cell line, to ionizing radiation [21]. Activation of

the NF-κB pathway by ionizing radiation induces antiapoptotic

genes and inhibits apoptosis by upregulation of NF-κB genes.

This was linked to proliferation and increased survival of B-CLL

[22]. B-CLL and HCL cells are known to be refractory to signals

activating normal B cells. B-CLL and HCL cells are stimulated by

tumor necrosis factor (TNF-α) [23]. TNF-α is involved in many

human tumors and associated with poor prognosis. TNF-α is

produced by B-CLL and HCL cells [24] and contributes to the

escape of HCL cells from apoptosis through NF-κB activation

[25]. Radiation exposure results in high levels of NF-κB gene

expression. We found upregulated levels of NF-κB1, NF-κB2, and

Rel genes in our patients. Our results were in concordance with

the previous findings above.

NF-κB expressions were found significantly higher than in the

controls in both AML and ALL by Kapelko-Słowik et al. before

[26]. They also found lower expression levels of NF-κB in AML

patients who reached complete remission compared with

patients with primary resistance to chemotherapy who did

not reach complete remission. These data indicated that high

expression levels of NF-κB might be involved in the pathogenesis

of AML and ALL [26]. There are few studies on radiation-induced

leukemia populations, such as in the Chernobyl area. However,

we cannot assess all etiological sources for our subjects. Levels of

exposure among subjects during the Chernobyl accident remain

unclear. Thus, we cannot conclude that the NF-κB pathway

is the main cause of AML and ALL pathogenesis in radiationinduced

forms of the disease.

Rel has the potential to transform cells in culture and is

expressed in high levels in both B-cell NHL [27] and large

granular lymphocytic leukemia [28]. Interestingly, we found

decreased levels of Rel, NF-κB1, and NF-κB2 in our T-cell LGLL

group.

In our study we found NF-κB2 significantly higher in MDS

cases. There is evidence in the literature that the degree of

NF-κB activity is correlated with the risk of progression to AML.

NF-κB activation is known to be a hallmark of high-risk MDS

[27].

We obtained increased levels of NF-κB2 in CML cases. Exposure

to ionizing radiation causes CML [29]. CML is characterized by

t(9;22), which leads to Bcr/Abl fusion oncoprotein expression.

This protein activates the NF-κB pathway. The NF-κB pathway,

in turn, leads to expression of antiapoptotic proteins such as

Bcl-X L and lets Bcr/Abl + cells grow [27].

Here we have defined a positive correlation between upregulated

levels of NF-κB genes in hematological malignancies related to

radiation exposure. However, the limited number of patients

and controls was an obstacle. Therefore, these experiments are

presented here as results of a preliminary study. Similar studies

should be extended to experiments in time- and dose-dependent

manners in cell lines or primary cultures. We think that our

results are a good starting point for drawing a network around

the NF-κB genes to investigate the life cycles of hematological

malignancies.

13



Conclusion

Moreover, it would be tempting to suggest that this gene region

may be used as a trace of early radiation exposure leading to

leukemia. Either way, the NF-κB pathway certainly deserves

more attention since its overexpression is almost a rule in many

solid tumors and hematopoietic malignancies.

Ethics

Ethics Committee Approval: Bioethics Committee of the R.E.

Kavetsky Institute of Experimental Pathology, Oncology and

Radiobiology of the National Academy of Sciences of Ukraine

(Approval number: 5/2008), Informed Consent: It was taken.


Concept: Hakan Savlı, Daniil F. Gluzman, Design: Hakan Savlı,

Daniil F. Gluzman, Michael P. Zavelevich, Data Collection or

Processing: Lilia M. Sklyarenko, Stella V. Koval, Analysis or

Interpretation: Deniz Sünnetçi, Naci Çine, Ramis Ufuk Akkoyunlu,

Lilia M. Sklyarenko, Literature Search: Deniz Sünnetçi, Ramis

Ufuk Akkoyunlu, Michael P. Zavelevich, Writing: Hakan Savlı,

Ramis Ufuk Akkoyunlu.

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.

Financial Disclosure: The authors declared that this study has

received a financial support by TÜBİTAK-NASU joint project

(M/32-2008).

References

1. Vose JM. Mantle cell lymphoma: 2012 update on diagnosis, riskstratification,

and clinical management. Am J Hematol 2012;87:604-609.

2. Nagase H, Visse R, Murphy G. Structure and function of matrix

metalloproteinases and TIMPs. Cardiovasc Res 2006;69:562-573.

3. Bendrik C, Robertson J, Gauldie J, Dabrosin C. Gene transfer of matrix

metalloproteinase-9 induces tumor regression of breast cancer in vivo.

Cancer Res 2008;68:3405-3412.

4. Kato K, Hara A, Kuno T, Kitaori N, Huilan Z, Mori H, Toida M, Shibata T. Matrix

metalloproteinases 2 and 9 in oral squamous cell carcinomas: manifestation

and localization of their activity. J Cancer Res Clin Oncol 2005;131:340-346.

5. Groblewska M, Siewko M, Mroczko B, Szmitkowski M. The role of matrix

metalloproteinases (MMPs) and their inhibitors (TIMPs) in the development

of esophageal cancer. Folia Histochem Cytobiol 2012;24:12-19.

6. Plummer SM, Holloway KA, Manson MM, Munks RJ, Kaptein A, Farrow S,

Howells L. Inhibition of cyclo-oxygenase 2 expression in colon cells by the

chemopreventive agent curcumin involves inhibition of NF-κB activation

via the NIK/IKK signalling complex. Oncogene 1999;18:6013-6020.

7. Barnes PJ, Karin M. Nuclear factor-κB, a pivotal transcription factor in

chronic inflammatory diseases. N Engl J Med 1997;336:1066-1071.

8. Savli H, Aalto Y, Nagy B, Knuutila S, Pakkala S. Gene expression analysis of

1,25(OH)2D3-dependent differentiation of HL-60 cells: a cDNA array study.

Br J Haematol 2002;118:1065-1070.

9. Savli H, Karadenizli A, Kolayli F, Gundes S, Ozbek U, Vahaboglu H. Expression

stability of six housekeeping genes: a proposal for resistance gene

quantification studies of Pseudomonas aeruginosa by real-time quantitative

RT-PCR. J Med Microbiol 2003;52:403-408.

10. Savli H, Szendröi A, Romics I, Nagy B. Gene network and canonical pathway

analysis in prostate cancer: a microarray study. Exp Mol Med 2008;40:176-

185.

11. Cine N, Tarkun I, Canturk N, Gunduz Y, Sunnetci D, Savli H. Whole genome

expression, canonical pathway and gene network analysis in the cases

of papillary thyroid cancer. In: European Society of Human Genetics

Conference Proceedings, 2012;20(Suppl 1):192.

12. Savli H, Sunnetci D, Cine N, Gluzman DF, Zavelevich MP, Sklyarenko LM,

Nadgornaya VA, Koval SV. Gene expression profiling of B-CLL in Ukrainian

patients in post-Chernobyl period. Exp Oncol 2012;34:57-63.

13. Galimberti S, Canestraro M, Savli H, Palumbo GA, Tibullo D, Nagy B, Piaggi S,

Guerrini F, Cine N, Metelli MR, Petrini M. ITF2357 interferes with apoptosis

and inflammatory pathways in the HL-60 model: a gene expression study.

Anticancer Res 2010;30:4525-4535.

14. Cine N, Baykal AT, Sunnetci D, Canturk Z, Serhatli M, Savli H. Identification

of ApoA1, HPX and POTEE genes by omic analysis in breast cancer. Oncol

Rep 2014;32:1078-1086.

15. Fuchs O. Transcription factor NF-κB inhibitors as single therapeutic agents

or in combination with classical chemotherapeutic agents for the treatment

of hematologic malignancies. Curr Mol Pharmacol 2010;3:98-122.

16. Rodríguez AE, Hernández JÁ, Benito R, Gutiérrez NC, García JL, Hernández-

Sánchez M, Risueño A, Sarasquete ME, Fermiñán E, Fisac R, de Coca AG,

Martín-Núñez G, de Las Heras N, Recio I, Gutiérrez O, De Las Rivas J,

González M, Hernández-Rivas JM. Molecular characterization of chronic

lymphocytic leukemia patients with a high number of losses in 13q14. PLoS

ONE 2012;7:48485.

17. Forconi F, Rinaldi A, Kwee I, Sozzi E, Raspadori D, Rancoita PM, Scandurra

M, Rossi D, Deambrogi C, Capello D, Zucca E, Marconi D, Bomben R, Gattei

V, Lauria F, Gaidano G, Bertoni F. Genome-wide DNA analysis identifies

recurrent imbalances predicting outcome in chronic lymphocytic leukaemia

with 17p deletion. Br J Haematol 2008;143:532-536.

18. Migliazza A, Lombardi L, Rocchi M, Trecca D, Chang CC, Antonacci R,

Fracchiolla NS, Ciana P, Maiolo AT, Neri A. Heterogeneous chromosomal

aberrations generate 3’ truncations of the NFKB2/lyt-10 gene in lymphoid

malignancies. Blood 1994;84:3850-3860.

19. Rayet B, Gélinas C. Aberrant rel/nfkb genes and activity in human cancer.

Oncogene 1999;18:6938-6947.

20. Richardson DB, Wing S, Schroeder J, Schmitz-Feuerhake I, Hoffmann W.

Ionizing radiation and chronic lymphocytic leukemia. Environ Health

Perspect 2005;113:1-5.

21. Lyng H, Landsverk KS, Kristiansen E, DeAngelis PM, Ree AH, Myklebost

O, Hovig E, Stokke T. Response of malignant B lymphocytes to ionizing

radiation: gene expression and genotype. Int J Cancer 2005;115:935-942.

22. Chavez JC, Sahakian E, Pinilla-Ibarz J. Ibrutinib: an evidence-based review

of its potential in the treatment of advanced chronic lymphocytic leukemia.

Core Evid 2013;8:37-45.

23. Jabbar SA, Hoffbrand AV, Wickremasinghe RG. Defects in signal transduction

pathways in chronic B lymphocytic leukemia cells. Leuk Lymphoma

1995;18:163-170.

24. Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G. Inflammation

and cancer: how hot is the link? Biochem Pharmacol 2006;72:1605-1621.

25. Tiacci E, Liso A, Piris M, Falini B. Evolving concepts in the pathogenesis of

hairy-cell leukaemia. Nat Rev Cancer 2006;6:437-448.

26. Kapelko-Słowik K, Urbaniak-Kujda D, Wołowiec D, Jazwiec B, Dybko J,

Jakubaszko J, Słowik M, Kuliczkowski K. Expression of PIM-2 and NF-κB

genes is increased in patients with acute myeloid leukemia (AML) and acute

lymphoblastic leukemia (ALL) and is associated with complete remission

rate and overall survival. Postepy Hig Med Dosw (Online) 2013;67:553-559.

27. Braun T, Carvalho G, Fabre C, Grosjean J, Fenaux P, Kroemer G. Targeting

NF-κB in hematologic malignancies. Cell Death Differ 2006;13:748-758.

28. Zhang D, Loughran TP Jr. Large granular lymphocytic leukemia: molecular

pathogenesis, clinical manifestations, and treatment. Hematology Am Soc

Hematol Educ Program 2012;2012:652-659.

29. Corso A, Lazzarino M, Morra E, Merante S, Astori C, Bernasconi P, Boni

M, Bernasconi C. Chronic myelogenous leukemia and exposure to

ionizing radiation--a retrospective study of 443 patients. Ann Hematol

1995;70:79-82.

14

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0197


Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin

Expression

BACH1 ve Hemoglobin E/Beta-Talasemi Globin Sunumu Korelasyonu

Tze Yan Lee 1 , Logeswaran Muniandy 1 , Lai Kuan Teh 1 , Maha Abdullah 1 , Elizabeth George 1,2 Jameela Sathar 3 , Mei I Lai 1,2

1Universiti Putra Malaysia Faculty of Medicine and Health Sciences, Department of Pathology, Serdang, Malaysia

2Universiti Putra Malaysia Faculty of Medicine and Health Sciences, Genetic and Regenerative Medicine Research Centre, Serdang, Malaysia

3Ampang Hospital, Clinic of Hematology, Selangor, Malaysia

Abstract

Objective: The diverse clinical phenotype of hemoglobin E (HbE)/βthalassemia

has not only confounded clinicians in matters of patient

management but has also led scientists to investigate the complex

mechanisms involved in maintaining the delicate red cell environment

where, even with apparent similarities of α- and β-globin genotypes,

the phenotype tells a different story. The BTB and CNC homology

1 (BACH1) protein is known to regulate α- and β-globin gene

transcriptions during the terminal differentiation of erythroid cells.

With the mutations involved in HbE/β-thalassemia disorder, we

studied the role of BACH1 in compensating for the globin chain

imbalance, albeit for fine-tuning purposes.

Materials and Methods: A total of 47 HbE/β-thalassemia samples

were analyzed using real-time quantitative polymerase chain reaction

and correlated with age, sex, red blood cell parameters, globin gene

expressions, and some clinical data.

Results: The BACH1 expression among the β-thalassemia intermedia

patients varied by up to 2-log differences and was positively

correlated to age; α-, β-, and γ-globin gene expression level; and

heme oxygenase 1 protein. BACH1 was also negatively correlated to

reticulocyte level and had a significant correlation with splenectomy.

Conclusion: This study indicates that the expression of BACH1 could

be elevated as a compensatory mechanism to decrease the globin

chain imbalance as well as to reduce the oxidative stress found in

HbE/β-thalassemia.

Keywords: BACH1, Gene expression, Hemoglobin E/β-thalassemia,

Oxidative stress, Red blood cell parameters

Öz

Amaç: Hemoglobin E (HbE)/β-talaseminin çeşitli klinik fenotipleri

klinisyenlerin hasta yönetimi esnasında zihinlerini karıştırmakla

kalmamış, α- ve β-globin genotiplerinde bariz benzerlikler

varken fenotiplerde farklılıklar bulunduğundan bilim insanlarının

hassas eritrosit çevrenin muhafaza edilmesinde yer alan karmaşık

mekanizmaları incelemelerine de ön ayak olmuştur. BTB ve CNC

homoloji 1 (BACH1) proteininin eritroid hücrelerin son farklılaşması

sırasında α- and β-globin gen transkripsiyonlarını ayarladığı

bilinmektedir. HbE/β-talasemi hastalığındaki mutasyonlar ile her ne

kadar ince ayar amaçlı ise de BACH1’in globin zincir dengesizliğini

kompanse etmedeki rolünü inceledik.

Gereç ve Yöntemler: Toplam 47 HbE/β-talasemi örneği gerçek

zamanlı kantitatif polimeraz zincir reaksiyonu ile incelendi ve yaş,

cinsiyet, eritrosit değişkenleri, globin gen sunumları ve bazı klinik

veriler ile korele edildi.

Bulgular: β-talasemi intermedia hastalarındaki BACH1 sunumu

2-log’a kadar farklılık göstermekteydi ve yaş; α-, β- ve γ-globin gen

sunum düzeyleri; ve hem oksijenaz 1 proteini ile pozitif korelasyonu

vardı. Ayrıca BACH1’in retikülosit düzeyi ile negatif korelasyonu vardı

ve splenektomi ile anlamlı korelasyonu bulunmaktaydı.

Sonuç: Bu çalışma hem HbE/β-talasemide bulunan oksidatif stresi

hem de globin zincir dengesizliğini azaltmak için BACH1 sunumunun

kompansasyon mekanizması olarak artabileceğini göstermiştir.

Anahtar Sözcükler: BACH1, Gen sunumu, Hemoglobin E/β-talasemi,

Oksidatif stres, Eritrosit değişkenleri

Address for Correspondence/Yazışma Adresi: Mei I LaI, PhD.,

Universiti Putra Malaysia Faculty of Medicine and Health Sciences, Department of Pathology, Serdang, Malaysia

Phone : +603-89472494

E-mail : lmi@upm.edu.my


Accepted/Kabul tarihi: August 12, 2014

15

Lee TY, et al: BACH1 in Hemoglobin E/Beta-Thalassemia


Introduction

Hemoglobin E (HbE) is a highly common hemoglobin variant

in Asia. The substitution of G>A at codon 26 of the β-globin

gene not only causes a reduced and structurally abnormal HbE

production but also activates a cryptic splice site, leading to

abnormal messenger ribonucleic acid (mRNA) processing. The

heterozygous state of HbE is asymptomatic with minimal

morphological red cell changes, and homozygous HbE at most

displays mild anemia. However, when this variant is co-inherited

with β-thalassemia, the clinical spectrum ranges from mild

anemia to severe thalassemia intermedia [1,2].

This complex phenotype is affected not only by free α-globin

chain precipitations causing membrane damage and increased

reactive oxygen species, but also by various secondary and

tertiary modifiers including variable compensatory mechanisms

available in the red cell environment. This is evident particularly

in families with seemingly similar α/β genotypes but discordant

phenotypes [3].

BACH1 is a heme-binding factor that regulates multiple gene

expressions. By forming a multivalent deoxyribonucleic acid

(DNA)-binding complex in the enhancer regions of targeted

genes, BACH1 represses transcription of its target genes.

However, this function is inhibited in the presence of heme

[4,5]. BACH1 plays an important role in the expression of

heme-responsive genes like α- and β-globin, which are the key

proteins in HbE/β-thalassemia, as well as in the regulation of

heme oxygenase 1 (HO-1), a stress-responsive protein [5,6,7].

As BACH1 is able to suppress the expression of α- and β-globin

genes under physiological conditions, the expression of BACH1

in HbE/β-thalassemia is of interest. A study by De Franceschi

et al. showed a reduced intracellular heme content in late

β-thalassemic precursors, which leads to an increased expression

of the BACH1 gene [8]. Perhaps the increased BACH1 expression

may play a compensatory role to reduce α/β-globin chain

imbalance, thereby decreasing the oxidative stress produced by

the imbalance. Thus, this study investigates the correlation of

BACH1 to HbE/β-thalassemia parameters, where any effects of

BACH1 would most likely be manifested.


Study Population

HbE/β-thalassemia patients from the Ampang Hospital

Thalassemia Clinic were recruited for this study. The HbE/βthalassemia

patients in our cohort were either transfusionindependent

or previously had transfusion record of not less than

3 months prior to recruitment for this study. Ethical approval

was obtained from the Medical Research and Ethics Committee,

Ministry of Health Malaysia (NMRR-10-1177-6947), and the

Medical Research Ethics Committee, Faculty of Medicine and

Health Sciences, Universiti Putra Malaysia (UPM/FPSK/PADS/

T7-MJKEtikaPer/F01). All subjects had given their informed

consents prior to blood collection and anonymity of all data

was made possible by numerical identification throughout the

study.

Laboratory Studies

Full Blood Count, High-Performance Liquid Chromatography,

and Plasma Ferritin Study

Collection of blood samples were done in BD Vacutainer spraydried

K2EDTA tubes and BD Vacutainer freeze-dried lithium

heparin tubes (Becton, Dickinson and Company, Franklin Lakes,

NJ, USA) and full blood count indices were analyzed using a

Sysmex 5000i Automated Hematology Analyzer (Sysmex, Kobe,

Japan) according to the manufacturer’s protocol. The screening

of HbE/β-thalassemia was performed using the VARIANT II

β-Thalassemia Short Program on the VARIANT II Hemoglobin

Testing System (Bio-Rad, Hercules, CA, USA). Plasma ferritin

analysis was performed using the Tina-Quant ferritin assay

(Cobas, Roche Diagnostics GmbH, Mannheim, Germany) on the

Hitachi 902 Automatic Chemistry Analyzer (Hitachi, Ibaraki,

Japan) according to the manufacturer’s protocol.

Genomic Studies

Genomic DNA was extracted using the QIAamp Blood Midi Kit

(QIAGEN GmbH, Hilden, Germany). Amplification refractory

mutation system polymerase chain reaction (PCR) using primer

sequences and PCR protocol modified from Old was performed

to identify and confirm HbE and β-thalassemia mutations for

each sample [9]. The β-thalassemia mutations characterized

were codon 19 (A>G), IVS I-5 (G>C), IVS I-1 (G>T), codon 41/42

(-TCTT), and IVS II-654 (C>T). Genomic sequencing was done

on uncharacterized samples. Co-inheritance of α-thalassemia

was determined by --SEA,/-α 3.7 , and /-α 4.2 mutation screening

to minimize the occurrence of confounding factors for this

study. Xmn1 polymorphisms were determined as previously

described by Wong et al. [10]. Primers covering the exons of the

BACH1 gene were used to sequence the gene. Primers and PCR

conditions for the BACH1 gene are available upon request.

Expression Analysis

Ribonucleic acid (RNA) extraction from peripheral reticulocytes

was performed as described previously by Lai et al. [11]. Expression

levels of BACH1, HO-1, and α-, β-, and γ-globin genes were

quantified using real-time quantitative reverse transcription-

PCR (Applied Biosystems, Warrington, UK). Glyceraldehyde

3-phosphate dehydrogenase (GAPDH) (TaqMan Gene Expression

Assay, Applied Biosystems) acts as the endogenous control in

this expression study.

16



Table 1. Correlation of BACH1 expression with demographic and hematological data and heme oxygenase 1 and globin gene

expressions.

Parameter n Mean ± 2 Standard Deviations Range (min-max) p-value (R 2 value)

Age, years 47 34.36±19.02 21-57 0.006 (0.155)*

Sex (M/F) 47 15/32 - 0.891

Ethnicity (M^/C^) 47 45/2 - NA

Transfusion (Y^/N^) 47 46/1 - NA

Splenomegaly (n/s/sp) 47 23/19/5 - 0.0085*

Iron overload (Y^/N^) 47 39/8 - 0.622

Iron chelation (Y^/N^) 47 33/14 - 0.174

RBC, x10 6 /mL 47 3.85±1.72 2.32-5.86 0.086 (0.064)

Hb, g/dL 47 7.40±2.56 4.8-10.8 0.157 (0.044)

Hct, % 47 23.66±7.70 15.8-36.1 0.438 (0.013)

MCV, fL 47 63.09±20.52 46.4-90.0 0.125 (0.052)

MCH, pg 47 19.61±5.96 14.5-30.4 0.491 (0.011)

MCHC, g/dL 47 31.12±4.12 26.0-34.6 0.051 (0.082)

RDW-SD, # 47 62.1±24.82 36.2-84.3 0.112 (0.055)

RDW-CV % 47 28.61±11.68 16.9-52.9 0.856 (0.001)

Retic #, x10 6 /µL 46 3.93±23 (1) 0-53.72 0.005 (0.16)*

Retic % 46 28.94±152.46 (1) 0-376.8 0.01 (0.138)*

NRBC #, x10 3 /µL 46 6.85±44.24 (1) 0.02-112.27 0.681 (0.004)

NRBC % 46 34.03±244.72 (1) 0.31-758.6 0.435 (0.014)

Plasma ferritin, ng/mL 46 2134.37±4141.14 (3) 153.5-8138.4 0.098 (0.06)

Hb F, % 47 28.79±29.36 3.8-62.2 0.569 (0.007)

Hb A 2 , % 47 11.07±34 1.3-77.8 0.721 (0.003)

Beta genotype † 47 0.6531

Xmn1 (+-/++) 47 31/13 - 0.139 (0.031)

Log BACH1 expression 47 0.418±1.932 -2.92 to 2.00 -

Log HO-1 expression 47 1.319±2.083 -2.22 to 3.95 0.001 (0.329)*

Log beta expression 47 -1.128±2.991 -7.04 to 1.64 0.001 (0.253)*

Log alpha expression 47 -1.487±3.264 -7.54 to 0.02 0.002 (0.192)*

Log gamma expression 47 0.1634±2.142 -2.92 to 2.56 0.001 (0.330)*

Min: Minimum, Max: maximum, M: male, F: female, M^: Malay, C^: Chinese, Y^: intermittent transfusion, N^: transfusion-independent, n: normal, s: splenomegaly, sp: splenectomized, SD:

standard deviation, RBC: red blood cell, Hb: hemoglobin, Hct: hematocrit, MCV: mean cell volume, MCH: mean cell hemoglobin, MCHC: mean cell hemoglobin concentration, RDW: red

cell distribution width, RDW-CV: red cell distribution width coefficient of variation, NRBC: nucleated red blood cell, Retic: reticulocytes, † HbE/IVSI-5, HbE/IVSI-1, HbE/Cd41-42, HbE/

IVSII-654, HO-1: heme oxygenase 1, (): number of samples with unknown related data, NA: not analyzed. *: All values are considered significant when p≤0.05.


IBM SPSS 20 was used to analyze the correlation of BACH1

expression to age, sex, red blood cell indices, HbF, HbA2 level,

Xmn1 genotype, β-thalassemia genotypes, HO-1 expression, and

α-, β-, and γ-globin genes expression. BACH1 was also correlated

to clinical data such as transfusion history, splenomegaly, iron

overloading, and iron chelation therapy. The parameters were

analyzed using analysis of variance, Student’s t-test, Pearson’s

correlation coefficient analysis, and simple linear regression

analysis.

Results

Correlation of BACH1 Gene Expression with Age, Sex, and Red

Blood Cell Parameters

A total of 47 unrelated HbE/beta-thalassemia samples were

collected (14 males and 33 females; 45 Malays and 2 Chinese;

21-57 years old). These individuals had thalassemia intermedia

with a mean hemoglobin level of 7.40±2.56 g/dL. Co-inheritance

of α-thalassemia or iron-deficiency anemia was not found in

our sample cohort. BACH1 expression in our samples varied

up to 100.43 with a mean of 1.601±42.775 fold change and

17



was positively skewed. To fit the normal distribution curve for

statistical analyses, the BACH1 expression results were logtransformed

with approximately 2 log-fold variation. Simple

linear regression analysis showed positive association of log

BACH1 expression with age (p=0.006; R2=0.155). Log BACH1

expression was not correlated to sex or most red blood cells

parameters, except reticulocyte count (p=0.005; R2=0.16) and

percentage (%) (p=0.01; R2=0.138). Detailed results can be found

in Table 1.

Correlation of BACH1 Gene Expression to Hemoglobin

+E/β-Thalassemia Genotypes, BACH1 Genotypes, and Xmn1

Polymorphisms

The β-thalassemia mutations present in these HbE/βthalassemia

individuals were 20 IVS I-5 (G>C), 18 IVS I-1 (G>T),

7 CD41/42 (-TCTT), and 2 IVS II-654. BACH1 expression was not

correlated to any β-thalassemia genotypes (p=0.6531) or Xmn1

polymorphisms (p=0.139; R 2 =0.031) (Table 1). The Xmn1 -/-

polymorphism was excluded from the analysis as the numbers

were too small (n=3). The efficiency of βE-globin mRNA splicing

could be the main factor of β-globin expression variations,

rather than β genotypes, to affect the BACH1 expression level

[12]. Five samples were randomly selected for re-sequencing of

the BACH1 gene. However, no polymorphisms could be detected

in these samples. Polymorphisms that affect the BACH1 gene

expression could be located upstream in the enhancer regions

that were not sequenced.

Correlation of BACH1 Gene Expression with Globin Gene

Expressions and Heme Oxygenase 1 Expression

All positively skewed expression data were log-transformed to

fit the normal distribution. Log BACH1 expression was positively

correlated to log α-globin (p=0.002; R 2 =0.192), log β-globin

(p=0.001; R 2 =0.253), log γ-globin (p=0.001; R 2 =0.330), and log

HO-1 (p=0.001; R 2 =0.329) gene expressions (Figure 1).

Correlation of BACH1 Gene Expression to Clinical Data

BACH1 was not affected by transfusion history (p=0.6298), iron

overloading (p=0.6216), or iron chelation therapy (p=0.1743).

However, BACH1 correlation was significant in splenectomized

individuals (p=0.0085) (Figure 2).

Discussion

The heme-BACH1 transcription activation pathway provides

a mechanism for the cellular environment to maintain a

balanced homeostasis during erythroid differentiation when

the production and assembly of hemoglobin actively comes

together. While BACH1 functions to maintain a dormant

expression of the α- and β-globin genes, it could quickly

change with the increase of heme [13]. However, in HbE/β-

Figure 1. Correlations of BACH1 expression with age; reticulocyte

count; α-, β-, and γ-globin gene expression; and heme oxygenase

1 gene expression. A) Correlation of BACH1 with age (p=0.006),

B) correlation of BACH1 with reticulocyte number (µL) (p=0.01),

C) correlation of BACH1 with α-globin expression (p=0.002),

D) correlation of BACH1 with β-globin expression (p=0.001),

E) correlation of BACH1 with γ-globin expression (p=0.001),

F) correlation of BACH1 expression with heme oxygenase 1

expression (p=0.001).

Figure 2. BACH1 expression correlation to spleen sizes and

splenectomized hemoglobin E/β-thalassemia individuals

(p=0.0085).

18



thalassemia, the expression of the β-globin gene is reduced. We

studied the BACH1 gene expression in this disorder to elucidate

the function of BACH1 when the α/β-globin chain ratio balance

is compromised.

HbE/β-thalassemia red cells are known to have increased levels

of oxidative stress, which is caused by the excess free α-globin

chains available and the increased labile iron pool [14,15].

Cell damage or death caused by increased available reactive

oxygen species could be related to the natural cellular aging

process or iron overload in HbE/β-thalassemia as excess iron

absorption from the intestinal tract accumulates slowly over

time [15,16,17,18]. A study by Dohi et al. showed that BACH1

is involved in the repression of premature cellular senescence

induced by oxidative stress [19]. Therefore, the rise of BACH1

expression as age increases is perhaps to delay premature

cellular aging due to excessive oxidative stress.

As reticulocyte numbers reflect the bone marrow erythropoietic

activity, the degree of ineffective erythropoiesis due to harmful

precipitation of excess free α-globin chains in β-thalassemia

can be phenotypically typed [20,21,22]. The negative correlation

of BACH1 and reticulocyte count could reflect the role of BACH1

in repressing the excess α-globin gene to reduce the rate of

ineffective erythropoiesis [6].

HbE/β-thalassemia does not only have lower βE-globin chain

synthesis but also the presence of aberrantly spliced β E -globin

mRNA. Higher levels of aberrant compared to correctly spliced

β E -globin mRNA have been linked to increased severity [12,23].

BACH1 could function to decrease the production of β E -globin

to reduce the burden of aberrantly spliced β E -globin mRNA. The

non-association of beta genotypes to BACH1 expression could

be due to the masking of the genotype expression by HbE.

In terms of the lack of β-globin gene expression in HbE/βthalassemia,

compensatory increase of γ-globin chains to form

HbF with excess α-globin chains explains the indirect correlation

to BACH1 expression. To date, there has not been any study

done to examine the effect of BACH1 on the γ-globin gene.

However, if BACH1 does have a repressive role on γ-globin gene

expression, then BACH1 is maintaining cellular homeostasis by

regulating the expression of the γ-globin chain. Wickramasinghe

and Lee showed that large production of γ-globin chains does

not necessarily protect against extensive precipitations of

α-globin monomers [24].

BACH1 is also a well-known repressor of HO-1. De Franceschi

et al. showed that heme and HO-1 levels were both reduced in

β-thalassemia precursors compared to controls [8]. BACH1 could

be repressing HO-1 in β-thalassemia to prevent the cytotoxic

effect of excess free heme due to the lack of normal hemoglobin

formation and also to prevent the excessive accumulation of

heme degradation products as they possess the potential to

be cytotoxic beyond a certain threshold [25]. However, this

mechanism could be overwhelmed in splenectomized patients.

Splenectomy is performed when accelerated transfusions are

required to maintain adequate hemoglobin levels in the patient

[26]. A study on thalassemia intermedia patients showed

severe iron decompartmentalization in the red blood cells of

splenectomized patients compared to non-splenectomized

patients with significantly higher levels of membrane-bound

free iron, non-heme iron, and heme compounds [27]. Heme not

only suppresses the function of BACH1 but has also been found

to promote BACH1 degradation [28].

Conclusion

BACH1 plays a role in maintaining the microcellular homeostasis

in HbE/β-thalassemia by repressing the excess α-globin chains

and aberrantly spliced β E -globin mRNA as well as preventing

cytotoxic effects of excess free heme, although the amount

expressed may not be sufficient to alleviate the severity of

the HbE/β-thalassemia phenotype and it is abolished in the

presence of excessive heme. Further investigations to confirm

the pathways involved are necessary, perhaps by using a mouse

model.

Acknowledgments

The authors wish to thank the Director General of Health,

Malaysia, for permission to publish this paper. Funding of this

work is supported by the Fundamental Research Grant Scheme,

Ministry of Higher Education, Malaysia (04-04-10-837FR), and

a Research University Grant Scheme, Universiti Putra Malaysia

(04-02-11-1378RU), awarded to MIL. TYL is a recipient of a

MyBrain scholarship from the Ministry of Higher Education

(MOHE), Malaysia.

Ethics

Ethics Committee Approval: NMRR-10-1177-6947 and UPM/

FPSK/PADS/T7-MJKEtikaPer/F01, Informed Consent: It was

taken.


Concept: Mei I Lai, Design: Mei I Lai, Data Collection or

Processing: Tze Yan Lee, Logeswaran Muniandy, Jameela Sathar,

Analysis or Interpretation: Mei I Lai, Elizabeth George, Maha

Abdullah, Lai Kuan Teh, Tze Yan Lee, Logeswaran Muniandy,

Literature Search: Tze Yan Lee, Writing: Mei I Lai, Tze Yan Lee.




included.

19



References

1. Fucharoen S, Weatherall DJ. The hemoglobin E thalassemias. Cold Spring

Harb Perspect Med 2012;2:a011734.

2. Olivieri NF, Muraca GM, O’Donnell A, Premawardhena A, Fisher C,

Weatherall DJ. Studies in hemoglobin E beta-thalassaemia. Br J Haematol

2008;141:388-397.

3. Thein SL. Genetic insights into the clinical diversity of β-thalassaemia. Br J

Haematol 2004;124:264-274.

4. Igarashi K, Hoshino H, Muto A, Suwabe N, Nishikawa S, Nakauchi H,

Yamamoto M. Multivalent DNA binding complex generated by small Maf

and BACH1 as a possible biochemical basis for β-globin locus control region

complex. J Biol Chem 1998;273:11783-11790.

5. Sun J, Hoshino H, Takaku K, Nakajima O, Muto A, Suzuki H, Tashiro S,

Takahashi S, Shibahara S, Alam J, Taketo MM, Yamamoto M, Igarashi K.

Hemoprotein Bach1 regulates enhancer availability of heme oxygenase-1

gene. EMBO J 2002;19:5216-5224.

6. Tahara T, Sun J, Igarashi K, Taketani S. Heme-dependent up-regulation

of the α-globin gene expression by transcriptional repressor BACH1 in

erythroid cells. Biochem Biophys Res Commun 2004;324:77-85.

7. Tahara T, Sun J, Nakanishi K, Yamamoto M, Mori H, Saito T, Fujita H, Igarashi

K, Taketani S. Heme positively regulates the expression of beta-globin at the

locus control region via the transcriptional factor BACH1 in erythroid cells.

J Biol Chem 2004;279:5480-5487.

8. De Franceschi L, Bertoldi M, De Falco L, Santos Franco S, Ronzoni L, Turrini

F, Colancecco A, Camaschella C, Cappellini MD, Iolascon A. Oxidative

stress modulates heme synthesis and induces peroxiredoxin-2 as a novel

cytoprotective response in β-thalassemic erythropoiesis. Haematologica

2011;96:1595-1604.

9. Old JM. Hematological applications: hemoglobinopathies. Methods Mol

Med 2004;92:203-219.

10. Wong YC, George E, Tan KL, Yap SF, Chan LL, Tan JA. Molecular

characterisation and frequency of G g Xmn I polymorphism in Chinese and

Malay beta-thalassaemia patients in Malaysia. Malays J Pathol 2006;28:17-

21.

11. Lai MI, Jiang J, Silver N, Best S, Menzel S, Mijovic A, Colella S, Ragoussis

J, Garner C, Weiss MJ, Thein SL. Alpha-hemoglobin stabilising protein is a

quantitative trait gene that modifies the phenotype of beta-thalassaemia.

Br J Haematol 2006;133:675-682.

12. Watanapokasin Y, Winichagoon P, Fuchareon S, Wilairat P. Relative

quantitation of mRNA in beta-thalassemia/Hb E using real-time polymerase

chain reaction. Hemoglobin 2000;24:105-116.

13. Igarashi K, Sun J. The heme-BACH1 pathway in the regulation of oxidative

stress response and erythroid differentiation. Antioxid Redox Signal

2006;8:107-118.

14. Amer J, Goldfarb A, Fibach E. Flow cytometric analysis of the oxidative

status of normal and thalassemic red blood cells. Cytometry A 2004;60:73-

80.

15. Prus E, Fibach E. The labile iron pool in human erythroid cells. Br J Haematol

2008;142:301-307.

16. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing.

Nature 2000;408:239-247.

17. Ginzburg Y, Rivella S. β-Thalassemia: a model for elucidating the dynamic

regulation of ineffective erythropoiesis and iron metabolism. Blood

2011;118:4321-4330.

18. Olivieri NF. The beta-thalassemias. N Engl J Med 1999;341:99-109.

19. Dohi Y, Ikura T, Hoshikawa Y, Katoh Y, Ota K, Nakanome A, Muto A, Omura S,

Ohta T, Ito A, Yoshida M, Noda T, Igarashi K. Bach1 inhibits oxidative stressinduced

cellular senescence by impeding p53 function on chromatin. Nat

Struct Mol Biol 2008;15:1246-1254.

20. Cortellazzi LC, Teixeira SM, Borba R, Gervásio S, Cintra CS, Grotto HZW.

Reticulocyte parameters in hemoglobinopathies and iron deficiency

anemia. Rev Bras Hematol Hemoter 2003;25:97-102.

21. Lamchiagdhase P, Pattanapanyasat K, Muangsup W. Reticulocyte counting

in thalassemia using different automated technologies. Laboratory

Hematology 2000;6:73-78.

22. Nathan DG, Gunn RB. Thalassemia: the consequences of unbalanced

hemoglobin synthesis. Am J Med 1966;41:815-830.

23. Tubsuwan A, Munkongdee T, Jearawiriyapaisarn N, Boonchoy C,

Winichagoon P, Fucharoen S, Svasti S. Molecular analysis of globin gene

expression in different thalassaemia disorders: individual variation of β(E)

pre-mRNA splicing determine disease severity. Br J Haematol 2011;154:

635-643.

24. Wickramasinghe SN, Lee MJ. Observations on the relationship between

gamma-globin chain content and globin chain precipitation in thalassaemic

erythroblasts and on the composition of erythroblastic inclusions in HbE/

beta-thalassaemia. Eur J Haematol 1997;59:305-309.

25. Shibahara S, Nakayama M, Kitamuro T, Udono-Fujimori R, Takahashi K.

Repression of heme oxygenase-1 expression as a defense strategy in

humans. Exp Biol Med (Maywood) 2003;228:472-473.

26. Smith CH, Erlandson ME, Stern G, Schulman I. The role of splenectomy in

the management of thalassemia. Blood 1960;15:197-211.

27. Tavazzi D, Duca L, Graziadei G, Comino A, Fiorelli G, Cappellini MD.

Membrane-bound iron contributes to oxidative damage of betathalassaemia

intermedia erythrocytes. Br J Haematol 2001;112:48-50.

28. Hada H, Shiraki T, Watanabe-Matsui M, Igarashi K. Hemopexin-dependent

heme uptake via endocytosis regulates the Bach1 transcription repressor and

heme oxygenase gene activation. Biochim Biophys Acta 2014;1840:2351-

2360.

20

RESEARCH ARTICLE

DOI: 10.4274/tjh.2015.0142


The Feasibility of Magnetic Resonance Imaging for Quantification of

Liver, Pancreas, Spleen, Vertebral Bone Marrow, and Renal Cortex R2*

and Proton Density Fat Fraction in Transfusion-Related Iron Overload

Manyetik Rezonans Görüntülemenin Transfüzyon İlişkili Demir Birikimi Bulunan Hastalarda

Karaciğer, Pankreas, Dalak, Vertebral Kemik İliği ve Böbrek Korteksi R2* ve Proton Dansite

Yağ Oranı Ölçümünde Uygulanabilirliği

İlkay S. İdilman 1,2 , Fatma Gümrük 3 , Mithat Haliloğlu 1 , Muşturay Karçaaltıncaba 1,2

1Hacettepe University Faculty of Medicine, Department of Radiology, Ankara, Turkey

2Hacettepe University Faculty of Medicine, Liver Imaging Team, Ankara, Turkey

3Hacettepe University Faculty of Medicine, Department of Pediatrics, Division of Pediatric Hematology, Ankara, Turkey

Abstract

Objective: We aimed to evaluate the feasibility of quantification of

liver, pancreas, spleen, vertebral bone marrow, and renal cortex R2*

and magnetic resonance imaging-proton density fat fraction (MRI-

PDFF) and to evaluate the correlations among them in patients with

transfusion-related iron overload.

Materials and Methods: A total of 9 patients (5 boys, 4 girls) who

were referred to our clinic with suspicion of hepatic iron overload

were included in this study. All patients underwent T1-independent

volumetric multi-echo gradient-echo imaging with T2* correction and

spectral fat modeling. MRI examinations were performed on a 1.5 T

MRI system.

Results: All patients had hepatic iron overload. Severe hepatic iron

overload was recorded in 5/9 patients (56%), and when we evaluated

the PDFF maps of these patients, we observed an extensive patchy

artifact in the liver in 4 of 5 patients (R2* greater than 671 Hz).

When we performed MRI-PDFF measurements despite these artifacts,

we observed artifactual high MRI-PDFF values. There was a close

correlation between average pancreas R2* and average pancreas MRI-

PDFF (p=0.003, r=0.860). There was a significant correlation between

liver R2* and average pancreas R2* (p=0.021, r=0.747), liver R2* and

renal cortex R2* (p=0.020, r=0.750), and average pancreas R2* and

renal cortex R2* (p=0.003, r=0.858). There was a significant negative

correlation between vertebral bone marrow R2* and age (p=0.018, r=-

0.759).

Conclusion: High iron content of the liver, especially with a T2*

value shorter than the first echo time can spoil the efficacy of

PDFF calculation. Fat deposition in the pancreas is accompanied by

pancreatic iron overload. There is a significant correlation between

hepatic siderosis and pancreatic siderosis. Renal cortical and pancreatic

siderosis are correlated, too.

Keywords: Iron overload, Liver, Pancreas, R2*, Magnetic resonance

imaging-proton density fat fraction

Öz

Amaç: Bu çalışmada, transfüzyon ilişkili demir birikimi bulunan

hastalarda karaciğer, pankreas, dalak, vertebral kemik iliği ve böbrek

korteksi R2* ve manyetik rezonans görüntüleme-proton dansite yağ

oranı (MRG-PDYO) ölçümünün uygulanabilirliği ve bunlar arasındaki

ilişkiyi değerlendirmeyi amaçladık.

Gereç ve Yöntemler: Çalışmaya, karaciğer demir birikimi şüphesi

ile kliniğimize gönderilmiş olan toplam 9 hasta (5 erkek, 4 kız) dahil

edildi. Tüm hastalara T2* düzeltmeli ve spektral yağ modellemeli T1

bağımsız volumetrik multi-eko gradient-eko görüntüleme yapıldı.

MRG incelemeleri 1.5 T MRG sistemi ile yapıldı.

Bulgular: Tüm hastalarda karaciğerde demir birikimi mevcuttu. Dokuz

hastanın beşinde (%56) ağır demir birikimi saptandı ve bu hastaların

PDYO haritaları incelendiğinde 4 hastada (R2* değeri 671 Hz’den fazla

olanlarda) karaciğerde yaygın yamasal artefaktlar olduğu saptandı.

Bu artefaktlara rağmen MRG-PDYO ölçümü yapıldığında normalden

çok yüksek MRG-PDYO değerleri belirlendi. Ortalama pankreas R2* ve

ortalama pankreas MRG-PDYO arasında (p=0,003, r=0,860), karaciğer

R2* and ortalama pankreas R2* arasında (p=0,021, r=0,747), karaciğer

R2* ve renal korteks R2* arasında (p=0,020, r=0,750) ve ortalama

pankreas R2* ve renal korteks R2* arasında (p=0,003, r=0,858) anlamlı

korelasyon saptandı. Vertebral kemik iliği R2* ve yaş arasında anlamlı

negatif korelasyon saptandı (p=0,018, r=-0,759).

Sonuç: Karaciğerdeki yüksek demir birikimi özellikle de T2* değeri

ilk eko zamanından daha kısa olduğunda PDYO hesaplamasını

bozabilmektedir. Pankreastaki yağ birikimi pankreatik demir birikimine

eşlik edebilmektedir. Karaciğer ve pankreas siderozisi birbiri ile ilişki

göstermektedir. Böbrek korteksi ve pankreas siderozisi arasında da

ilişki mevcuttur.

Anahtar Sözcükler: Demir birikimi, Karaciğer, Pankreas, R2*, Manyetik

rezonans görüntüleme-proton dansite yağ oranı

Address for Correspondence/Yazışma Adresi: Muşturay Karçaaltıncaba, M.D.,

Hacettepe University Faculty of Medicine,

Department of Radiology, Ankara, Turkey

E-mail : musturayk@yahoo.com

Received/Geliş tarihi: April 01, 2015

Accepted/Kabul tarihi: June 15, 2015

21

İdilman İS, et al: Magnetic Resonance IDEAL-IQ in Transfusion-Related Iron Overload


Introduction

Iron is an essential nutrient for all human cells [1,2]. Under

normal circumstances, intake and excretion of iron is balanced

within a daily range of 1-2 mg [3]. There is no effective way of

excretion of iron from the body; therefore, if the total amount

of income exceeds outcome, such as in cases of increased

intestinal absorption, long-term transfusion therapies, or

excess parenteral iron treatment, total body iron increases.

Transfusion-related iron overload is one of the leading causes

of iron overload. It primarily depends on repetitive transfusions

that bring a burden of excess iron, especially after 40 to 50

transfusions that saturate the capacity of reticuloendothelial

system [4]. After saturation, iron accumulates in parenchymal

organs like the liver, pancreas, myocardium, and endocrine

glands, which leads to tissue damage and fibrosis [3].

Iron is mainly stored in the liver and the iron content of the liver

is an indirect marker of total body iron [5]. Hence, quantification

of hepatic iron content is used for guiding and monitoring iron

chelation therapies in transfusion-related iron overload [1]. Current

methods for quantification of hepatic iron consist of liver biopsy

and imaging. Percutaneous liver biopsy is the current reference

method for quantification of hepatic iron content. However, it

is an invasive procedure that can bring serious complications,

and acquired small sample sizes in liver biopsy are insufficient

to represent the whole organ. In addition, iron overload can be

organ-specific and estimation of iron accumulation in different

organs such as the pancreas, spleen, vertebral bone marrow, and

kidneys requires noninvasive techniques.

Magnetic resonance imaging (MRI) methods including signal

intensity ratio and relaxometry are promising for quantification

of hepatic iron overload compared with liver biopsy [6,7,8].

However, coexistence of fat influences the measurement of

hepatic iron content because of the spectral complexity of fat

[9]. Vice versa, hepatic iron overload influences the measurement

of hepatic fat content in chemical shift techniques due to its

T2* shortening effect [10]. A recent technique, MR IDEAL-IQ

(Iterative Decomposition of water and fat with Echo Asymmetry

and Least square estimation), which was defined primarily

for quantification of the fat fraction of tissue, quantifies R2*

by correcting the fat-dependent confounding factors [11].

However, the former technique’s accuracy was found to be

lower, especially in patients with T2* values below 1 ms [12].

Additionally, the feasibility of this technique was not evaluated

in the estimation of pancreas, spleen, vertebral bone marrow,

and renal cortex iron overload. In the present study, we aimed

to evaluate the feasibility of MR IDEAL-IQ in the quantification

of liver, pancreas, spleen, vertebral bone marrow, and renal

cortex R2* and MRI-proton density fat fraction (MRI-PDFF)

and to evaluate the correlations among them in patients with

transfusion-related iron overload.


Patients

This was a retrospective cross-sectional study. A total of 9 patients

(5 boys, 4 girls) who were referred to our clinic with suspicion of

hepatic iron overload and examined with MR IDEAL-IQ between

August 2010 and November 2010 were included in the study.

All of the patients had a history of repetitive transfusions, 8 of

them with a diagnosis of beta thalassemia major and 1 of them

with non-Hodgkin lymphoma (NHL). Body mass index (BMI) was

calculated as weight in kilograms divided by height in meters

squared.

Magnetic Resonance Imaging Examination

MR images were acquired with a 1.5 T HDxt MRI system (GE

Healthcare, Milwaukee, WI, USA). The subjects were examined

in the supine position. An 8-channel phased array body coil

was used for acquisition. A 3-plane gradient echo localizer

sequence was performed at the beginning of the examination.

The MRI protocol included the IDEAL-IQ sequence. This is a 3D

volumetric imaging sequence used to create T2* and triglyceride

fat fraction maps from a single breath-hold acquisition. The

technique was used to estimate R2* (1/T2*) and PDFF (watertriglyceride

fat separation) in the liver simultaneously in a single

acquisition. Afterwards, a correction was applied to the resulting

PDFF maps to correct for T2* effects. Six gradient echoes were

applied to reconstruct water and triglyceride fat images,

relative triglyceride fat fractions, and R2* maps. The IDEAL-IQ

sequence uses a novel “complex field map” to incorporate the

T2* and field inhomogeneity effects into the overall multi-echo

acquisition signal model. It was shown by Yu et al. [11] that,

by acquiring a 6-echo image and estimating a complex field

map using an iterative least square estimation algorithm, it is

possible to achieve fat-water separation and T2* estimation in a

single breath-hold acquisition.

The parameters of this sequence were TR: 12.9 ms, FOV: 35-

40 cm, matrix: 224x160, 125 kHz bandwidth, flip angle: 5 °,

and slice thickness: 5 mm. A single 3D slab with 44 to 56 slices

was acquired. We acquired data sets with 6 different echoes

ranging from 1.6 ms to 9.8 ms. A 2D self-calibrated parallel

imaging technique called auto calibrating reconstruction of

Cartesian sampling was used with an acceleration factor of 2.

The images were processed using the software provided by the

manufacturer to create water, fat, in-phase, opposed-phase,

R2*, and fat fraction maps.

Image Analysis

By using a work station (AW 4.4, GE Healthcare), a radiologist

placed an elliptic region of interest (ROI) of approximately

4 cm 2 in Couinaud segments 5-6 on the PDFF maps and the

22



R2* maps, avoiding blood vessels, bile ducts, and artifacts. An

elliptic ROI of approximately 1 cm2 for the pancreatic head,

body, and tail on the PDFF maps and the R2* maps was placed

for pancreatic measurements and the arithmetic mean was

calculated. The same procedure was performed for the spleen

with a ROI of 2 cm2, for the L1 or L2 vertebral corpus with a

ROI of 2 cm2, and for the renal cortex with a ROI of 1 cm 2 .

The patients with a T2* value under 18 ms were included in the

hepatic iron overload group [13]. The patients with a T2* value

under 2 ms were included in the severe hepatic iron overload

group. The patients with pancreatic R2* values between 30 and

100 Hz were included in the mild pancreatic siderosis group,

those between 100 and 400 Hz were included in the moderate

pancreatic siderosis group, and those >400 Hz were included in

the severe pancreatic siderosis group [14].

in the study population. The results are summarized in Table 1.

All of the patients had hepatic iron overload. Severe hepatic

iron overload was recorded in 5/9 patients (56%). When we

evaluated the PDFF maps of severe hepatic iron overload

patients, we observed an extensive patchy artifact in the

liver in the majority of them (4/5) (Figures 1 and 2). When we

performed MRI-PDFF measurements even with these artifacts,

we observed unexpectedly high MRI-PDFF values. The liver

R2* values of these patients were higher (range: 671.1-773.9

Hz) when compared with the patient with severe hepatic iron

overload without artifacts (528.70 Hz).

Mean average pancreas R2* value was 236.8 Hz (47.5-496.9

Hz) and mean average pancreas MRI-PDFF value was 14.7%

The degree of association between continuous and/or ordinal

variables was calculated by Pearson correlation coefficient (r)

and p<0.05 was considered significant.

Results

Eight patients with beta thalassemia major and 1 patient with

NHL were included in this study (male/female: 5/4). The mean age

of the patients was 17.4 years (range: 13-22 years). Thalassemia

patients had a diagnosis of beta thalassemia major since infancy

and were treated with repetitive red blood cell transfusion and

iron chelation therapy. The patient with NHL had an 8-year

disease history with two bone marrow transplantations and

multiple red blood cell transfusion therapies. Three of the

patients had a history of myocardial iron overload, and two of

them had hypogonadotropic hypogonadism. The mean BMI of

the patients was 19.1 kg/m2 (range: 17.6-20.7 kg/m 2 ).

The mean ferritin level was 5933.1 ng/mL (range: 600-29950

ng/mL), mean liver R2* value was 424.8 Hz (range: 60.9-773.9

Hz), and mean liver MRI-PDFF was 22.4% (range: 1.5%-63.9%)

Figure 1. The magnetic resonance imaging-proton density fat

fraction (top row) and R2* (bottom row) maps of the patients

with severe hepatic iron overload. Except for Patient 1, all patients

with severe hepatic iron overload demonstrated a patchy artifact

in proton density fat fraction maps.

Figure 2. The magnetic resonance imaging-proton density fat

fraction (top row) and R2* (bottom row) maps of the patients

with milder hepatic iron overload. There is no artifact in patients

with milder hepatic iron overload.

Table 1. The characteristics and magnetic resonance imaging findings of patients with transfusion-related iron overload.

Subject Sex Age

(Years)

Primary

Diagnosis

Serum Ferritin

Level (ng/mL)

SHIO Liver R2*

(Hz)

Liver T2* (ms)

1 M 19 NHL 29950 + 528.7 1.89 1.5

2 F 19 β-TM 600 - 68.7 14.56 2.1

3 F 22 β-TM 1232 - 60.9 16.42 2.2

4 F 19 β-TM 8177 + 722.6 1.38 32.6

5 F 14 β-TM 4709 + 671.1 1.49 30.5

6 M 13 β-TM 3106 - 105.4 9.49 2.7

7 M 17 β-TM 1381 - 155.9 6.41 3.6

8 M 16 β-TM 1931 + 773.9 1.29 63.9

9 M 18 β-TM 2312 + 736.4 1.36 62.6

Liver MRI-PDFF

(%)

MRI-PDFF: Magnetic resonance imaging-proton density fat fraction, NHL: non-Hodgkin lymphoma, β-TM: beta thalassemia major, SHIO: severe hepatic iron overload, M: male, F:

female.

23



(1.2%-42.2%). All patients had pancreatic siderosis; 2 of them

had mild siderosis, 5 of them had moderate siderosis, and 2 of

them had severe siderosis (Table 2). There was a close correlation

between average pancreas R2* and average pancreas MRI-PDFF

(p=0.003, r=0.860) (Figure 3). We could not perform splenic

measurements in 3 patients as they were splenectomized. Mean

spleen R2* value was 142.1 Hz (29.9-224.2 Hz) and mean spleen

MRI-PDFF was 2.1% (1.3%-3.1%). Mean vertebral bone marrow

R2* value was 289 Hz (151.4-548.8 Hz) and mean vertebral bone

marrow MRI-PDFF was 13.3% (0.1%-60%). Mean average renal

cortex R2* value was 21.9 Hz (11.2-42.2 Hz) and mean average

renal cortex MRI-PDFF was 0.8% (0%-1.5%).

Figure 3. Scatterplot shows the correlation between pancreatic

R2* and magnetic resonance imaging-proton density fat fraction

(p=0.003, r=0.860).

When we evaluated the correlations among patient age, serum

ferritin level, and MRI findings, we observed a significant

correlation between liver R2* and average pancreas R2* (p=0.021,

r=0.747) (Figure 4). There was also a significant correlation among

liver R2* and renal cortex R2* (p=0.020, r=0.750) (Figure 5) and

Figure 4. Scatterplot shows the correlation between liver R2* and

average pancreas R2* (p=0.021, r=0,747).

Figure 5. Scatterplot shows the correlation between liver R2* and

renal cortex R2* (p=0.020, r=0.750).

Table 2. Pancreas, spleen, vertebral bone marrow, and renal cortical R2* and magnetic resonance imaging-proton density fat

fraction of the patients.

Subject

Average

Pancreas

R2* (Hz)

Average

Pancreas

MRI-PDFF

(%)

Average

Spleen R2*

(Hz)

Average

Spleen MRI-

PDFF (%)

Vertebral Bone

Marrow

R2* (Hz)

Vertebral Bone

Marrow

MRI-PDFF (%)

Renal

Cortex R2*

(Hz)

1 154.8 1.9 215.7 1.6 206.7 60 19.7 0.7

2 177 12.7 N/A N/A 151.4 11.4 11.8 1.6

3 76.4 1.6 29.9 1.3 238.6 1.7 11.2 0.7

4 350.6 42.2 224.4 2.4 308 2.6 24.8 1.1

5 178.5 7.4 184 2.2 467.3 1.9 19.5 1.4

6 216.9 9 164.5 3.1 548.8 40 18.4 0.4

7 47.5 1.2 N/A N/A 196.3 0.1 20 0

8 432.9 25 33.8 1.7 252.6 0.8 29.7 0.6

9 496.9 31.5 N/A N/A 231 1.2 42.2 1.1

MRI-PDFF: Magnetic resonance imaging-proton density fat fraction, N/A: not applicable.

Renal Cortex

MRI-PDFF

(%)

24



average pancreas R2* and renal cortex R2* (p=0.003, r=0.858)

(Figure 6). There was a significant negative correlation between

vertebral bone marrow R2* and age (p=0.018, r=-0.759) (Figure

7). No other significant correlation was observed between patient

age, serum ferritin level, and liver, pancreas, spleen, vertebral

bone marrow, and renal cortex R2* and MRI-PDFF values.

Discussion

Noninvasive assessment of hepatic iron content in transfusionrelated

iron overload, which predominantly involves the

pediatric population, is an important issue that was studied

before by many investigators [6,7,8]. However, within the

obesity epidemic, fat accumulation can coexist in the liver in

such patients. The presence of both fat and iron in the liver has

a confounding effect on the quantification of each one with

MRI techniques [15]. In the present study we evaluated the

Figure 6. Scatterplot shows the correlation between average

pancreas R2* and renal cortex R2* (p=0.003, r=0.858).

Figure 7. Scatterplot shows the correlation between vertebral

bone marrow R2* and age (p=0.018, r=-0.759).

feasibility of a recently described method, IDEAL-IQ, for liver,

pancreas, spleen, vertebral bone marrow, and renal cortex iron

and fat quantification in patients with transfusion-related iron

overload.

Previously, Liau et al. evaluated the effect of changes in R2*

caused by an intravenous infusion of super paramagnetic iron

oxide contrast agent in quantification of liver fat fraction

with IDEAL-IQ and observed that the IDEAL-IQ method of fat

quantification is robust to changes in R2* [16]. However, the

highest liver R2* value observed in this study was 212 Hz, which

was distinctly lower than our patients with severe hepatic iron

overload. Another study by Vasanawala et al. evaluated the

clinical feasibility of weighted least squares T2* IDEAL, which

is a similar technique to the one that we used, in transfusionrelated

iron overload and concluded that this technique is

feasible in clinical applications [12]. In that study, investigators

also observed significant hepatic steatosis in the liver in three

patients that had milder hepatic iron overload compared with

our study.

In our study, we observed artifacts and unexpectedly high

MRI-PDFF values in PDFF maps of patients with a T2* value

approximately under 1.6 ms with the MR-IDEAL technique. As

discussed by Vasanawala et al. [12], estimation of a T2* value

under the first echo time used in the technique is challenging.

However, they did not mention that there is a problem in PDFF

maps in such patients. In previous studies that evaluated the

utility of this technique in quantification of hepatic steatosis

in adults [17] and individuals predominantly consisting of

pediatric patients [18] with biopsy-proven nonalcoholic fatty

liver disease, the authors did not observe such high MRI-PDFF

values, even in patients with severe steatosis, as we observed in

the present study. Additionally, the patients in our study had

no risk factors for hepatic steatosis or any hepatic steatosis,

supporting additional imaging findings and confirming the

inability of this technique in quantification of fat fraction in

severe hepatic iron overload.

All of the patients in our study had pancreatic siderosis with

variable degrees. Diabetes mellitus is one of the most common

endocrine problems in thalassemia patients and pathogenesis

depends on beta-cell dysfunction according to increased iron

deposition [19]. It has been shown that early application of

chelation therapy is protective for diabetes [20]. Pancreatic

iron overload causes cell death and fatty transformation in

pancreatic tissue [21]. An MRI technique that is feasible for both

fat fraction quantification and R2* values would be valuable in

assessing the pancreas in patients with transfusion-related iron

overload. In our study, we could demonstrate both pancreatic

siderosis and steatosis with MRI-PDFF. In addition, we observed

a significant correlation between pancreatic siderosis and

pancreatic steatosis, which was presumed but not demonstrated

in previous studies.

25

İdilman İS, et al: Magnetic Resonance IDEAL-IQ in Transfusion-Related Iron Overload Turk J Hematol 2016;33:21-27

Papakonstantinou et al. previously investigated the correlations

between hepatic, splenic, pancreatic, vertebral bone marrow,

and myocardial siderosis and did not find a correlation between

pancreatic siderosis and hepatic and splenic siderosis [22].

Another study by Argyropoulou et al. did not find a correlation of

T2 among the liver, bone marrow, pancreas, and pituitary gland

[23]. However, in our study, we observed significant correlation

between hepatic siderosis and pancreatic siderosis, hepatic

siderosis, and renal cortical siderosis and pancreatic siderosis

and renal cortical siderosis. We could not find a correlation

between liver R2* and either spleen or bone marrow R2*. On

the contrary, we observed a significant negative correlation

between vertebral bone marrow R2* and age, which was not

mentioned in previous studies.

One of the limitations of our study is the small sample size.

However, there were patients with severe hepatic iron overload,

which demonstrates the inability of the technique in fat

quantification in short T2* values. Another limitation is the

absence of histology assessment for accurate quantification of

iron and fat content of the liver and other tissues. Furthermore,

it is not feasible to evaluate iron and fat content of all organs

with biopsy. On the other hand, we could easily demonstrate

R2* values of different tissues just with an ROI placement

with this technique, which is valuable in transfusion-related

iron overload. In addition, we could demonstrate relationships

between different tissues’ R2* values and the relationships

between R2* values and fat fractions in tissues with a R2* value

below approximately 671 Hz.

Conclusion

High iron content of the liver, especially with a T2* value shorter

than the first echo time can spoil the efficacy of PDFF calculation.

Fat deposition in the pancreas is accompanied by pancreatic

iron overload. There is a significant correlation between hepatic

siderosis and pancreatic siderosis. Renal cortical and pancreatic

siderosis are correlated, too.

Acknowledgments

Muşturay Karçaaltıncaba was supported by the Turkish Academy

of Sciences (TÜBA) in the framework of the Young Scientist

Award Program (EA-TÜBA-GEBİP/2011).

Ethics

Ethics Committee Approval: Hacettepe University Ethics

Committee, Informed Consent: It was taken.


Concept: Muşturay Karçaaltıncaba, Design: Muşturay

Karçaaltıncaba, Data Collection or Processing: İlkay S. İdilman,

Fatma Gümrük, Mithat Haliloğlu, Muşturay Karçaaltıncaba,

Analysis or Interpretation: İlkay S. İdilman, Literature Search:

İlkay S. İdillman, Muşturay Karçaaltıncaba, Writing: İlkay S.

İdilman, Muşturay Karçaaltıncaba.




included.

References

1. Brittenham GM, Cohen AR, McLaren CE, Martin MB, Griffith PM, Nienhuis

AW, Young NS, Allen CJ, Farrell DE, Harris JW. Hepatic iron stores and plasma

ferritin concentration in patients with sickle cell anemia and thalassemia

major. Am J Hematol 1993;42:81-85.

2. Alustia JM, Castiella A, De Juan MD, Emparanza JI, Artetxe J, Uranga

M. Iron overload in the liver diagnostic and quantification. Eur J Radiol

2007;61:499-506.

3. Andrews NC. Disorders of iron metabolism. N Engl J Med 1999;341:1986-

1995.

4. Siegelman ES, Mitchell DG, Semelka RC. Abdominal iron deposition:

metabolism, MR findings, and clinical importance. Radiology 1996;199:13-22.

5. Angelucci E, Brittenham GM, McLaren CE, Ripalti M, Baronciani D, Giardini

C, Galimberti M, Polchi P, Lucarelli G. Hepatic iron concentration and total

body iron stores in thalassemia major. N Eng J Med 2000;343:327-331.

6. Gandon Y, Olivie D, Guyader D, Aubé C, Oberti F, Sebille V, Deugnier Y. Noninvasive

assessment of hepatic iron stores by MRI. Lancet 2004;363:357-

362.

7. St Pierre TG, Clark PR, Chua-Anusorn W, Fleming AJ, Jeffrey GP, Olynyk JK,

Pootrakul P, Robins E, Lindeman R. Noninvasive measurement and imaging

of liver iron concentrations using proton magnetic resonance. Blood

2005;105:855-861.

8. Wood JC, Enriquez C, Ghugre N, Tyzka JM, Carson S, Nelson MD, Coates TD.

MRI R2 and R2* mapping accurately estimates hepatic iron concentration

in transfusion-dependent thalassemia and sickle cell disease patients. Blood

2005;106:1460-1465.

9. Sirlin CB, Reeder SB. Magnetic resonance imaging quantification of liver

iron. Magn Reson Imaging Clin N Am 2010;18:359-381.

10. Westphalen AC, Qayyum A, Yeh BM, Merriman RB, Lee JA, Lamba A, Lu Y,

Coakley FV. Liver fat: effect of hepatic iron deposition on evaluation with

opposed-phase MR imaging. Radiology 2007;242:450-455.

11. Yu H, McKenzie CA, Shimakawa A, Vu AT, Brau AC, Beatty PJ, Pineda AR,

Brittain JH, Reeder SB. Multiecho reconstruction for simultaneous water-fat

decomposition and T2* estimation. J Magn Reson Imaging 2007;26:1153-

1161.

12. Vasanawala SS, Yu H, Shimakawa A, Jeng M, Brittain JH. Estimation of liver

T2 in transfusion-related iron overload in patients with weighted least

squares T2 IDEAL. Magn Reson Med 2012;67:183-190.

13. Westwood M, Anderson LJ, Firmin DN, Gatehouse PD, Charrier CC, Wonke

B, Pennell DJ. A single breath-hold multiecho T2* cardiovascular magnetic

resonance technique for diagnosis of myocardial iron overload. J Magn

Reson Imaging 2003;18:33-39.

14. Noetzli LJ, Papudesi J, Coates TD, Wood JC. Pancreatic iron loading predicts

cardiac iron loading in thalassemia major. Blood 2009;114:4021-4026.

15. Sharma P, Altbach M, Galons JP, Kalb B, Martin DR. Measurement of liver fat

fraction and iron with MRI and MR spectroscopy techniques. Diagn Interv

Radiol 2014;20:17-26.

26



16. Liau J, Shiehmorteza M, Girard OM, Sirlin CB, Bydder M. Evaluation of MRI

fat fraction in the liver and spine pre and post SPIO infusion. Magn Reson

Imag 2013;31:1012-1016.

17. Idilman IS, Aniktar H, Idilman R, Kabacam G, Savas B, Elhan A, Celik A, Bahar

K, Karcaaltincaba M. Hepatic steatosis: quantification by proton density fat

fraction with MR imaging versus liver biopsy. Radiology 2013;267:767-775.

18. Tang A, Tan J, Sun M, Hamilton G, Bydder M, Wolfson T, Gamst AC, Middleton

M, Brunt EM, Loomba R, Lavine JE, Schwimmer JB, Sirlin CB. Nonalcoholic

fatty liver disease: MR imaging of liver proton density fat fraction to assess

hepatic steatosis. Radiology 2013;267:422-431.

19. Tiosano D, Hochberg Z. Endocrine complications of thalassemia. J Endocrinol

Invest 2001;24:716-723.

20. Brittenham GM, Griffith PM, Nienhuis AW, McLaren CE, Young NS, Tucker

EE, Allen CJ, Farrell DE, Harris JW. Efficacy of deferoxamine in preventing

complications of iron overload in patients with thalassemia major. N Engl J

Med 1994;331:567-573.

21. Midiri M, Lo Casto A, Sparacia G, D’Angelo P, Malizia R, Finazzo M, Montalto

G, Solbiati L, Lagalla R, De Maria M. MR imaging of pancreatic changes

in patients with transfusion dependent beta-thalassemia major. AJR Am J

Roentgenol 1999;173:187-192.

22. Papakonstantinou O, Alexopoulou E, Economopoulos N, Benekos O,

Kattamis A, Kostaridou S, Ladis V, Efstathopoulos E, Gouliamos A, Kelekis

NL. Assessment of iron distribution between liver, spleen, pancreas, bone

marrow and myocardium by means of R2 relaxometry with MRI in patients

β-thalassemia major. J Magn Reson Imag 2009;29:853-859.

23. Argyropoulou MI, Kiortsis DN, Astrakas L, Metafratzi Z, Chalissos N, Efremidis

SC. Liver, bone marrow, pancreas and pituitary gland iron overload in

young and adult thalassemic patients: a T2 relaxometry study. Eur Radiol

2007;17:3025-3030.

27

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0181


Freezing of Apheresis Platelet Concentrates in 6% Dimethyl

Sulfoxide: The First Preliminary Study in Turkey

Aferez Trombosit Konsantrelerinin Yüzde 6’lık Dimetil Sülfoksitte Dondurulması: İlk Türkiye

Çalışmasının Başlatılması

Soner Yılmaz 1 , Rıza Aytaç Çetinkaya 1 , İbrahim Eker 2 , Aytekin Ünlü 3 , Metin Uyanık 4 , Serkan Tapan 4 , Ahmet Pekoğlu 1 , Aysel Pekel 5 ,

Birgül Erkmen 6 , Uğur Muşabak 5 , Sebahattin Yılmaz 1 , İsmail Yaşar Avcı 7 , Ferit Avcu 6 , Emin Kürekçi 2 , Can Polat Eyigün 7

1Gülhane Military Medical Academy, Blood Training Center and Blood Bank, Ankara, Turkey

2Gülhane Military Medical Academy, Division of Pediatric Hematology, Ankara, Turkey

3Gülhane Military Medical Academy, Department of General Surgery, Ankara, Turkey

4Gülhane Military Medical Academy, Department of Medical Biochemistry, Ankara, Turkey

5Gülhane Military Medical Academy, Division of Immunology and Allergy, Ankara, Turkey

6Gülhane Military Medical Academy, Division of Hematology, Ankara, Turkey

7Gülhane Military Medical Academy, Department of Infectious Disease and Clinical Microbiology, Ankara, Turkey

Abstract

Objective: Transfusion of platelet suspensions is an essential part of patient

care for certain clinical indications. In this pioneering study in Turkey, we aimed

to assess the in vitro hemostatic functions of platelets after cryopreservation.

Materials and Methods: Seven units of platelet concentrates were obtained

by apheresis. Each apheresis platelet concentrate (APC) was divided into 2

equal volumes and frozen with 6% dimethyl sulfoxide (DMSO). The 14 frozen

units of APCs were kept at -80 °C for 1 day. APCs were thawed at 37 °C and

diluted either with autologous plasma or 0.9% NaCl. The volume and residual

numbers of leukocytes and platelets were tested in both before-freezing and

post-thawing periods. Aggregation and thrombin generation tests were used

to analyze the in vitro hemostatic functions of platelets. Flow-cytometric

analysis was used to assess the presence of frozen treated platelets and their

viability.

Results: The residual number of leukocytes in both dilution groups was

<1x106. The mean platelet recovery rate in the plasma-diluted group

(88.1±9.5%) was higher than that in the 0.9% NaCl-diluted group (63±10%).

These results were compatible with the European Directorate for the Quality

of Medicines quality criteria. Expectedly, there was no aggregation response

to platelet aggregation test. The mean thrombin generation potential of postthaw

APCs was higher in the plasma-diluted group (2411 nmol/L per minute)

when compared to both the 0.9% NaCl-diluted group (1913 nmol/L per

minute) and the before-freezing period (1681 nmol/L per minute). The flowcytometric

analysis results for the viability of APCs after cryopreservation

were 94.9% and 96.6% in the plasma and 0.9% NaCl groups, respectively.

Conclusion: Cryopreservation of platelets with 6% DMSO and storage at -80

°C increases their shelf life from 7 days to 2 years. Besides the increase in

hemostatic functions of platelets, the cryopreservation process also does not

affect their viability rates.

Keywords: Frozen platelets, Flow-cytometric analysis, In vivo thrombin

generation test

Öz

Amaç: Trombosit süspansiyonlarının transfüzyonu, belirli klinik

endikasyonlarda hastaların tedavisinin önemli bir parçasıdır. Bu çalışma

ile Türkiye’de ilk kez olmak üzere trombositlerin in vitro hemostatik

fonksiyonlarının kriyopreservasyon işleminden sonra değerlendirilmesi

amaçlanmıştır.

Gereç ve Yöntemler: Çalışmamızda 7 ünite trombosit süspansiyonu aferez

yöntemiyle elde edildi. Her aferez trombosit konsantresi (ATK) iki eşit hacime

ayrıldıktan sonra %6 dimetil sülfoksit (DMSO) kullanılarak donduruldu.

Dondurulmuş 14 ünite ATK -80 °C’de bir gün süre ile bekletildi. ATK’lar 37

°C’de çözdürüldükten sonra otolog plazma veya %0,9 NaCl kullanılarak dilüe

edildi. ATK’ların dondurma öncesi ve çözülme işlemi sonrası; hacim, rezidüel

lökosit ve trombosit sayıları incelendi. Trombositlerin in vitro hemostatik

fonksiyonların incelenmesinde agregasyon ve trombin jenerasyon testleri

kullanıldı. Dondurma işlemine maruz kalan ATK’lardaki trombositlerin varlığı

ve bu hücrelerin canlılığını değerlendirmek için akım sitometri yöntemi

kullanıldı.

Bulgular: Her iki dilüsyon grubunda yer alan ATK’ların residüel lökosit sayısı

1x106’nın altındaydı. Plazma ile dilüe edilen grubun ortalama trombosit geri

kazanım oranı %0,9 NaCl ile dilüe edilen gruptan daha yüksekti (%88,1±9,5’e

karşılık %63±10). Bu sonuçlar Avrupa İlaç Kalite ve Sağlık Hizmetleri

Direktörlüğü’nün kalite kriterlerine uygundu. Trombosit agregasyon testine

beklenildiği üzere yanıt alınamadı. Dondurulup çözülerek otolog plazma

ile dilüe edilen ATK’ların ortalama trombin oluşturma potansiyeli (2411

nmol/L×dakika), %0,9 NaCl ile dilüe edilenlere (1913 nmol/L×dakika) ve

dondurma işlemi öncesine göre (1681 nmol/L×dakika) daha yüksek saptandı.

Kriyopreservasyon işlemi sonrası plazma ve %0,9 NaCl ile dilüe edilen ATK’ların

akım sitometri yöntemi ile canlılığı sırasıyla %94,9 ve %96,6 olarak bulundu.

Sonuç: Trombositlerin DMSO ile kriyopreserve edilerek -80 °C saklanmaları,

raf ömürlerini 7 günden 2 yıla çıkarmaktadır. Kriyopreservasyon işlemi

trombositlerin in vitro hemostatik fonksiyonlarını arttırmanın yanında canlılık

oranlarını da etkilememektedir.

Anahtar Sözcükler: Dondurulmuş trombositler, Akım-sitometri testi, İn vivo

thrombin jenerasyon testi

Address for Correspondence/Yazışma Adresi: Soner YILMAZ, M.D.,

Gülhane Military Medical Academy, Blood Training Center and Blood Bank, Ankara, Turkey

Phone : +90 312 304 49 02

E-mail : soyilmaz@gata.edu.tr



28


Yılmaz S, et al: Cryopreservation of Platelet Concentrates

Introduction

Currently in blood banking applications, platelet concentrates

(PCs) prepared through apheresis or from buffy coat should be

used within 5-7 days after preparation. In order to overcome

the short shelf life-related problems, studies on frozen PCs

have continued since the 1970s [1]. Although the literature

data on the use of cryopreserved platelets showed that in vivo

cryopreserved platelet suspensions have hemostatic activities

superior to those of fresh apheresis suspensions, they showed

delayed responses to in vitro platelet aggregation tests. Initially,

this delay was attributed to the loss of aggregation capability

of platelets during the cryopreservation process. However,

recent studies proved that the in vitro failure of aggregation

response to agonists occurred in response to the transformation

of platelets into a procoagulant phenotype by the activationdegranulation

process [2,3,4].

In the last decade, the demand for frozen platelets that have

a long shelf life has increased for the treatment of military

casualties in the Iraq and Afghanistan campaigns. As for Turkey,

freezing and storing PCs as a part of contingency plans and

prevention of this valuable blood product’s disposal due to short

shelf life has an importance beyond emphasis.

The most commonly used cryopreservation protocol is the

addition of dimethyl sulfoxide (DMSO) to PCs at a final

concentration of 4%-6%, followed by removal of DMSO

involving supernatant before the freezing process and finally

freezing of the hyperconcentrated low volume of PCs. After

the thawing process, PCs can be diluted by adding 0.9% NaCl,

autologous plasma, or platelet additive solutions. This protocol

can attain a gain of platelets between 70% and 80% [5]. In

this study, we aimed to assess the in vitro hemostatic activity of

cryopreserved platelets using different dilution methods (0.9%

NaCl and autologous plasma).


In August 2013, Gülhane Military Medical Academy Ethics

Committee Approval was received for the assessment of in

vitro hemostatic activity of cryopreserved apheresis platelet

concentrates (APCs). Written informed consent was obtained

from all participants.

PCs were obtained using the apheresis method (Trima, Caridian

BCT, Inc., Lakewood, CO, USA) from donors that met the National

Blood and Blood Products criteria for the donation of APCs.

APCs at a total of 200 mL were collected in acid-citrate-dextrose

(ACD, NIH, Formula A, Baxter Healthcare Corp., Deerfield, IL,

USA) at a ratio of 1 volume of ACD to 10 volumes of blood. APCs

from all 7 donors were divided into 2 packs of 100 mL in volume

each. One of each of the 100-mL packs was included in either

the plasma-diluted group or the 0.9% NaCl-diluted group. Each

group comprised 7 APCs. Before the freezing process, APCs were

preserved in an automatic shaker on a horizontal plane at 20-

24 °C for 1 day. Each of the APCs’ volume and weight were

calculated at all stages of the procedure.

Apheresis Platelet Concentrate Freezing Process

A 41-mL sample of plasma collected by apheresis from each

donor and 0.9% NaCl were mixed with 9 mL of 27% DMSO in

an empty blood bag located on a rigid ice pack for the plasmadiluted

group and 0.9% NaCl-diluted groups, respectively. The

resultant 50-mL mixture and 100 mL of APC were collected

in a 750-mL ethyl vinyl acetate freezing bag (CryoMACS®

Freezing Bag 750, Miltenyi Biotec, Teterow,Germany) through

a sterile hose combining device. The final DMSO concentration

in the freezing bag was 6% and the bag was centrifuged at 22

°C and 1250x g for 10 min (Thermo Fisher Scientific RC12BP,

Asheville, NC, USA). A platelet pellet of 20-25 mL was obtained

after removal of the supernatant and the bag was put into a

cardboard freezing box and stored at -80 °C.

Thawing of Frozen Apheresis Platelet Concentrates

The 1-day-old frozen APCs were thawed through immersion in

37 °C water within 10 min. Either 20 mL of autologous plasma

or 0.9% NaCl was added to the APCs depending on the dilution

group and they were kept at room temperature for 30 min.

In Vitro Measurements

All analyses were repeated in the fresh state and after diluting

the APCs in the post-thaw state.

Residual Leukocyte and Platelet Counts

The frozen APCs were analyzed for the determination of platelet

and residual leukocyte counts with a whole-blood analyzer

device (ABX Pentra XL80, HORIBA ABX SAS, Montpellier, France).

Platelet Aggregation Test

Platelet aggregation tests were performed with a Chrono-log

platelet aggregometer by the same laboratory technician and

thrombocyte agonist (ADP, epinephrine, collagen, and ristocetin)

responses were assessed for both dilution groups.

Thrombin Generation Test

Thrombin generation test (TGT) was performed with a

calibrated automated thrombogram device (Thrombinoscope

BV, Maastricht, the Netherlands) [6]. In this test, thrombin

generation occurs in the co-presence of phospholipid and

tissue factor present in the platelet supernatant and/or added

reagents. The platelet-rich plasma reagent (Thrombinoscope

BV) used in our test involves 1 pmol/L tissue factor. However,

this reagent does not involve phospholipid and is used for

assessing the presence of phospholipid in the medium. A sample

of 80 µL was collected from both dilution groups. Each sample

29



was transferred to 3 different microtitrated plates (Immulon

2 HB, Thermo Electron Corporation, Milford, MA, USA) that

involved 20 µL of platelet-rich plasma reactant and 20 µL of

thrombin calibrator. After the incubation of the mixture at 37

°C for 15 min, a 20-µL sample was collected and added to 20

µL of Fluo-buffer solution, and the reaction was monitored

with a fluorometer. Using the Thrombinoscope program, the

thrombogram curve, endogenous thrombin potential, and peak

height were measured. The endogenous thrombin potential,

which indicates the total amount of endogenous thrombin

generated, was recorded as nmol/L per minute. The peak height,

which indicates the highest measured value of thrombin, was

shown as nmol/L.

Flow Cytometry Analysis

Platelet samples were transferred to tubes containing K3

EDTA. CD41a FITC (BD Biosciences, San Jose, CA, USA) and

7-aminoactinomycin D (7-AAD) were used to determine

viable platelets. The incubated cells were analyzed using the

FACSDiva software for FACSCanto II model flow cytometry (BD

Biosciences).


Quantitative results were presented as mean ± standard deviation

and minimum-maximum. Categorical results were presented as

number and percentage. All statistical analyses were processed

using SPSS 14.0 for Windows (SPSS Inc., Chicago, IL, USA).

Results

The mean volume of APCs after dilution with autologous

plasma or 0.9% NaCl was 45±3 mL. The mean platelet counts

of the plasma and 0.9% NaCl groups were (123.6±13.7)x1011

(range: (106.9-143.5)x1011) and (84.6±7.6)x10 11 (range: (77.8-

100.8)x1011), respectively (Table 1). The freeze-thaw percentage

recovery was calculated according to the standard operating

procedure of the Naval Blood Research Laboratory [7]. The

residual leukocyte counts of all APCs were <1x106. In the 0.9%

NaCl-diluted group, one sample was excluded from the study

due to damage to the plastic bag. The platelet and residual

leukocyte counts and the rate of platelet recovery of APCs are

shown in Table 1.

In all 13 fresh APC samples, platelet aggregation tests with

ADP, epinephrine, and collagen were normal. There was no

aggregation response to a variety of dilution ratios in any of the

frozen treated platelet samples.

TGT revealed that post-thaw APCs diluted with autologous

plasma (Figure 1A) had higher endogenous thrombin potentials

when compared to fresh-state samples (Figure 1B) and post-

Table 1. Residual leukocyte and platelet counts, and platelet recovery rates of fresh apheresis platelet concentrates and

apheresis platelet concentrates after thawing.

Plasma-Diluted Group

Residual Leukocytes

(x10 9 /L)

Fresh

Platelet Count (x10 9 /L)

Post-thaw

Platelet Recovery

(%)

Sample 1 0.10 115.7 113.6 98

Sample 2 0.10 129 128.2 99

Sample 3 0.30 139.8 106.9 76

Sample 4 0.20 139.5 117.8 83

Sample 5 0.20 148.3 139.1 93

Sample 6 0.10 149 116.1 77

Sample 7 0.10 156.7 143.5 91

Mean ± SD 0.15±0.07 139.7±13.7 123.6±13.7 88.1±9.5

0.9% NaCl-Diluted Group

Sample 1 0.10 115.7 100.8 86

Sample 2 0.10 129 79.6 62

Sample 3 0.30 139.8 84.4 60

Sample 4 0.20 139.5 85.3 61

Sample 5 0.20 139.1 77.8 56

Sample 6 0.10 149 80 53

Sample 7* - - - -

Mean ± SD 0.16±0.07 135.3±10.5 84.6±7.6 63±10

SD: Standard deviation.

*This sample was excluded from the study due to a damaged plastic bag.

30



thaw APC samples diluted with 0.9% NaCl (Figure 1C) (2411 vs.

1681 and 1913 nmol/L per minute). The peak height values were

also higher in post-thaw APC samples diluted with autologous

plasma (609 vs. 350 and 338 nmol/L, respectively).

As a result of flow-cytometric analysis, 99.2% of fresh APCs were

stained with the CD41A thrombocyte indicator, while 97.9%

were determined as viable when tested with 7-AAD nucleic acid

dye. The viability rates of the post-thaw APC samples diluted

with plasma and 0.9% NaCl are shown in Table 2.

Discussion

In 1956, Klein et al. reported the use of previously frozen

platelets in an actively bleeding thrombocytopenic patient, and

since then numerous studies have been reported on both the in

vitro and in vivo efficacies of cryopreserved platelets [8]. Since

Schiffer et al.’s 1976 study on the use of autologous platelets

for the treatment of patients with leukemia, relevant studies

until the 1990s showed that the platelets were damaged to

a significant extent by the freezing process, which decreased

their efficacy when compared to fresh platelets [1]. These

results were supported by other in vitro studies that assessed

the platelets’ primary hemostatic functions [1,9,10]. However,

Khuri et al.’s 1999 report caused a shift in this paradigm; they

showed that the in vivo hemostatic functions of cryopreserved

APCs were superior to those of fresh preserved platelets [3].

Almost simultaneously, Bernard et al. reported the procoagulant

changes in frozen treated platelet membrane surfaces [4].

Recently, it was also reported that fresh PCs with almost expired

shelf lives contained platelet-derived microparticles with 50 to

100 times more potent procoagulant activity than activated

platelets and they had a significant impact on the activation

and continuation of the coagulation cascade [11,12]. In 2014,

Johnson et al. demonstrated increased phosphatidylserine

expression on cryopreserved platelet membranes and

also showed that these cryopreserved APCs contained

Figure 1B. Thrombin generation test results of apheresis platelet

concentrates before freezing (sample). Endogenous thrombin

potential and peak height values of apheresis platelet concentrates

were 1681 nmol/L per minute and 350 nmol/L, respectively.

Figure 1A. Thrombin generation test results of plasma diluted

group (sample). Endogenous thrombin potential and peak height

values of apheresis platelet concentrates were 2411 nmol/L per

minute and 609 nmol/L, respectively.

Figure 1C. Thrombin generation test results of 0.9% NaCl-diluted

group (sample). Endogenous thrombin potential and peak height

values of apheresis platelet concentrates were 1913 nmol/L per

minute and 338 nmol/L, respectively.

Table 2. Viability rates of fresh and post-thaw apheresis platelet concentrates.

Fresh Platelets Plasma-Diluted Group 0.9% NaCl-Diluted Group

Total Platelets

(%)

Viability

(%)

Viability

(%)

Sample 1 98.7 94.3 87.3 97.1

Sample 2 98.5 99.9 91.7 98.4

Sample 3 99.3 96.6 95.4 94.7

Sample 4 99.5 99.9 93.1 95.4

Sample 5 99.5 94.8 98.8 95.6

Sample 6 99.6 99.9 99.7 98.9

Sample 7* 99.8 99.9 98.5 -*

SD: Standard deviation.

*This sample was excluded from the study due to a damaged plastic bag.

Viability

(%)

31

Yılmaz S, et al: Cryopreservation of Platelet Concentrates Turk J Hematol 2016;33:28-33

phosphatidylserine microparticles that might contribute to the

increased hemostatic activity. They also presented the first in

vitro phosphatidylserine-dependent coagulation and thrombin

generation potentials of cryopreserved APCs by using the TGT

[13].

The use of autologous plasma was the most common method

for resuspending PCs after thawing until 2006 when Valeri et

al. claimed that 0.9% NaCl could be used instead of autologous

plasma [14]. The design of this study includes 2 different

dilution groups (0.9% NaCl and autologous plasma), aimed to

better delineate the dilution method that meets the quality

control criteria.

According to the Guide to the Preparation, Use and Quality

Assurance of Blood Components: European Directorate for the

Quality of Medicines & Healthcare of the Council of Europe

(EDQM), frozen APCs have 3 quality control criteria (platelet

recovery, residual leukocyte count, and volume) [15]. The platelet

recovery rate should be higher than 40%. In our study, the mean

platelet recovery rate in the plasma-diluted group (88.1±9.5%)

was higher than in the 0.9% NaCl-diluted group (63±10%).

However, the platelet recovery rate of both dilution groups met

the EDQM quality criteria. Compared to other studies, the results

of the 0.9% NaCl-diluted group were lower than in Valeri et al.’s

study (74±11%), but those of the plasma-diluted group were

better than in Lelkens et al.’s (77±15%) [16,17]. One potential

weakness in the current study is the lower number of samples

assessed.

The EDQM’s criteria require that the volume of post-thaw APCs

be ≥50 mL. In our study, the mean volume of APCs after thawing

and dilution was 45±3 mL, which was due to the division of

200 mL of APCs into 2 packs of equal volume prior to freezing.

Another quality standard requires the presence of <1x10 6

residual leukocytes in post-thaw APCs, which was met by all the

samples in both dilution groups [15].

The maximum shelf life of PCs stored at 22 °C is 5-7 days.

Below this temperature, toxic effects begin to appear in the

cells. Moreover, ice crystal formation occurs at low-temperature

storage (<0 °C). This formation can puncture the platelet

membrane, leading to cell death. In this study, we aimed to

show the effect of low temperature on the viability of platelets

by flow-cytometric analysis. The mean viability rates of postthaw

APC samples diluted with plasma and 0.9% NaCl were

determined as 94.9% and 96.6%, respectively (Table 2). These

results revealed that toxic effects of temperature could be

prevented using DMSO as a cryoprotective agent.

The TGT is an assay that measures the overall tendency of

thrombin formation after initiation of coagulation [18]. The

use of autologous plasma for the dilution of frozen thawed

platelets could affect the TGT test results. Frozen APCs diluted

with autologous plasma (Figure 1A) had correspondingly higher

thrombin generation potentials, as in Johnson et al.’s study [13],

when compared to both fresh platelets (Figure 1B) and the

APC group diluted with 0.9% NaCl (Figure 1C). These findings

suggest that cryopreservation increases the platelet hemostatic

activities independently from the plasma content. Due to the

presence of coagulation factors, the plasma content may also

provide an additional hemostatic stimulus when compared to

the 0.9% NaCl-diluted group.

Platelet aggregation responses were negative as expected. In

Valeri et al.’s study, frozen treated platelets had a significant

decrease in aggregation response irrespective of the dilution or

resuspension method when compared to fresh platelets (p<0.001)

[2]. On the other hand, Hornsey et al. reported that frozen treated

platelets demonstrated no aggregation response [19]. However,

DMSO-treated platelets were effective on kidney bleeding time

in a study that investigated the correlation of in vivo and in vitro

functions of fresh and stored human platelets [20].

DMSO-treated frozen platelets have been used successfully

since the 1970s [1,17,21]. Khuri et al. compared the clinical

effects and hemostatic efficiency of frozen and liquid-preserved

platelets in patients undergoing cardiopulmonary bypass in

1999 [3]. They reported that cryopreserved platelet transfusions

were superior to liquid-preserved platelets in reducing blood

loss and blood transfusion requirements after cardiopulmonary

bypass. They concluded that these results were probably related

to the improved in vivo hemostatic activity of cryopreserved

platelets [3].

In 2001, Özsan et al. studied the cryopreservation of platelets

by using a cryopreserving agent and showed that sialic acid was

not an alternative compound for cryopreservation [22]. In 2003,

Kurt Yüksel et al. presented a case report that demonstrated that

the autologous transfusion of cryopreserved platelets could be

a reasonable approach in bleeding alloimmunized patients [23].

In 2001, the Netherlands Military Blood Bank implemented

the use of frozen platelets in Bosnia and abandoned the

walking blood bank concept. Within a 6-month period, 2

thrombocytopenic casualties with exsanguinating hemorrhage

were treated with frozen platelets. After those reports, frozen

platelets and frozen blood bank facilities became an essential

part of military hospital standard equipment deployed by the

military of the Netherlands [17]. In 2008, the Australian Defence

Force embedded a surgical and intensive care team into the

Netherlands-led forward health facility in Afghanistan. Twentytwo

units of frozen platelets were used by these teams for 17

casualties undergoing surgery. Except in one patient, there

was no clinical evidence of coagulopathy in patients treated

with frozen platelets [24]. Between 2006 and 2012, 6246

cryopreserved blood products were transfused in Afghanistan;

2175 of them were erythrocyte concentrations, 3001 were fresh

frozen plasma, and 1070 were frozen platelets. No transfusion

32



reactions were reported related to the use of these cryopreserved

blood components [25].

The cryopreservation of platelets increases their shelf life

from 7 days to 2 years when they are stored at -80 °C with

the cryoprotective agent DMSO. Difficulties in the preparation

of APCs and the cost of sets could be prevented with the use

of cryopreserved blood products. Moreover, the availability

of autologous cryopreserved platelets for patients likely to

develop refractoriness to platelets or allogenic ABO- and human

leukocyte antigen-compatible cryopreserved platelets is crucial

for the treatment of these patients. The strategic location

of Turkey mandates the urgent collaboration of the Turkish

Armed Forces, the Red Crescent, and other governmental

medical organizations in establishing both frozen platelet and

erythrocyte stocks.

Ethics

Ethics Committee Approval: Gülhane Military Medical Academy

Ethics Committee (Approval number: 06-05-14/37), Informed

Consent: It was taken.


Concept: Soner Yılmaz, İbrahim Eker, Design: Soner Yılmaz,

İbrahim Eker, Data Collection or Processing: Aytekin Ünlü, Metin

Uyanık, Ahmet Pekoğlu, Aysel Pekel, Birgül Erkmen, Analysis or

Interpretation: Rıza Aytaç Çetinkaya, Literature Search: Serkan

Tapan, Uğur Muşabak, Sebahattin Yılmaz, İsmail Yaşar Avcı, Ferit

Avcu, Emin Kürekçi, Can Polat Eyigün, Writing: Aytekin Ünlü,

Rıza Aytaç Çetinkaya.




included.

References

1. Schiffer CA, Aisner J, Wiernik PH. Clinical experience with transfusion of

cryopreserved platelets. Br J Haematol 1976;34:377-385.

2. Valeri CR, Macgregor H, Ragno G. Correlation between in vitro aggregation

and thromboxane A2 production in fresh, liquid-preserved, and

cryopreserved human platelets: effect of agonists, pH, and plasma and

saline resuspension. Transfusion 2005;45:596-603.

3. Khuri SF, Healey N, MacGregor H, Barnard MR, Szymanski IO, Birjiniuk

V, Michelson AD, Gagnon DR, Valeri CR. Comparison of the effects of

transfusions of cryopreserved and liquid preserved platelets on hemostasis

and blood loss after cardiopulmonary bypass. J Thorac Cardiovasc Surg

1999;117:172-183.

4. Barnard MR, MacGregor H, Ragno G, Pivacek LE, Khuri SF, Michelson AD,

Valeri CR. Fresh, liquid preserved, and cryopreserved platelets: adhesive

surface receptors and membrane procoagulant activity. Transfusion

1999;39:880-888.

5. Johnson LN, Winter KM, Reid S, Hartkopf-Theis T, Marks DC. Cryopreservation

of buffy-coat-derived platelet concentrates in dimethyl sulfoxide and

platelet additive solution. Cryobiology 2011;62:100-106.

6. Dargaud Y, Wolberg AS, Luddington R, Regnault V, Spronk H, Baglin

T, Lecompte T, Ten Cate H, Negrier C. Evaluation of a standardized

protocol for thrombin generation measurement using the calibrated

automated thrombogram: an international multicenter study. Thromb Res

2012;130:929-934.

7. Naval Blood Research Laboratory. Standard Operating Procedure:

Glycerolization and Deglycerolization of Red Blood Cells in a Closed System

Using the Haemonetics ACP215. Plymouth, MA, USA, NBRL. Available at

http://www.nbrl.org/SOP/ACP215/ACP215All.html.

8. Klein E, Toch R, Farber S, Freeman G, Fiorentino R. Hemostasis in

thrombocytopenic bleeding following infusion of stored, frozen platelets.

Blood 1956;11:693-699.

9. Lazarus HM, Kaniecki-Green EA, Warm SE, Aikawa M, Herzig RH. Therapeutic

effectiveness of frozen platelet concentrates for transfusion. Blood

1981;57:243-249.

10. Towell BL, Levine SP, Knight WA 3rd, Anderson JL.A comparison of frozen

and fresh platelet concentrates in the support of thrombocytopenic

patients. Transfusion 1986;26:525-530.

11. Keuren JF, Magdeleyns EJ, Govers-Riemslag JW, Lindhout T, Curvers J.

Effects of storage-induced platelet microparticles on the initiation and

propagation phase of blood coagulation. Br J Haematol 2006;134:307-313.

12. Sinauridze EI, Kireev DA, Popenko NY, Pichugin AV, Panteleev MA, Krymskaya

OV, Ataullakhanov FI. Platelet microparticle membranes have 50- to 100-

fold higher specific procoagulant activity than activated platelets. Thromb

Haemost 2007;97:425-434.

13. Johnson L, Coorey CP, Marks DC. The hemostatic activity of cryopreserved

platelets is mediated by phosphatidylserine-expressing platelets and

platelet microparticles. Transfusion 2014;54:1917-1926.

14. Valeri CR, Ragno G, Khuri S. Freezing human platelets with 6 percent

dimethyl sulfoxide with removal of the supernatant solution before

freezing and storage at -80 °C without postthaw processing. Transfusion

2005;45:1890-1898.

15. Ayhan Y, Karakoç E, Pelit NB. Guide to the Preparation, Use and Quality

Assurance of Blood Components. Strasbourg, France, Council of Europe

Publishing, 2008.

16. Valeri CR, Srey R, Lane JP, Ragno G. Effect of WBC reduction and storage

temperature on PLTs frozen with 6 percent DMSO for as long as 3 years.

Transfusion 2003;43:1162-1167.

17. Lelkens CC, Koning JG, de Kort B, Floot IB, Noorman F. Experiences with

frozen blood products in the Netherlands military. Transfus Apher Sci

2006;34:289-298.

18. Castoldi E, Rosing J. Thrombin generation tests. Thromb Res 2011;127

(Suppl 3):21-25.

19. Hornsey VS, McMillan L, Morrison A, Drummond O, Macgregor IR, Prowse

CV. Freezing of buffy coat-derived, leukoreduced platelet concentrates in 6

percent dimethyl sulfoxide. Transfusion 2008;48:2508-2514.

20. Rothwell SW, Maglasang P, Reid TJ, Gorogias M, Krishnamurti C. Correlation

of in vivo and in vitro functions of fresh and stored human platelets.

Transfusion 2000;40:988-993.

21. Schiffer CA, Aisner J, Dutcher JP, Daly PA, Wiernik PH. A clinical program of

platelet cryopreservation. Prog Clin Biol Res 1982;88:165-180.

22. Özsan GH, Pişkin Ö, Demirkan F, Ateş H, Özcan MA, Ündar B. Effect of sialic

acid on platelet cryopreservation. Turk J Hematol 2001;18:251-257.

23. Kurt Yüksel M, Arat M, Arslan Ö, Beksaç M, İlhan O. Autologous platelet

collection and storage to support thrombocytopenia in a leukemia patient

with platelet alloimmunization undergoing chemotherapy. Turk J Hematol

2003;20:233-236.

24. Neuhaus SJ, Wishaw K, Lelkens C. Australian experience with frozen blood

products on military operations. Med J Aust 2010;192:203-205.

25. Holley A, Marks DC, Johnson L, Reade MC, Badloe JF, Noorman F. Frozen

blood products: clinically effective and potentially ideal for remote

Australia. Anaesth Intensive Care 2013;41:10-19.

33

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0159


Is It Possible to Predict Pulmonary Complications and Mortality in

Hematopoietic Stem Cell Transplantation Recipients from

Pre-Transplantation Exhaled Nitric Oxide Levels?

Hematopoetik Kök Hücre Nakli Uygulanan Hastalarda Nakil Öncesi Ekshale Nitrik Oksit

Düzeyi ile Pulmoner Komplikasyonları ve Mortaliteyi Öngörmek Mümkün müdür?

Nurdan Köktürk1, Fatma Yıldırım1, Müge Aydoğdu1, Şahika Zeynep Akı2, Zeynep Arzu Yeğin2, Zübeyde Nur Özkurt2, Elif Suyanı2,

İpek Kıvılcım Oğuzülgen1, Gülsan Türköz Sucak2

1Gazi University Faculty of Medicine, Department of Pulmonary Medicine, Ankara, Turkey

2Gazi University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Abstract

Objective: Chemo/radiotherapy-induced free oxygen radicals and

reactive oxygen derivatives contribute to the development of early

and late transplantation-related pulmonary and extra-pulmonary

complications in hematopoietic stem cell transplantation (HSCT)

recipients. It has been proposed that an increase in fractional exhaled

nitric oxide (FeNO) level indicates oxidative stress and inflammation in

the airways. The aim of this prospective study is to evaluate the pretransplantation

FeNO levels in HSCT patients and to search for its role in

predicting post-transplantation pulmonary complications and mortality.

Materials and Methods: HSCT patients were included in the study

prospectively between October 2009 and July 2011. Pre-transplantation

FeNO levels were measured with a NIOX MINO® device prior to

conditioning regimens. All patients were monitored prospectively for

post-transplantation pulmonary complications with medical history,

physical examination, chest X-ray, and pulmonary function tests.

Results: A total of 56 patients (33 autologous, 23 allogeneic) with mean

age of 45±13 years were included in the study, among whom 40 (71%)

were male. Pre-transplantation FeNO level of the whole study group was

found to be 24±13 (mean ± standard deviation) parts per billion (ppb).

The FeNO level in allogeneic HSCT recipients was 19±6 ppb while it

was 27±15 ppb in autologous HSCT recipients (p=0.042). No significant

correlation was found between the pre-transplantation chemotherapy

and radiotherapy protocols and baseline FeNO levels (p>0.05). Posttransplantation

pulmonary toxicity was identified in 12 (21%) patients

and no significant relationship was found between baseline FeNO levels

and pulmonary toxicity. The survival rate of the whole study group for

1 year after transplantation was 70%. No significant relationship was

identified between baseline FeNO values and survival (FeNO 19±7 ppb

in patients who died and 26±15 ppb in the survivors; p=0.114).

Conclusion: Pre-transplantation FeNO measurement does not

seem to have a role in predicting post-transplantation pulmonary

complications and mortality.

Keywords: Hematopoietic stem cell transplantation, Exhaled nitric

oxide, Pulmonary complications, Mortality

Öz

Amaç: Hematopoetik kök hücre nakli (HKHN) hastalarında, nakil öncesi

kemoterapi/radyoterapi etkisi ile oluşan serbest oksijen radikalleri ve

reaktif oksijen türevleri pek çok nakil ilişkili erken ve geç pulmoner

ve nonpulmoner komplikasyonun oluşmasında rol oynamaktadır.

Ekshale nitrik oksit (NO) düzeyindeki artışın hava yollarındaki oksidatif

stresi ve enflamasyonu yansıttığı ileri sürülmektedir. Bu prospektif

çalışmada amacımız HKHN hastalarında nakil öncesi bakılan ekshale

NO düzeylerinin nakil sonrası gelişen pulmoner komplikasyonlar ve

sağkalım üzerine etkisinin araştırılması idi.

Gereç ve Yöntemler: Ekim 2009-Temmuz 2011 tarihleri arasında

HKHN uygulanan hastalar prospektif olarak çalışmaya alındılar. Nakil

öncesi ekshalasyon havasında NO ölçümleri NIOX MINO® cihazı ile

yapıldı. Bütün hastalar transplantasyon sonrası prospektif olarak

anemnez, fizik muayene, akciğer filmi ve solunum fonksiyon testleri

ile pulmoner komplikasyon açısından takip edildi.

Bulgular: Çalışmaya yaş ortalamaları 45±13 yıl olan 40’ı (71%) erkek

toplam 56 hasta (33 otolog, 23 allojeneik) dahil edildi. Nakil öncesi

ekshale NO düzeyi tüm grupta 24±13 ppb (ortalama ± standart

sapma) (median: 22; minimum-maksimum: 5-75) bulundu. Allojeneik

HKHN uygulanan hastalarda ekshale NO düzeyi 19±6 ppm; otolog

nakil uygulananlarda 27±15 ppm olarak bulundu (p=0,042). Nakil

öncesi uygulanan kemoterapi ve radyoterapi rejimleri ile bazal NO

düzeyleri arasında anlamlı korelasyon bulunmadı (p>0,05). Nakil

sonrası pulmoner toksisite 12 (%21) hastada saptandı. Bazal NO değeri

ile pulmoner toksisite arasında anlamlı ilişki saptanmadı. Tüm çalışma

grubunda sağkalım oranı %70 olarak bulundu. Nakil öncesi ekshale

NO düzeyinin sağ kalım üzerinde etkisinin olmadığı görüldü (eksitus

grubunda 19±7 ppm, taburcu grubunda 26±15 ppm, p=0,114).

Sonuç: HKHN hastalarında nakil öncesi ekshale NO ölçümü nakil

sonrası pulmoner komplikasyon gelişimini ve mortaliteyi öngörmede

kullanılabilir gözükmemektedir.

Anahtar Sözcükler: Hematopoetik kök hücre nakli, Ekshale nitrik

oksit, Pulmoner komplikasyonlar, Mortalite

Address for Correspondence/Yazışma Adresi: Fatma YILDIRIM, M.D.,

Gazi University Faculty of Medicine, Department of Pulmonary Medicine, Ankara, Turkey

Phone : +90 312 202 61 19

E-mail : fatma_bodur2000@yahoo.com


Accepted/Kabul tarihi: December 16, 2014

34


Köktürk N, et al: Exhaled NO in Predicting Post-Transplantation Pulmonary Complications and Mortality in HSCT Patients

Introduction

Hematopoietic stem cell transplantation (HSCT) is an important

treatment option for several malignant and non-malignant

hematological diseases. However, pulmonary complications

such as idiopathic pulmonary syndromes, bronchiolitis

obliterans organizing pneumonia (BOOP), and infections and

graft-versus-host disease (GVHD) developing after bone marrow

transplantation have a negative impact on outcome. Chemo/

radiotherapy-induced oxidative stress occurring prior to HSCT

is claimed to contribute to development of many early and late

transplantation-related pulmonary complications [1,2,3,4,5]. A

marker of bronchial inflammation might guide in predicting

HSCT-related pulmonary pathology.

Nitric oxide (NO) is an endogenous regulator molecule that

is synthesized in the body from L-arginine by the enzyme NO

synthase. NO in the airways is measured after reaction with

ozone by chemiluminescence method. Fractional exhaled NO

(FeNO) has been shown to increase as a non-invasive marker

of inflammation especially in bronchiectasis, bronchial asthma,

tuberculosis, acute exacerbation of chronic obstructive

pulmonary disease (COPD), and many other systemic and

autoimmune diseases such as systemic lupus erythematosus,

systemic sclerosis, and cirrhosis of the liver. Its level increases in

parallel with the increase in the level of inflammation and tends

to decline in a short period of time after anti-inflammatory

therapies [6,7,8].

Our group demonstrated a significant relationship between

pre-transplantation diffusion capacity of the lungs for carbon

monoxide adjusted for hemoglobin (DLCO adj) levels and

development of post-transplantation sinusoidal obstruction

syndrome after transplantation in a previous study [9]. We

hypothesized whether exhaled FeNO, a simple and non-invasive

measurement that has been shown to be increased in many

inflammatory conditions, might show pre-transplantation

inflammation and endothelial injury and could possibly predict

post-transplantation pulmonary complications and mortality.


Study Subjects

After receiving the approval of our institutional review board,

56 patients were enrolled in the study prospectively between

October 2009 and July 2011. Inclusion criteria were age above

18 years, being a candidate for allogeneic or autologous stem

cell transplantation, and signing an informed consent form to

participate in the study and for the use of their medical records.

Patients who were younger than 18 years of age; who smoked

through the last 6 months; who had asthma, COPD, bronchial

hyperreactivity, or upper or lower respiratory tract infection in

the last 4 weeks; who were diagnosed to have had activation

of the underlying disease in the last 4 weeks; and who used

L-arginine, phosphodiesterase inhibitors, or nitrate were

excluded from the study.

Graft-Versus-Host Disease

Cyclosporine and methotrexate was the standard prophylaxis

regimen for GVHD. The assessment and grading of acute and

chronic GVHD was primarily based on clinical findings and

pulmonary function test (PFT) results. Overall grade of acute

GVHD and severity of organ involvement was assessed on a

0-to-4 scale according to the original Seattle criteria. Acute

GVHD was considered present if a grade of at least 2 was

assigned. Pulmonary toxicities were also graded on a 0-to-4

scale according to these criteria. Chronic GVHD was defined as

GVHD occurring 100 days or more after HSCT. GVHD was treated

with 1-2 mg/kg/day of prednisolone.

Antimicrobial Prophylaxis

Antimicrobial prophylaxis, which was performed with acyclovir

and fluconazole, was given from the beginning of the

conditioning regimen until day +180 post-transplantation for

autologous and until discontinuation of the immunosuppressive

therapy for allogenic HSCT recipients. All patients received

trimethoprim-sulfamethoxazole orally as prophylaxis against

Pneumocystis jirovecii beginning from the conditioning regimen

until 1 day before stem cell infusion and from neutrophil

engraftment to 6 months post-transplantation for autologous

and until the discontinuation of the immunosuppressive therapy

for allogenic HSCT.

Fractional Exhaled Nitric Oxide Measurements

FeNO levels were measured with a NIOX MINO® device prior

to HSCT. Each subject inhaled NO-free air to the total lung

capacity (TLC) and expired as long as possible at 4 different flow

rates (50, 100, 150, and 200 mL/s) against a resistance of 10 cm

H2O/L/s as previously described. The respiratory tract contains

both forms of nitric oxide synthetase enzymes, inducible and

endothelial. The FeNO measurement represents the total NO of

the respiratory tract [10].

Pulmonary Follow-Up

PFTs were routinely performed for detection of underlying

ventilatory abnormalities and for assessment of baseline lung

function. The following parameters were measured: forced

expiratory volume in the first second (FEV1), forced vital

capacity (FVC), FEV1/FVC ratio, and TLC. In order to determine

non-infectious pulmonary complications, serial PFTs of patients

were also performed at 3, 6, 9, and 12 months after HSCT.

DLCO adj, DLCO per unit alveolar volume (DLCO VA), and DLCO

adjusted for alveolar volume (DLCO VA/adj) were also measured.

35



Pulmonary Toxicity Definitions

Infectious pulmonary complications were defined as pulmonary

infections with clinical signs of fever, dyspnea, and crackles,

proven by radiologic infiltrates on chest X-ray and in

microbiological samples. Microbiological samples were obtained

from the data of microbiological analyses, including direct

microscopy or culture of sputum or bronchoalveolar lavage

fluid.

Non-infectious pulmonary complications were defined as

the new onset of an obstructive pulmonary defect clinically

manifested by dyspnea on exertion, cough, or wheezing.

Evidence of obstructive defect is revealed in PFTs. Non-infectious

pulmonary complication was accepted as bronchiolitis obliterans

syndrome (BOS), because BOS is a clinical term defined by

pulmonary function changes rather than histology. Patients

were classified as having BOS if they showed FVC % predicted

of >80% and FEV1/FVC of <70% [11,12].


Statistics were calculated using SPSS 15.0 for Windows.

Continuous variables are presented as mean±SD and categorical

variables as percentages. Patients with and without pulmonary

complications were compared using the chi-square test for

categorical variables and the t-test and Mann-Whitney U test

for continuous variables. Pearson and Spearman correlation

tests were used to determine the relationship between basal

FeNO levels and pulmonary complications. Values of p<0.05

were considered statistically significant.

The allogeneic and autologous HSCT groups were compared

according to their basal FeNO levels as high and low FeNO

groups, but there was no cut-off point. Therefore, we divided

the groups according to their median basal FeNO group. Pearson

and Spearman correlation tests were used to determine the

relationships between groups.

Kaplan-Meier survival analysis was performed for comparing

the progression-free survival (PFS) and overall survival among

allogenic and autologous patients with low and high basal FeNO

levels. A difference was considered statistically significant when

p<0.05 by log-rank.

Results

Patient Characteristics

Fifty-six patients were included in the study; 33 received

autologous and 23 received allogeneic stem cells. Forty (71%)

patients were male, and the mean age of the patients was

45±13 years. The median age of the autologous HSCT group

was higher than that of the allogenic HSCT group (53 vs. 34,

p=0.001). The basal FeNO level of the autologous HSCT group

was higher than that of the allogenic HSCT group (26.8±15.4 vs.

18.9±6.2 ppb, p=0.042) (Tables 1 and 2).

In the allogeneic and autologous HSCT groups there were no

significant relations with regard to age, sex, diagnosis, exitus

rate, progression rate, and total and infectious pulmonary

complications for low and high basal FeNO levels (p>0.05)

(Table 3).

Basal PFT values of the groups with pulmonary complications

and without pulmonary complications were similar (Table 4).

Infectious Pulmonary Toxicities

In the allogeneic HSCT group, 3 of the infections were bacterial,

2 were fungal, and 1 was viral. In the autologous HSCT group, the

infectious agent was bacterial in one patient and Pneumocystis

jirovecii in the other patient. All of the pulmonary infections

were with febrile neutropenia and pneumonia. The incidence of

invasive pulmonary aspergillosis was 3.7%.

Non-Infectious Pulmonary Toxicities

In the allogeneic HSCT group, BOS was diagnosed in 2 (9%)

patients, and in the autologous HSCT group BOS was detected

in 2 (6%) patients.

In the autologous HSCT group, one patient had acute GVHD.

This patient was a woman and had 2 children. This is a rare

complication and it was thought to be the result of maternal

antigen activation [13,14].

Survival

There were no deaths in the allogeneic and autologous groups

within the first 100 days after HSCT. The survival rate of the

whole study group for 1 year after transplantation was 70%.

The mean value of the pre-transplantation FeNO was 19±7 ppb

in the exitus group, while it was 26±15 ppb in the survivors

in the total study group. There was no significant relationship

between baseline FeNO and survival (p=0.114).

The median follow-up of 33 patients with autologous HSCT was

424 days (154-766 days). Survival rate of these patients at the

end of the follow-up period was 78.8%. Six patients succumbed

to their underlying disease after autologous HSCT, whereas 1

patient died due to transplant-related causes. None of patients

died due to only pulmonary complications. Pre-transplantation

basal FeNO levels did not have an impact on survival (p>0.05).

After autologous HSCT, 4 of the 33 patients (12.1%) developed

grade 3-4 pulmonary toxicity. Basal FeNO levels had no impact

on pulmonary toxicity (p>0.05).

The median follow-up of the 23 allogeneic HSCT recipients was

203 days (10-774 days). The survival rate of these patients at the

36



end of the follow-up period was 56.5%. While 6 patients died

due to transplant-related causes, 4 patients died of diseaserelated

causes. Pre-transplantation FeNO level was 19±6 ppb

in allogeneic HSCT recipients. Pre-transplantation basal FeNO

levels had no impact on survival, as well (p>0.05). Seven of the

23 patients (30.4%) developed grade 3-4 pulmonary toxicity

after allogeneic HSCT. One patient had grade 1-2 toxicities and

this patient was excluded from analysis. No relationship was

found between the basal FeNO levels and the development of

pulmonary toxicity (p>0.05).

Mean PFS was 246.3±210.1 days in the allogeneic HSCT group;

for autologous HSCT patients, mean PFS was 366.7±199.1. There

were no significant associations between PFS and basal FeNO

levels of low and high basal FeNO group patients in either the

Table 1. Baseline clinical characteristics of patients undergoing hematopoietic stem cell transplantation.

Characteristics

Autologous HSCT,

Allogeneic HSCT,

Total,

p

n=33,

n (%)

n=23,

n (%)

n=56,

n (%)

Sex

Male

Age, years

Median (range)

25 (75.7)

53 (27-64)

15 (65.2)

34 (19-62)

40 (71.0)

45 (19-64)

0.287

0.001

Average CD34 (x106/kg) 4.04±0.92 4.34±0.81 0.214

Diagnosis

Multiple myeloma

Hodgkin disease

Non-Hodgkin lymphoma

Acute myeloid leukemia

Acute lymphoblastic leukemia

SAA/MDS/PNH*

Myeloproliferative disease

Preparation regimes

BEAM

Melphalan

Busulfan-cyclophosphamide

TBI-cyclophosphamide

Non-myeloablative regimes

Others

21 (63.6)

4 (12.1)

7 (21.2)

1 (3.1)

-

-

-

9 (27.3)

20 (60.6)

-

-

-

4 (12.1)

2 (8.7)

-

1 (4.3)

8 (34.8)

4 (17.4)

5 (21.7)

3 (13.0)

-

-

8 (34.8)

4 (17.4)

11 (47.8)

-

23 (41.1)

4 (7.2)

8 (14.3)

9 (16.1)

4 (7.2)

5 (8.9)

3 (5.4)

9 (16.1)

20 (35.7)

8 (14.3)

4 (7.2)

11 (19.6)

4 (7.2)

*SAA: Severe aplastic anemia, MDS: myelodysplastic syndrome, PNH: paroxysmal nocturnal hemoglobinuria, HSCT: hematopoietic stem cell transplantation, TBI: total body

irradiation.

Table 2. Fractional exhaled nitric oxide levels, pulmonary function test measurements, and pulmonary complications.

Characteristics

Autologous HSCT, Allogenic HSCT, p

n=33

n=23

Basal FeNO, ppb (mean ± SD) 26.8±15.4 18.9±6.2 0.042

Basal FEV1, % (mean ± SD) 94.3±12.1 96.1±15.2 0.613

Basal TLC, L (mean ± SD) 7.9±11.4 5.7±0.9 0.383

DLCO/VA adj, % 86.7±16.9 84.1±10.9 0.535

Exitus, n (%) 7 (21.0) 10 (44.0) 0.069

Progression, n (%) 10 (30.0) 4 (17.0) 0.218

Total pulmonary toxicity, n (%) 6 (18.2) 6 (26.2) 0.054

Infectious pulmonary complications 2 (6.1) 3 (13.1) 0.044

Non-infectious pulmonary complications 4 (12.1) 3 (13.1) 0.058

Acute GVHD, n (%) 1 (3.0) 7 (30.0) -

FeNO: Fractional exhaled nitric oxide, FEV1: forced expiratory volume in the first second, TLC: total lung capacity, DLCO/VA adj: DLCO adjusted for alveolar volume, GVHD: graftversus-host

disease, HSCT: Hematopoietic stem cell transplantation, SD: standard deviation.

37



allogeneic (p=0.460) or the autologous (p=0.52) HSCT group

when examined with Kaplan-Meier analysis and log-rank test.

Discussion

We hypothesized that pre-transplantation FeNO could be a

surrogate biomarker demonstrating pre-existing pulmonary

inflammation and/or pre-transplantation injury caused by the

oxidative stress due to previous chemotherapies, radiation, and

infections. We therefore measured pre-transplantation FeNO

levels and investigated whether they had an impact on posttransplantation

pulmonary toxicity and mortality. However,

our results failed to demonstrate an impact of FeNO levels

on transplantation outcomes. There was neither a correlation

between baseline FeNO levels and pre-transplantation

induction chemotherapy and radiotherapy protocols nor posttransplantation

pulmonary complications and mortality.

Pulmonary complications are major causes of morbidity and

mortality after HSCT. Unfortunately, there is currently no

established marker that is non-invasive and can predict the

pulmonary complications and guide preventive strategies or

risk-stratified transplant techniques. A significant correlation

has been observed between the levels of serum nitrite/nitrate

and host-versus-graft and graft-versus host reactions in

rats [15] and humans [4,5] in previous studies. Furthermore,

measurement of NO in exhaled air has been proposed for the

assessment of individual “pulmonary” risk status amongst

various other factors in adults.

Haddad et al. [16] studied a mouse model of idiopathic

pneumonia syndrome following bone marrow transplantation.

They showed that alveolar macrophages, after being stimulated

by allogeneic T cells of graft origin, express higher NO synthase

levels and thus produce more NO. Cyclophosphamide in

Table 3. Comparison of allogeneic and autologous hematopoietic stem cell transplantation groups according to basal fractional

exhaled nitric oxide.

Characteristics Allogeneic HSCT (n=23) Autologous HSCT (n=33)

Low Basal

FeNO, n=12

High Basal FeNO,

n=11

Table 4. Characteristics of patients with and without pulmonary toxicities.

Characteristics

Pulmonary Toxicities

Pulmonary Toxicities

p

(+), n=12

(-), n=44

Basal FeNO, ppb (mean ± SD) 20±10 25±14 0.252

Basal FEV1, % 96±13 95±14 0.837

Basal TLC, L 6±1 7±10 0.551

DLCO/VA adj, % 86±16 86±15 0.980

FeNO: Fractional exhaled nitric oxide, FEV1: forced expiratory volume in the first second, TLC: total lung capacity, DLCO/VA adj: DLCO adjusted for alveolar volume, SD: standard

deviation.

p

Low Basal

FeNO, n=17

High Basal

FeNO, n=16

Age, years (mean ± SD) 34.6±14.3 36.0±12.4 0.367 50.1±10.6 52.3±6.7 0.871

Sex, male, n (%) 7 (58.0) 8 (72.7) 0.389 14 (82.4) 11 (68.6) 0.438

Diagnosis, n (%) 0.487 0.125

Multiple myeloma 1 (8.3) 1 (9.1) 8 (47.1) 13 (81.3)

Hodgkin disease - - 2 (11.8) 2 (12.5)

Non-Hodgkin lymphoma - 1 (9.1) 6 (35.3) 1 (6.3)

Acute myeloid leukemia 3 (25.0) 5 (45.5) 1 (5.9) -

Acute lymphoblastic leukemia 4 (33.3) - - -

SAA/MDS/PNH* 2 (16.7) 3 (27.3) - -

Myeloproliferative disease 2 (16.7) 1 (9.1) - -

Exitus, n (%) 5 (41.7) 5 (45.5) 0.593 6 (35.3) 1 (6.3) 0.085

Progression, n (%) 4 (33.3) 0 (0.0) 0.056 5 (29.4) 5 (31.3) 0.603

Total pulmonary toxicity, n (%) 5 3 0.389 3 (17.7) 1 (6.3) 0.485

Infectious pulmonary

4 2 0.203 1 (5.9) 1 (6.3) 0.601

complications, n (%)

Acute GVHD*, n (%) 3 (25.0) 4 (36.4) 0.444 1 (5.9) - 0.515

*SAA: Severe aplastic anemia, MDS: myelodysplastic syndrome, PNH: paroxysmal nocturnal hemoglobinuria, GVHD: graft-versus-host disease, HSCT: hematopoietic stem cell

transplantation, SD: standard deviation.

p

38



their model stimulated superoxide production by alveolar

macrophages. They concluded that the resultant higher NO and

superoxide levels might have led to production of peroxynitrites

and nitrotyrosines, which mediated lung damage.

Several investigators assessed the usefulness of FeNO for the

early detection of chronic GVHD in HSCT recipients. Kanamori

et al. [17] published a case series of adults with BOOP after

HSCT with FeNO levels above 36 ppb, suggesting that elevated

FeNO may be indicative of pulmonary complications after HSCT.

Increased FeNO production in their cases also suggests that bone

marrow transplantation-related BOOP might be a manifestation

of chronic GVHD. They proposed that the FeNO measurement

was useful in monitoring inflammatory complications after

HSCT.

Two previous reports studied the concentration of exhaled

NO in patients after lung transplantation [8,18]. Neurohr et

al. studied 166 consecutive lung transplantation recipients

[18]. Those patients received no induction therapy and were

maintained with standard care on triple immunosuppression

with corticosteroids, tacrolimus, and mycophenolate mofetil.

A total of 611 FeNO measurements were classified depending

on BOS stage at the time of assessment and course during a

minimum follow-up of 3 months: stable non-BOS, unstable

non-BOS, stable BOS, and unstable BOS. FeNO was significantly

increased prior to the unstable course in comparison to the

stable counterparts (non-BOS: 28.9±1.2 ppb, 16.4±0.8 ppb, and

BOS: 32.5±1.3 ppb, 15.3±0.8 ppb, respectively). Their report

demonstrated that elevated levels of FeNO constituted an

increased risk for future BOS and preceded further deterioration

in transplant recipients.

Another report studying FeNO in patients after autologous

HSCT is that of Qureshi et al. [19]. In their study, FeNO was

significantly increased following autologous peripheral HSCT and

correlated with reduction in DLCO. Mean FeNO increased from

12.54±1.32 ppb before HSCT to 21.26±1.94 ppb at 6 weeks and

25.28±3.31 ppb at 24 weeks. The exhaled FeNO was determined

before and after the conditioning regimen and showed a

significant and progressive increase after the conditioning,

suggesting chemotherapy-related pulmonary toxicity. FeNO

was also measured prior to the conditioning regimen in the

current study in order to determine whether it could be used

as a biomarker that defines pre-transplantation pulmonary risk

status. However, FeNO unfortunately failed to demonstrate such

a predictive value. Fazekas et al. [20] evaluated the correlation

of FeNO and pulmonary complications in 30 pediatric HSCT

patients. They measured FeNO 10 days before HSCT and at

day 0, day 28, and day 60 of HSCT. Similar to our results pretransplantation,

FeNO levels were not different in patients with

and without post-transplantation pulmonary complications.

However, children with any kind of pulmonary complications

until day 100 of HSCT had higher FeNO levels at day 0 than

children without early respiratory pathology, suggesting the

role of conditioning chemotherapy rather than the induction

regimens prior to transplantation.

In previous studies, age was noted as a predictor of FeNO in

children [21,22]. However, the influence of age is controversial

in healthy adults [23,24,25]. In our study, the autologous HSCT

group was older than the allogenic HSCT group, and the basal

FeNO level of the autologous HSCT group was higher than

that of allogenic HSCT group. We attributed this difference to

advanced age.

The limitation of our study is the lack of consecutive FeNO

measurements within the long-term follow-up. By serial

measurement of FeNO, its potential as a non-invasive marker for

continuous risk stratification of HSCT patients for determining

pulmonary complications might be better identified.

Conclusion

We conclude that pre-transplantation FeNO levels do not seem

to be of value as a marker of post-transplantation pulmonary

complications and mortality pre-transplantation. Further studies

are required to designate a pre-transplantation surrogate

marker of post-transplantation pulmonary toxicity.

Ethics

Ethics Committee Approval: Gazi University Ethics Committee

(Approval number: 43/10.01.2009), Informed Consent: It was

taken.


Concept: Nurdan Köktürk, İpek Kıvılcım Oğuzülgen, Gülsan

Türköz Sucak, Design: Nurdan Köktürk, İpek Kıvılcım Oğuzülgen,

Gülsan Türköz Sucak, Data Collection or Processing: Fatma

Yıldırım, Müge Aydoğdu, Şahika Zeynep Akı, Zeynep Arzu Yeğin,

Zübeyde Nur Özkurt, Elif Suyanı, Analysis or Interpretation:

Fatma Yıldırım, Müge Aydoğdu, Şahika Zeynep Akı, Zeynep Arzu

Yeğin, Zübeyde Nur Özkurt, Elif Suyanı, İpek Kıvılcım Oğuzülgen,

Gülsan Türköz Sucak, Literature Search: Fatma Yıldırım, Müge

Aydoğdu, Şahika Zeynep Akı, Zeynep Arzu Yeğin, Zübeyde Nur

Özkurt, Elif Suyanı, Writing: Nurdan Köktürk, Fatma Yıldırım,

Müge Aydoğdu.




included.

Financial Disclosure: The authors declared that this study has

received financial support from Glaxosmithkline.

39



References

1. Khurshid I, Anderson LC. Non-infectious pulmonary complications after

bone marrow transplantation. Postgrad Med J 2002;78:257-262.

2. Sakaida E, Nakaseko C, Harima A, Yokota A, Cho R, Saito Y, Nishimura

M. Late-onset noninfectious pulmonary complications after allogeneic

stem cell transplantation are significantly associated with chronic graftversus-host

disease and with the graft-versus-leukemia effect. Blood

2003;102:4236-4242.

3. Soubani AO, Miller KB, Hassoun PM. Pulmonary complications of bone

marrow transplantation. Chest 1996;109:1066-1077.

4. Vora A, Monaghan J, Nuttall P, Crowther D. Cytokine mediated nitric oxide

release: a common cytotoxic pathway in host-versus-graft and graftversus-host

reactions? Bone Marrow Transplant 1997;20:385-389.

5. Weiss G, Schwaighofer H, Herold M, Nachbaur D, Wachter H, Niederwieser D,

Werner ER. Nitric oxide formation as predictive parameter for acute graftversus

host-disease after human allogeneic bone marrow transplantation.

Transplantation 1995;60:1239-1244.

6. Kharitonov SA, Barnes PJ. Exhaled biomarkers. Chest 2006;130:1541-1546.

7. Smith AD, Cowan JO, Brassett KP, Herbison GP, Taylor DR. Use of exhaled

nitric oxide measurements to guide treatment in chronic asthma. N Engl J

Med 2005;352:2163-2173.

8. Antus B, Csiszer E, Czebe K, Horvath I. Pulmonary infections increase

exhaled nitric oxide in lung transplant recipients: a longitudinal study. Clin

Transplant 2005;19:377-382.

9. Aydoğdu M, Özyilmaz E, Köktürk N, Yeğin ZA, Özkurt ZN, Aki ZŞ, Sucak

G. Is there any relationship between pulmonary function tests and

post-transplant complications of allogeneic hematopoetic stem cell

transplantation? Minerva Med 2012;103:189-198.

10. American Thoracic Society Workshop. ATS Workshop Proceedings: Exhaled

nitric oxide and nitric oxide oxidative metabolism in exhaled breath

condensate: Executive summary. Am J Respir Crit Care Med 2006;173:811-

813.

11. Clark JG, Crawford SW, Madtes DK, Sullivan KM. Obstructive lung disease

after allogeneic marrow transplantation. Clinical presentation and course.

Ann Intern Med 1989;111:368-376.

12. Colby T, Myers J. Clinical and histologic spectrum of bronchiolitis obliterans,

including bronchiolitis obliterans with organizing pneumonia. Semin Respir

Med 1992;13:119-133.

13. Wolin MJ, Rigor RL. Acute graft-versus-host disease in a recipient of a twin

blood cell transplant. Bone Marrow Transplant 1996;17:125-126.

14. Fidler C, Klumpp T, Mangan K, Martin M, Sharma M, Emmons R, Lu M,

Kropf P. Spontaneous graft versus host disease occurring in a patient with

multiple myeloma after autologous stem cell transplant. Am J Hematol

2012;87:219-221.

15. Langrehr JM, Murase N, Markus PM, Cai X, Neuhaus P, Schraut W, Simmons

RL, Hoffman RA. Nitric oxide production in host-versus-graft and graftversus-host

reactions in the rat. J Clin Invest 1992;90:679-683.

16. Haddad IY, Panoskaltsis-Mortari A, Ingbar DH, Yang S, Milla CE, Blazar BR.

High levels of peroxynitrite are generated in the lungs of irradiated mice given

cyclophosphamide and allogeneic T cells: a potential mechanism of injury

after marrow transplantation. Am J Respir Cell Mol Biol 1999;20:1125-1135.

17. Kanamori H, Fujisawa S, Tsuburai T, Yamaji S, Tomita N, Fujimaki K,

Miyashita A, Suzuki S, Ishigatsubo Y. Increased exhaled nitric oxide in

bronchiolitis obliterans organizing pneumonia after allogeneic bone

marrow transplantation. Transplantation 2002;74:1356-1358.

18. Neurohr C, Huppmann P, Leuschner S, von Wulffen W, Meis T, Leuchte H,

Ihle F, Zimmermann G, Baezner C, Hatz R, Winter H, Frey L, Ueberfuhr P,

Bittmann I, Behr J, Munich Lung Transplant Group. Usefulness of exhaled

nitric oxide to guide risk stratification for bronchiolitis obliterans syndrome

after lung transplantation. Am J Transplant 2011;11:129-137.

19. Qureshi MA, Girgis RE, Dandapantula HK, Abrams J, Soubani AO. Increased

exhaled nitric oxide following autologous peripheral hematopoietic stemcell

transplantation: a potential marker of idiopathic pneumonia syndrome.

Chest 2004;125:281-287.

20. Fazekas T, Eickhoff P, Lawitschka A, Knotek B, Pötschger U, Peters C.

Exhaled nitric oxide and pulmonary complications after paediatric stem cell

transplantation. Eur J Pediatr 2012;171:1095-1101.

21. Kovesi T, Kulka R, Dales R. Exhaled nitric oxide concentration is affected

by age, height, and race in healthy 9- to 12-year-old children. Chest

2008;133:169-175.

22. Buchvald F, Baraldi E, Carraro S, Gaston B, De Jongste J, Pijnenburg MW,

Silkoff PE, Bisgaard H. Measurements of exhaled nitric oxide in healthy

subjects age 4 to 17 years. J Allergy Clin Immunol 2005;115:1130-1136.

23. Travers J, Marsh S, Aldington S, Williams M, Shirtcliffe P, Pritchard A,

Weatherall M, Beasley R. Reference ranges for exhaled nitric oxide derived

from a random community survey of adults. Am J Respir Crit Care Med

2007;176:238-242.

24. Olivieri M, Talamini G, Corradi M, Perbellini L, Mutti A, Tantucci C, Malerba

M. Reference values for exhaled nitric oxide (reveno) study. Respir Res

2006;7:94.

25. Liu HC, Hsu JY, Cheng YW, Chou MC. Exhaled nitric oxide in a Taiwanese

population: age and lung function as predicting factors. J Formos Med

Assoc 2009;108:772-777.

40

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0010


A Quasi-Experimental Study Analyzing the Effectiveness of

Portable High-Efficiency Particulate Absorption Filters in

Preventing Infections in Hematology Patients during Construction

İnşaat Esnasında Hematoloji Hastalarında Gelişen Enfeksiyonların Önlenmesinde Portabl Yüksek

Etkinlikte Partikül Emici Filtrelerin Etkinliğinin Değerlendirildiği Bir Öncesi Sonrası Çalışması

Mehmet Özen 1 , Gülden Yılmaz 2 , Belgin Coşkun 2 , Pervin Topçuoğlu 1 , Bengi Öztürk 3 , Mehmet Gündüz 1 , Erden Atilla 1 , Önder Arslan 1 ,

Muhit Özcan 1 , Taner Demirer 1 , Osman İlhan 1 , Nahide Konuk 1 , İsmail Balık 2 , Günhan Gürman 1 , Hamdi Akan 1

1Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey

2Ankara University Faculty of Medicine, Department of Infectious Diseases, Ankara, Turkey

3Ankara University Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey

Abstract

Objective: The increased risk of infection for patients caused by

construction and renovation near hematology inpatient clinics is a major

concern. The use of high-efficiency particulate absorption (HEPA) filters

can reduce the risk of infection. However, there is no standard protocol

indicating the use of HEPA filters for patients with hematological

malignancies, except for those who have undergone allogeneic

hematopoietic stem cell transplantation. This quasi-experimental study

was designed to measure the efficacy of HEPA filters in preventing

infections during construction.

Materials and Methods: Portable HEPA filters were placed in the rooms

of patients undergoing treatment for hematological malignancies

because of large-scale construction taking place near the hematology

clinic. The rates of infection during the 6 months before and after the

installation of the portable HEPA filters were compared. A total of 413

patients were treated during this 1-year period.

Results: There were no significant differences in the antifungal

prophylaxis and treatment regimens between the groups. The rates

of infections, clinically documented infections, and invasive fungal

infections decreased in all of the patients following the installation of

the HEPA filters. When analyzed separately, the rates of invasive fungal

infections were similar before and after the installation of HEPA filters

in patients who had no neutropenia or long neutropenia duration. HEPA

filters were significantly protective against infection when installed in

the rooms of patients with acute lymphocytic leukemia, patients who

were undergoing consolidation treatment, and patients who were

neutropenic for 1-14 days.

Conclusion: Despite the advent of construction and the summer

season, during which environmental Aspergillus contamination is more

prevalent, no patient or patient subgroup experienced an increase

in fungal infections following the installation of HEPA filters. The

protective effect of HEPA filters against infection was more pronounced

in patients with acute lymphocytic leukemia, patients undergoing

consolidation therapy, and patients with moderate neutropenia.

Keywords: HEPA filter, Infection, Invasive fungal infection

Öz

Amaç: Hematoloji yatan hasta kliniklerinin yakınındaki inşaat ve

tamiratların neden olduğu, hastalardaki artmış enfeksiyon riski büyük

bir endişe kaynağıdır. Yüksek etkinlikte partikül emici (YEPE) filtrelerin

kullanımı enfeksiyon riskini azaltabilir. Bununla birlikte allojenik

hematopoetik kök hücre nakli yapılanlar dışında, YEPE filtrelerin

hematolojik malignite hastalarında kullanımı konusunda standart bir

öneri yoktur. Bu öncesi sonrası çalışma inşaat esnasında enfeksiyonların

engellenmesinde YEPE filtrelerin etkinliğini ölçmek için kurgulandı.

Gereç ve Yöntemler: Hematoloji kliniğinin yanında meydana gelen

geniş çaplı bir inşaat nedeniyle hematolojik tümörlerin tedavisinin

yapıldığı hasta odalarına portabl YEPE filtreler yerleştirildi. Portabl YEPE

filtrelerin yerleştirilmesinden önceki ve sonraki 6 aydaki enfeksiyon

oranları karşılaştırıldı. Bu 1 yıllık dönemde toplam 413 hasta tedavi

edildi.

Bulgular: Gruplar arasında antifungal profilaksi ve tedavi rejimleri

açısından farklılık yoktu. YEPE filtrelerin yerleştirilmesini takiben tüm

hastalarda enfeksiyon, klinik gösterilmiş enfeksiyon ve invaziv fungal

enfeksiyon oranları azaldı. Ayrı ayrı analiz edildiğinde ise YEPE filtre

yerleştirilmesi sonrası ve öncesi invaziv fungal enfeksiyon oranları

nötropenisi olmayan veya uzun nötropeni süresi olan hastalarda

benzerdi. Akut lenfoblastik lösemi hastalarının, konsolidasyon tedavisi

alan hastaların ve 1 ile 14 gün arasında nötropenik kalan hastaların

odalarına yerleştirilen YEPE filtreler enfeksiyonlara karşı belirgin bir

şekilde koruyucuydu.

Sonuç: Çevresel Aspergillus kontaminasyonunun daha sık olduğu yaz

sezonuna ve ortaya çıkan inşaata rağmen, YEPE filtrelerin yerleştirilmesi

sonrası dönemde ne hastalarda ne de hasta alt gruplarında fungal

enfeksiyonlarda artış gözlenmedi. YEPE filtrelerin enfeksiyonlara karşı

koruyucu etkisi akut lenfoblastik lösemi hastalarında, konsolidasyon

tedavisi alan hastalarda ve orta derecede nötropenisi olanlarda daha

belirgindi.

Anahtar Sözcükler: YEPE filtre, Enfeksiyon, İnvaziv fungal enfeksiyon

Address for Correspondence/Yazışma Adresi: Mehmet ÖZEN, M.D.,

Ankara University Faculty of Medicine, Department of Hematology, Ankara, Turkey

Phone : +90 312 595 70 99

E-mail : kanbilimci@gmail.com

Received/Geliş tarihi: January 10, 2014


41

Özen M, et al: High-Efficiency Particulate Absorption and Invasive Fungal Infections


Introduction

Infectious diseases are the most common cause of mortality

and morbidity in hematology inpatient clinics. The use of

high-efficiency particulate absorption (HEPA) filters in bone

marrow transplantation units reduces the rates of infection

and transplant-related mortality in allogeneic hematopoietic

stem cell transplantation (AlloHSCT) recipients [1]. The use of

HEPA systems is recommended because of the high infection

rates in these units [2]. Although the rates of infection are high

in all neutropenic patients [3], there are no recommendations

regarding the use of HEPA filters to prevent infections in non-

AlloHSCT hematologic patients.

Construction near hospitals is an important contributing factor

in the development of invasive fungal infections (IFIs) in patients

due to environmental fungal contamination, and HEPA filters

are effective in preventing IFIs [4,5,6]. The use of HEPA filters

can also prevent bacterial infections [7,8,9]. To our knowledge,

ours is the first study to compare the ability of HEPA filters to

prevent infections in various patient groups.


Demolition and construction occurring near a 6-story hospital

located 10 m from the hematology ward at our university

provided us with the opportunity to conduct a non-randomized

retrospective quasi-experimental study to evaluate the ability

of HEPA filters to prevent infections in patients being treated

for hematologic malignancies during the construction. All of

the patients in the hematopoietic stem cell transplantation unit

were excluded from the study because that unit already had

HEPA filters installed. Portable H14-type HEPA filters (99.9995%

effective; Uvion Air Aseptizör, Teknomar, Turkey) were installed

in all the patients’ rooms on 5 May 2011.

We compared the infection rates in the 6-month periods

before and after the installation of the HEPA filters to evaluate

whether the filters prevented infections. A total of 413 patients

were treated in our hematology ward during this 1-year period.

All patients were admitted to private rooms, and preventative

measures against infection were taken with all patients. The 210

patients treated between 5 November 2010 and 4 May 2011

served as the control group and the 203 patients treated between

5 May 2011 and 26 October 2011 served as the intervention

group. The patients in the control group were housed in rooms

without HEPA filters, and the patients in the intervention group

were housed in rooms with HEPA filters. We excluded patients

from the study if they acquired IFIs in other wards prior to being

admitted to our inpatient hematology department.

We also randomly measured the level of airborne particulates

in patients’ rooms to evaluate HEPA filter efficiency. The levels

of particulates in the patients’ rooms were within acceptable

limits.

Data were assembled from patients’ files, digital records, and

records of infection from the control team.

Definitions of Infections

Infections were classified as microbiologically documented

infections, clinically documented infections, and fevers of

unknown origin (FUOs).

Microbiologically documented infections were defined

microbiologically in cultures either as bloodstream infections or

infected foci [10,11].

Clinically documented infections in patients were defined by

the presence of clinical signs of infections in the absence of

positive cultures for pathogenic microorganisms [10,11].

FUOs were defined as isolated fevers with no clinical or

microbiological signs of infection [10,11].

IFIs were defined according to EORTC/MSG (European

Organization for Research and Treatment of Cancer/Mycoses

Study Group) criteria [12]. Although candidemia results were

given, Candida-associated yeast infections were not considered

as IFIs in this study because HEPA filters are not effective in

preventing yeast infections [13]. Therefore, in our study, all

cases of IFIs were mold-related. Although severe neutropenia

is classically defined as neutropenia persisting for more than 7

or 10 days, many experts extend this to 14 days for IFIs [2,14].

Thus, we defined severe neutropenia as neutropenia that lasted

for more than 14 days for IFIs.


Numeric variables are given as medians or mean and range. The

non-parametric Mann-Whitney U test was used to compare

nominal variables. The categorized variables were compared

using the chi-square or Fisher exact test. Data were analyzed

using SPSS 16.0 for Windows and p-values of less than 0.05

were considered to be significant.

Results

The control and intervention groups were similar in sex

distribution, underlying hematological disease, history of fungal

infections, presence of central catheter, granulocyte colonystimulating

factor usage, minimum albumin levels, and severity

of neutropenia (Table 1). However, patients in the intervention

group tended to have a higher mean age (p=0.053).

Mean hospitalization durations were longer in the control

group than in the intervention group at 20 days and 15 days,

respectively (p<0.05) (Table 2). The intervention group had lower

incidences of IFIs, clinically documented infections, clinically

documented pneumonia, and overall infections than the control

group (Table 2). The rates of FUOs, all pneumonias, bacterial

42



pneumonias, fungal infections, probable IFIs, possible IFIs,

microbiologically documented infections, gram-positive and

gram-negative bacterial infections, candidemia, and infectionrelated

mortality were similar between the groups (Table 2).

The most common bacterial infections were Streptococcus

in 8 patients, Escherichia coli in 6 patients, Pseudomonas in 4

patients, Staphylococcus in 4 patients, Klebsiella in 3 patients,

and Salmonella, Pneumococcus, and Acinetobacter baumannii

in 1 patient each in the control group, and Streptococcus in 4

patients, E. coli in 11 patents, Pseudomonas in 2 patients, Klebsiella

in 4 patients, Pneumococcus in 2 patients, and Enterococcus and

Staphylococcus in 1 patient each in the intervention group.

HEPA filters seemed to be effective in preventing IFIs in all

neutropenic patients during construction. Careful evaluation

of the data revealed that HEPA filters were more effective in

preventing infections in particular subgroups of hematology

patients during construction. When the subgroups were

analyzed separately, the IFI-preventive effect of HEPA filters was

most marked in acute lymphoid leukemia patients, especially

during consolidation treatment and moderate neutropenia (1-

14 days) (Table 3). HEPA filters did not appear to reduce the

rates of IFIs in non-neutropenic patients or in patients with >14

days of neutropenia, patients undergoing induction treatment,

or patients with either acute myeloid leukemia or non-acute

leukemia (multiple myeloma, solid tumors, lymphoma, etc.)

(Table 3).

We also evaluated the patients’ hospital bills per group. The total

cost of the HEPA filters, including costs of installment and service

over the 6-month intervention period, was 50,975 Turkish lira

(TL; equivalent to 29,809 US$ or 21,328 €) [15]. We found that

all costs as given in dollars and euros per patient were decreased

after HEPA filter installation, but costs as expressed in TL were

not significantly different between these groups (Table 4).

Table 1. Patient characteristics.

Control Group

n=210 (%)

Intervention Group

n=203 (%)

Median age, years (range) 47 (18-87) 53 (20-84) 0.053

Sex, M/F (n) 137/73 118/85 0.1

Diagnosis

AML

ALL

Bone marrow failure (SAA/PNH/MDS)

Lymphoma (NHL/HL)

Plasma cell disease

CMPD/CML

Other (solid/benign)

Treatment

Induction

Consolidation

Other chemotherapy

No chemotherapy

60 (28.6)

31 (14.8)

15 (7.1)

53 (25.2)

31 (14.8)

6 (2.9)

14 (6.7)

36 (17.1)

40 (19.0)

80 (38.1)

54 (25.7)

53 (26.1)

29 (14.3)

20 (9.9)

51 (25.1)

35 (17.2)

6 (3.0)

9 (4.4)

27 (13.3)

46 (22.7)

77 (37.9)

53 (26.1)

Catheter 38 (18.1) 36 (17.7) 0.9

Neutropenia, present

>15 days neutropenia

1-14 days neutropenia

116 (54.7)

69 (59.5)

47 (40.5)

96 (45.3)

60 (62.5)

36 (37.5)

Median neutropenia duration, days (range) 11.5 (1-72) 12 (1-50) 0.1

G-CSF usage 58 (27.6) 51 (25.1) 0.6

Previous fungal infection 12 (5.7) 10 (4.9) 0.7

Antibacterial prophylaxis 5 (2.3) 4 (2.0) 1.0

Antifungal prophylaxis 18 (8.5) 12 (5.9) 0.3

Minimum albumin level 2.85±0.617 2.84±0.623 1.0

p-value

M: Male, F: female, AML: acute myeloid leukemia, ALL: acute lymphoid leukemia, SAA: severe aplastic anemia, PNH: paroxysmal nocturnal hemoglobinuria, MDS: myelodysplastic

syndrome, NHL: non-Hodgkin lymphoma, HL: Hodgkin lymphoma, CMPD: chronic myeloproliferative disease, CML: chronic myeloid leukemia, CLL: chronic lymphocytic leukemia,

G-CSF: granulocyte colony-stimulating factor.

0.6

0.9

0.3

1

0.5

1

0.3

0.3

0.4

1.0

0.9

0.1

0.5

0.2

43



Table 2. The effect of high-efficiency particulate absorption filters on infection rates.

Control Group

n (%)

Intervention Group

n (%)

Hospitalization days, median (range) 20 (2-130) 15 (1-130) 0.02*

Infections 121 (57.6) 94 (46.3) 0.02*

FUOs 40 (19) 45 (22.2) 0.4

p-value

Clinically documented infections 49 (23.3) 19 (9.4) <0.001*

Microbiologically documented infections (bacteria and fungi) 32 (15.2) 30 (14.8) 0.9

Pneumonia 39 (18.6) 30 (14.8) 0.3

Clinically documented pneumonia 27 (12.9) 13 (6.4) 0.03*

Bacterial pneumonia 21 (10) 21 (10.3) 0.9

All fungal infections 27 (12.9) 18 (8.9) 0.2

IFIs 20 (9.5) 9 (4.4) 0.04*

Probable IFIs 7 (3.3) 3 (1.5) 0.3

Possible IFIs 13 (6.2) 6 (3) 0.1

Bacterial infections (according to culture) 28 (13.3) 25 (12.3) 0.8

Gram-positive infections 18 (8.6) 15 (7.4) 0.7

Gram-negative infections 12 (5.7) 10 (4.9) 0.7

Candidemia 7 (3.3) 9 (4.4) 0.6

Infection-related mortality 17 (8.1) 10 (4.9) 0.2

FUO: Fever of unknown origin, IFI: invasive fungal infection. *: Statistically significant.

Table 3. The effect of high-efficiency particulate absorption filters on invasive fungal infections.

Control Group

n (%)

Intervention Group

n (%)

IFIs in all neutropenic patients 20 (17.2) 7 (7.3) 0.03*

IFIs in non-neutropenic patients 0 (0) 2 (1.9) 0.5

IFIs in neutropenic patients, >14 days 13 (27.7) 7 (19.4) 0.4

IFIs in neutropenic patients, 1-14 days 7 (10.1) 0 (0) 0.02*

IFIs in AML 9 (15) 6 (11.3) 0.6

IFIs in ALL 9 (29) 1 (3.4) 0.01*

IFIs in AML induction 7 (25.9) 5 (23.8) 0.9

IFIs in AML consolidation 2 (7.7) 0 (0) 0.5

IFIs in ALL induction 2 (22.2) 1 (16.7) 1.0

IFIs in ALL consolidation 6 (42.9) 0 (0) 0.002*

IFIs in all induction 9 (25) 6 (22.2) 0.8

IFIs in all consolidation 8 (20) 0 (0) 0.001*

IFIs in non-AML non-ALL 2 (1.7) 2 (1.7) 1.0

AML: Acute myeloid leukemia, ALL: acute lymphoid leukemia, IFIs: invasive fungal infections. *: Statistically significant.

p-value

44

Turk Turk J Hematol 2016;33:41-47


Table 4. Financial analysis of the patients.

Control Group Intervention Group p-value

Hospital bill per patient, TL, mean ± SD 9688±13,454 9345±15,372 0.11

All costs including HEPA per patient, TL, mean ± SD 9688±13,454 9596±15,540 0.22

All costs including HEPA per patient, $, mean ± SD 6590±9152 5611±9087 0.01*

All costs including HEPA per patient, €, mean ± SD 4548±6316 4014±6502 0.02*

TL: Turkish lira, SD: standard deviation, $: US dollar, €: Euro, HEPA: high-efficiency particulate absorption. *: Statistically significant.

Discussion

Hospital construction is a significant source of serious hospitalacquired

infections due to aspergillosis, with nosocomial

aspergillosis outbreaks occurring primarily among neutropenic

patients [16]. The period before the construction, when the HEPA

filters had not yet been installed, was winter and spring, while

the installed HEPA filters were used in summer and autumn. The

use of HEPA filters was associated with a lack of increase in IFI

rates despite both the construction and the summer months

[17]. We conclude that HEPA filter installation in hematology

wards is a safe option to prevent IFIs during construction. The

use of HEPA filters most likely prevented the rates of infectionrelated

mortality from increasing in patients treated during

construction.

Clinically documented infections originate from either IFIs or

bacterial infections. The pulmonary system is the origin of most

infections, but other systems may also be involved [18,19]. The

most common bacterial agents observed in our study were

similar to those reported to be most common in the literature

[20]. HEPA filters prevented both IFIs and bacterial infections

[21]. In our study, the rates of clinically documented pneumonia

were also reduced, which may explain why the use of HEPA

filters decreased the rates of clinically documented infections.

The literature contains few reports about the effects of HEPA

filters on patients with hematological malignancies and either

clinically documented infections or clinically documented

pneumonia; to our knowledge, our study is the first to report

this clinical finding.

During construction, the IFI rates did not increase in the subsets

of patients who were at higher risk of IFIs, including those with

acute myeloid leukemia, those undergoing remission-induction

therapy, and/or those with neutropenia that lasted >14 days.

This outcome was most likely due to the ability of HEPA filters

to prevent IFIs. However, HEPA filters appeared to be most

effective in preventing infections in patients with moderate

duration of neutropenia, patients with acute lymphoid leukemia,

and patients undergoing consolidation therapy. These groups

are reported in the literature to have lower rates of IFIs [22].

This might result from a balance between the protective effects

of the HEPA filters and the deleterious effects of neutropenia

duration on developing IFIs. To our knowledge, this finding has

not yet been reported in the literature. In multi-center studies,

the effect of HEPA filters in preventing infections may be a

confounding variable, and HEPA filter effects should be taken

into account.

HEPA filters can reduce the exposure to Aspergillus from

unfiltered air and contaminated dust by reducing the number

of Aspergillus organisms in the air [23]. Aspergillus has been

cultured from numerous hospital sources including horizontal

surfaces, food, water supplies, and ventilation systems [24].

HEPA filters may not completely prevent IFI in high-risk patients

[16]. As a result, antifungal prophylaxis should be considered as

another preventive option in high-risk patient groups [6,25,26].

The effect of season on IFI is controversial. It has been reported

that aspergillosis infections are most commonly seen in the

summer [7]. However, one study found no seasonal effect on

the rate of IFIs [27]. In our study, we were not able to evaluate

seasonal effects on the incidence of IFIs because of the study

design. However, Bénet et al. reported that the incidence of IFIs

in hematological patients during the summer months in the

absence of HEPA filters was 13.2% (9/68) [28]. We observed

that the IFI incidence during the winter months in the absence

of HEPA filters was 9.5% (20/210). Our study population and

that of Bénet et al. [28] were similar. Thus, we compared the

findings of our study with those of Bénet et al. [28] to evaluate

seasonal effect on the rate of IFIs. There was no significant

difference between the summer and winter IFI rates in these

studies (p=0.4). In other words, the protective effects of HEPA

filters against infections were independent of season.

The duration of hospitalization was longer before the installation

of HEPA filters than after installation. Lower incidences of

infection in the intervention group during construction may

have led to shorter hospital stays.

Adal et al. reported that HEPA filters may be cost-effective [29].

We did not evaluate the cost-effectiveness of HEPA filters in our

patients. However, we found that HEPA filter installation lowered

all costs per patient in euro and dollar currencies, although costs

as expressed in TL were not significantly different between these

groups, probably due to the changes in exchange rates. Thus,

we propose that HEPA filters may be a cost-effective option for

45



preventing infections in hematology patients, especially when

construction is taking place nearby.

Our study had several limitations, including its retrospective

nature, a small sample size, the fact that it was conducted at a

single center, and the lack of cost-benefit analysis. In addition,

our confirmed IFIs rates were low, because they were not

evaluated by pathology.

Some studies found hypoalbuminemia to be a risk factor for

Aspergillus infections [30,31]. Therefore, we evaluated minimum

albumin levels in patients treated in HEPA and non-HEPA rooms.

However, we did not observe any differences in albumin levels

between these 2 patient groups.

Conclusion

In conclusion, after the implementation of infection control

measures during construction, we found that keeping

immunocompromised patients in single-bed rooms with air

filtration through a HEPA system could significantly reduce

IFIs in low-risk patient groups. However, additional protective

measurements such as antifungal prophylaxis are required to

reduce the rate of infection in high-risk patient groups.

Footnote

The preliminary data included in this study were previously

presented at the 2013 American Society of Clinical Oncology

(ASCO) congress: Gurman G, Ozen M, Yilmaz G, Coskun B,

Topcuoglu P, Ozturk B, Ozcan M, Arslan O, Ilhan O, Beksac M,

Ismail B, Akan H. Hepa systems in hematology clinic to ameliorate

the increased fungal infection risk owing to environmental

changing. J Clin Oncol 2013;31 (Suppl; abstr e18009).

Ethics

Ethics Committee Approval: Retrospective study, Informed

Consent: It was taken.


Concept: Günhan Gürman, Hamdi Akan, Design: Mehmet Özen,

Gülden Yılmaz, Belgin Coşkun, Data Collection or Processing:

Mehmet Özen, Gülden Yılmaz, Belgin Coşkun, Pervin Topçuoğlu,

Bengi Öztürk, Mehmet Gündüz, Erden Atilla, Analysis or

Interpretation: Mehmet Özen, Literature Search: Önder Arslan,

Muhit Özcan, Taner Demirer, Osman İlhan, Nahide Konuk, İsmail

Balık, Writing: Mehmet Özen, Gülden Yılmaz, Belgin Coşkun,

Hamdi Akan.




included.

References

1. Passweg JR, Rowlings PA, Atkinson KA, Barrett AJ, Gale RP, Gratwohl A,

Jacobsen N, Klein JP, Ljungman P, Russell JA, Schaefer UW, Sobocinski KA,

Vossen JM, Zhang MJ, Horowitz MM. Influence of protective isolation on

outcome of allogeneic bone marrow transplantation for leukemia. Bone

Marrow Transplant 1998;21:1231-1238.

2. Freifeld AG, Bow EJ, Sepkowitz KA, Boeckh MJ, Ito JI, Mullen CA, Raad II,

Rolston KV, Young JA, Wingard JR, Infectious Diseases Society of America.

Clinical practice guideline for the use of antimicrobial agents in neutropenic

patients with cancer: 2010 update by the infectious diseases society of

america. Clin Infect Dis 2011;52:56-93.

3. Cumbo TA, Segal BH. Prevention, diagnosis, and treatment of invasive

fungal infections in patients with cancer and neutropenia. J Natl Compr

Canc Netw 2004;2:455-469.

4. Nihtinen A, Anttila VJ, Richardson M, Meri T, Volin L, Ruutu T. The utility of

intensified environmental surveillance for pathogenic moulds in a stem cell

transplantation ward during construction work to monitor the efficacy of

HEPA filtration. Bone Marrow Transplant 2007;40:457-460.

5. Krüger WH, Zöllner B, Kaulfers PM, Zander AR. Effective protection of

allogeneic stem cell recipients against Aspergillosis by HEPA air filtration

during a period of construction--a prospective survey. J Hematother Stem

Cell Res 2003;12:301-307.

6. Oren I, Haddad N, Finkelstein R, Rowe JM. Invasive pulmonary aspergillosis

in neutropenic patients during hospital construction: before and

after chemoprophylaxis and institution of HEPA filters. Am J Hematol

2001;66:257-262.

7. Wald A, Leisenring W, van Burik JA, Bowden RA. Epidemiology of

Aspergillus infections in a large cohort of patients undergoing bone marrow

transplantation. J Infect Dis 1997;175:1459-1466.

8. Sautour M, Sixt N, Dalle F, L’ollivier C, Calinon C, Fourquenet V, Thibaut

C, Jury H, Lafon I, Aho S, Couillault G, Vagner O, Cuisenier B, Besancenot

JP, Caillot D, Bonnin A. Prospective survey of indoor fungal contamination

in hospital during a period of building construction. J Hosp Infect

2007;67:367-373.

9. Korves TM, Piceno YM, Tom LM, Desantis TZ, Jones BW, Andersen GL,

Hwang GM. Bacterial communities in commercial aircraft high-efficiency

particulate air (HEPA) filters assessed by PhyloChip analysis. Indoor Air

2013;23:50-61.

10. Febril Nötropeni Çalışma Grubu. Febril nötropenik hastalarda tanı ve tedavi

kılavuzu. Flora 2004;9:5-28 (in Turkish).

11. Hughes WT, Armstrong D, Bodey GP, Bow EJ, Brown AE, Calandra T, Feld R,

Pizzo PA, Rolston KV, Shenep JL, Young LS. 2002 guidelines for the use of

antimicrobial agents in neutropenic patients with cancer. Clin Infect Dis

2002;15;34:730-751.

12. De Pauw B, Walsh TJ, Donnelly JP, Stevens DA, Edwards JE, Calandra T,

Pappas PG, Maertens J, Lortholary O, Kauffman CA, Denning DW, Patterson

TF, Maschmeyer G, Bille J, Dismukes WE, Herbrecht R, Hope WW, Kibbler

CC, Kullberg BJ, Marr KA, Muñoz P, Odds FC, Perfect JR, Restrepo A,

Ruhnke M, Segal BH, Sobel JD, Sorrell TC, Viscoli C, Wingard JR, Zaoutis T,

Bennett JE; European Organization for Research and Treatment of Cancer/

Invasive Fungal Infections Cooperative Group; National Institute of Allergy

and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus

Group. Revised definitions of invasive fungal disease from the European

Organization for Research and Treatment of Cancer/Invasive Fungal

Infections Cooperative Group and the National Institute of Allergy and

Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.

Clin Infect Dis 2008;46:1813-1821.

13. Bodey GP, Freireich EJ. Influence of high-efficiency particulate air filtration

on mortality and fungal infection: a rebuttal. J Infect Dis 2006;194:1621-

1622.

14. Barberán J, Mensa J, Llamas JC, Ramos IJ, Ruiz JC, Marín JR, Tello PB,

Massana MB, Vidal JB, Viñas JM,Huelva FJ, Pons EC, Mediavilla JD, Morfa

ML, Barrigón FE, Avellán PF, López SG, García CG, Maraver DH, Guía AL,

46



Jiménez JL, Chacón EM, Rubio MO, Oteyza JP, Ramírez GR, Contreras RR,

Barbero AR, Tarrats MR, Félix DR, Godoy PS, Salinas AS, Alonso MA, Torroba

Jde L, Ferreiras DV, López LV, García JM, Perea JR, Moreno RC, Cáncer

RC, Abete JF, Rodríguez JG, Gómez JG, Pedrosa EG, Baranda JM, García

FJ, Camps IR, Lleti MS, Cisneros Jde L; Spanish Society of Chemotherapy.

Recommendations for the treatment of invasive fungal infection caused

by filamentous fungi in the hematological patient. Rev Esp Quimioter

2011;24:263-270.

15. http://paracevirici.com/doviz-arsiv/merkez-bankasi/tcmb-gecmis-tarihlidoviz-kurlari-cevirici.php

(accessed 29 September 2014).

16. Haiduven D. Nosocomial aspergillosis and building construction. Med Mycol

2009;47:210-216.

17. Menegueti MG, Ferreira LR, Silva MF, Silva AS, Bellissimo-Rodrigues F.

Assessment of microbiological air quality in hemato-oncology units and

its relationship with the occurrence of invasive fungal infections: an

integrative review. Rev Soc Bras Med Trop 2013;46:391-396.

18. Ascioglu S, Rex JH, de Pauw B, Bennett JE, Bille J, Crokaert F, Denning DW,

Donnelly JP, Edwards JE, Erjavec Z, Fiere D, Lortholary O, Maertens J, Meis JF,

Patterson TF, Ritter J, Selleslag D, Shah PM, Stevens DA, Walsh TJ; Invasive

Fungal Infections Cooperative Group of the European Organization for

Research and Treatment of Cancer; Mycoses Study Group of the National

Institute of Allergy and Infectious Diseases. Defining opportunistic invasive

fungal infections in immunocompromised patients with cancer and

hematopoietic stem cell transplants: an international consensus. Clin Infect

Dis 2002;34:7-14.

19. Young RC, Bennett JE, Vogel CL, Carbone PP, DeVita VT. Aspergillosis. The

spectrum of the disease in 98 patients. Medicine (Baltimore) 1970;49:147-173.

20. Digiorgio MJ, Fatica C, Oden M, Bolwell B, Sekeres M, Kalaycio M, Akins P,

Shane C, Bako J, Gordon SM, Fraser TG. Development of a modified surveillance

definition of central line-associated bloodstream infections for patients with

hematologic malignancies. Infect Control Hosp Epidemiol 2012;33:865-868.

21. Furuhashi M. Efficiency of bacterial filtration in various commercial air filters for

hospital air conditioning. Bull Tokyo Med Dent Univ 1978;25:147-155.

22. Camps IR. Risk factors for invasive fungal infections in haematopoietic stem

cell transplantation. Int J Antimicrob Agents 2008;32(Suppl 2):119-123.

23. Sherertz RJ, Belani A, Kramer BS, Elfenbein GJ, Weiner RS, Sullivan ML,

Thomas RG, Samsa GP. Impact of air filtration on nosocomial Aspergillus

infections. Unique risk of bone marrow transplant recipients. Am J Med

1987;83:709-718.

24. Anaissie EJ, Stratton SL, Dignani MC, Lee CK, Summerbell RC, Rex JH,

Monson TP, Walsh TJ. Pathogenic molds (including Aspergillus species)

in hospital water distribution systems: a 3-year prospective study and

clinical implications for patients with hematologic malignancies. Blood

2003;101:2542-2546.

25. Vehreschild JJ, Böhme A, Buchheidt D, Arenz D, Harnischmacher U, Heussel

CP, Ullmann AJ, Mousset S, Hummel M, Frommolt P, Wassmer G, Drzisga

I, Cornely OA. A double-blind trial on prophylactic voriconazole (VRC) or

placebo during induction chemotherapy for acute myelogenous leukaemia

(AML). J Infect 2007;55:445-449.

26. Racil Z, Toskova M, Kocmanova I, Buresova L, Kouba M, Drgona L, Masarova

L, Guman T, Tothova E, Gabzdilova J, Forsterova K, Haber J, Ziakova B,

Bojtarova E, Rolencova M, Timilsina S, Cetkovsky P, Mayer J. Micafungin

as empirical antifungal therapy in hematological patients: a retrospective,

multicenter study in the Czech and Slovak Republics. Leuk Lymphoma

2013;54:1042-1047.

27. Hospenthal DR, Kwon-Chung KJ, Bennett JE. Concentrations of airborne

Aspergillus compared to the incidence of invasive aspergillosis: lack of

correlation. Med Mycol 1998;36:165-168.

28. Bénet T, Nicolle MC, Thiebaut A, Piens MA, Nicolini FE, Thomas X, Picot

S, Michallet M, Vanhems P. Reduction of invasive aspergillosis incidence

among immunocompromised patients after control of environmental

exposure. Clin Infect Dis 2007;45:682-686.

29. Adal KA, Anglim AM, Palumbo CL, Titus MG, Coyner BJ, Farr BM. The use

of high-efficiency particulate air-filter respirators to protect hospital

workers from tuberculosis. A cost-effectiveness analysis. N Engl J Med

1994;331:169-173.

30. Baddley JW, Andes DR, Marr KA, Kontoyiannis DP, Alexander BD, Kauffman CA,

Oster RA, Anaissie EJ, Walsh TJ, Schuster MG, Wingard JR, Patterson TF, Ito JI,

Williams OD, Chiller T, Pappas PG. Factors associated with mortality in transplant

patients with invasive aspergillosis. Clin Infect Dis 2010;50:1559-1567.

31. Perfect JR, Cox GM, Lee JY, Kauffman CA, de Repentigny L, Chapman SW,

Morrison VA, Pappas P, Hiemenz JW, Stevens DA; Mycoses Study Group. The

impact of culture isolation of Aspergillus species: a hospital-based survey of

aspergillosis. Clin Infect Dis 200;33:1824-1833.

47

RESEARCH ARTICLE

DOI: 10.4274/tjh.2014.0029


Comparison of the Efficiencies of Buffers Containing Ankaferd

and Chitosan on Hemostasis in an Experimental Rat Model with

Femoral Artery Bleeding

Femoral Arter Kanamasında Ankaferd ve Chitosan İçeren Tamponların Hemostaz Üzerine

Etkilerinin Deneysel Fare Modelinde Karşılaştırılması

Serkan Abacıoğlu 1 , Kemal Aydın 2 , Fatih Büyükcam 2 , Ural Kaya 3 , Bahattin Işık 4 , Muhammed Evvah Karakılıç 5

1Osmaniye State Hospital, Clinic of Emergency, Osmaniye, Turkey

2Dışkapı Yıldırım Beyazıt Training and Research Hospital, Clinic of Emergency, Ankara, Turkey

3Bülent Ecevit University Faculty of Medicine, Department of Emergency, Zonguldak, Turkey

4Keçiören Training and Research Hospital, Clinic of Emergency, Ankara, Turkey

5Ankara Numune Training and Research Hospital, Clinic of Emergency, Ankara, Turkey

Abstract

Objective: In the first assessment of trauma patients with major

vascular injuries, we need effective and rapid-acting homeostatic

materials. In this study we compare the efficiencies of Ankaferd Blood

Stopper® and a chitosan linear polymer (Celox®) in an experimental

rat model with femoral artery bleeding.

Materials and Methods: Thirty male Wistar albino rats weighing 200-

250 g were divided into 3 groups: control, Ankaferd, and chitosan.

The femoral artery and vein were visualized and bleeding was started

by an incision. The bleeding time was recorded and categorized as

‘bleeding stopped at the second minute’, ‘bleeding stopped at the

fourth minute’, and ‘unsuccessful’ if bleeding continued after the

fourth minute.

Results: In the control group, 60% of the bleeding did not stop. In

the first 4 min in the Ankaferd group, the bleeding stopped in all rats;

only in 1 of the rats in the chitosan group did the bleeding not stop.

In stopping the bleeding in the first 4 min, Ankaferd was similar to

chitosan but better than the control group; the chitosan group was

similar to the control, but the p-value was close to significance.

Conclusion: For major arterial bleeding, the main treatment is surgical

bleeding control, but outside of the hospital we can use buffers

containing Ankaferd and chitosan on the bleeding region. The results

of this study should be supported with larger studies. Furthermore, in

our study, healthy rats were used. New studies are needed to evaluate

the results of hypovolemic and hypotensive cases with major artery

bleeding.

Keywords: Bleeding, Ankaferd, Chitosan, Hemostasis

Öz

Amaç: Büyük damar yaralanması olan travma hastalarının ilk

müdahalesinde etkin ve hızlı etkili kanama durdurucu malzemelere

ihtiyaç duyulmaktadır. Biz bu çalışmamızda, femoral arter kanamalı

deneysel fare modelinde Ankaferd Blood Stopper® ve chitosan lineer

polymerin (Celox®) etkinliklerini karşılaştırdık.

Gereç ve Yöntem: Ağırlıkları 200-250 gram olan 30 erkek Wistar

albino fare, kontrol, Ankaferd ve chitosan olmak üzere üç gruba

ayrıldı. Femoral arter ve ven görünür hale getirildi ve bir kesi ile

kanama başlatıldı. Kanama zamanı kaydedildi ve ‘kanaması ilk iki

dakikada duranlar’, ‘kanaması ilk dört dakikada duranlar’ şeklinde

ve eğer dördüncü dakikadan sonra kanama halen devam ediyorsa

‘başarısız’ olarak kaydedildi.

Bulgular: Kontrol grubunda, kanamaların %60’ı durmadı. İlk dört

dakikada Ankaferd grubunda tüm farelerde kanama durdu; chitosan

grubunda sadece bir farede kanama durmadı. İlk dört dakikada

kanamayı durdurmada Ankaferd, chitosan ile benzerdi fakat kontrol

grubundan daha iyiydi; ayrıca chitosan kontrol grubuna benzerdi

fakat p değeri anlamlılığa yakındı.

Sonuç: Büyük arteryel kanamaların kontrolü için asıl tedavi cerrahi

kanama kontrolüdür, fakat hastane dışı alanlarda Ankaferd ve chitosan

içeren tamponlar kanama bölgesinde kullanılabilir. Araştırmamızın

sonuçları daha geniş çalışmalarla desteklenmelidir. Ayrıca

araştırmamızda sağlıklı fareler kullanılmıştır. Büyük arter kanamalı

hipotansif ve hipovolemik olgulardaki sonuçları değerlendirmek için

yeni çalışmalara ihtiyaç vardır.

Anahtar Sözcükler: Kanama, Ankaferd, Chitosan, Hemostaz

Address for Correspondence/Yazışma Adresi: Serkan ABACIOĞLU, M.D.,

Osmaniye State Hospital, Clinic of Emergency,

Osmaniye, Turkey

E-mail : serkanabacioglu@gmail.com



48


Abacıoğlu S, et al: Comparison of Ankaferd and Chitosan on Hemostasis

Introduction

Injury is the most frequent cause of death before the age of

45 years [1]. Major vascular injury is one of the major causes

of death after trauma [2]. In the first assessment of trauma

patients, in the circulation step, direct pressure should be applied

to the sites of brisk external bleeding [2]. In this process, we

need effective and rapid-acting materials to stop the bleeding.

Some of the procedures that can be used locally are direct

pressure on bleeding, fibrin glues, microporous polysaccharide

hemosphere (TraumaDEX®), poly-N-acetylglucosamine (Chitin®),

microporous hydrogel forming polyacrylamide (BioHemostat®),

chitosan linear polymer (Celox®), and oxidized cellulose

(Bloodcare®) [3,4,5,6].

In this study, we compare the efficiencies of Ankaferd Blood

Stopper® (ABS) and a chitosan linear polymer (Celox®) in an

experimental rat model with femoral artery bleeding.

ABS is composed of folkloric herbal extracts that have been

traditionally used in Anatolia as hemostatic agents (5), including

Thymus vulgaris, Glycyrrhiza glabra, Vitis vinifera, Alpinia

officinarum, and Urtica dioica. ABS, which contributes to the

conventional methods to control bleeding, has been launched

as a novel topical hemostatic agent for the management of

visible hemorrhages [6,7,8,9,10]. ABS works by creating a

protein network. It induces a very rapid (<1 s) formation of a

cellular protein network, particularly including red blood cells

and activated leukocytes within the whole blood sample, as

well as within plasma and serum samples. It also induces the

very rapid (<1 s) formation of vital erythroid aggregations as

red blood cells clustered together to aggregate rapidly, thereby

inducing a protein network formation. High-resolution scanning

electron microscopy images accompanied by morphological

analysis following the topical application of ABS revealed

very rapid (<1 s) protein network formation within concurrent

vital erythroid aggregation covering the classical coagulation

cascade [11]. The overall hemostatic effects of ABS depend on

the protein agglutination and polymerization modulating the

erythroid aggregation and vascular endothelium. ABS also

affects the distinct steps of cellular proliferation [12]. As an

important advantage, ABS is also effective in patients with

deficient primary and/or secondary hemostasis [13,14,15,16].

In addition to its anti-hemorrhagic properties, ABS has been

shown to act as a topical biological response modifier [16]. All

of these abilities not only make ABS an effective hemostatic

agent, but they also confer anti-infective, anti-neoplastic, and

healing modulator properties [17]. ABS has been used in a wide

range of applications, from dental bleedings to gastrointestinal

bleedings [18].

Chitosan (Celox®) is a non-toxic biological polysaccharide

polymer of deacetylated chitin (poly [(1,4)-N-acetyl-D-glucose-

2-amine)]) (19). It was approved by the United State Food and

Drug Administration in June 2006 with ‘Generally Recognized

as Safe’ status. The positive loaded NH 3 + groups interact with

negative loaded platelets and red blood cells, binding them with

an ionic bond [20]. This causes the aggregation of platelets in the

formation of thrombus. In vitro studies have shown its positive

effects in wound healing on activation of polymorphonuclear

neutrophils, macrophages, and fibroblasts [21,22,23]. Chitosan

has antimicrobial activity against fungi and gram-positive and

gram-negative bacteria that accelerates wound healing [22,24].

Celox® is a topical compound of chitosan that is used to stop

bleeding of surface injuries [25].


The study was carried out with approval from the local

experimental animals ethics committee (Ankara Numune

Education and Research Hospital, 31.01.2011, protocol number:

2011/5). This study was performed in the Ankara Numune

Education and Research Hospital animal laboratory and 30 male

Wistar albino rats weighing 200-250 g were used. Rats were

all fed with the same amount of feed and were fasted for 12

h before the study. Rats were divided into 3 groups as follows:

in the control group (n=10), direct compression was applied to

the bleeding without medication; in the Ankaferd group (n=10),

direct compression was applied with ABS; and in the chitosan

group (n=10), direct compression was applied with Celox®.

Before the experiment, xylazine hydrochloride and ketamine

were used for anesthesia. At that time, arterial blood pressure

monitorization was done with a KMA®250 monitor (Petaş,

İstanbul, Turkey).

The right inguinal regions of the rats were wiped with Batticon

and shaved, and the skin and subcutaneous tissues were cut into

to reveal the femoral vein and artery. Bleeding was started with

a total incision of the femoral artery and vein. Another person

collected the accumulated blood with a sponge by pressing for

10 s. The sponge was removed and immediately the homeostatic

material was applied (Celox® or ABS), and a constant 50 g of

weight was put on this material. At this time, the timer was

started. After the first minute, the bleeding was checked. If the

bleeding had stopped, it was recorded as ‘bleeding stopped at

the first minute’; if not, compression was continued with the

same amount of material up to 2 min. After 2 min, the bleeding

was checked. If the bleeding had stopped, it was recorded as

‘bleeding stopped at the second minute’; if not, compression

was applied again with the same amount of material for 2 min.

After these additional 2 min, the bleeding was checked. If the

bleeding had stopped, it was recorded as ‘bleeding stopped at

the fourth minute’. If the bleeding was still continuing, it was

recorded as ‘unsuccessful’.

49



Before the rats were sacrificed under anesthesia with 100 mg/kg

sodium thiopental (Pental Sodyum®, İ.E. Ulagay, İstanbul, Turkey),

3-mL blood samples were taken from the abdominal aorta in

order to measure the levels of hemoglobin (Hb), hematocrit

(Hct), coagulation parameters (activated partial thromboplastin

time [APTT], prothrombin time [PT], and international normalized

ratio [INR]), potassium (K), and calcium (Ca).


Statistical analysis was performed with SPSS 18.0 for Windows.

Continuous variables were expressed as mean ± standard

deviation and categorical parameters were given as numbers

and percentages. For comparing continuous variables among

more than 2 groups, the Kruskal-Wallis test was used. For

comparison of categorical variables, Fisher’s exact test was

used. All calculations were 2-tailed and p<0.05 was accepted

as significant.

Results

Two rats were excluded from the study because their mean

arterial pressures fell below 50 mmHg. Two new rats were added

to the study in their place. At the end of the study, the rats

that survived were sacrificed by 100 mg/kg intravenous sodium

thiopental (Pental Sodyum®, İ.E. Ulagay).

Mean plasma K, Ca, Hb, Hct, and platelet levels; APTT, PT, and

INR values; and weights of the groups are expressed in Table 1.

These parameters were similar in all groups (p>0.05).

In the control group, 60% of the bleeding did not stop. In the

Ankaferd group, the bleeding stopped in the first 4 min in all

rats; only in 1 rat of the chitosan group did the bleeding not

stop (Table 2). The bleeding did not stop in any rats in the first

minute.

Among rats in which the bleeding stopped in the first 2 min,

results with ABS were similar to those with chitosan (p=1.000)

and to the control group (p=0.087), but the p-values were not

statistically significant; chitosan results were also similar to

those of the control group (p=0.211).

In stopping the bleeding in the first 4 min, ABS was similar to

chitosan (p=1.000) and better than the control group (p=0.011);

chitosan was similar to the control group (p=0.057), but the

p-value was close to significance.

Discussion

Various procedures, such as direct compression, tourniquets,

and clamps, are used to stop bleeding, but these methods do

not always end in success. Homeostatic materials are now

produced to deal with severe bleeding due to trauma. In this

study, we compared 2 known homeostatic materials and direct

compression without medication. There are limited studies

that have compared ABS and Celox®. Aktop et al. evaluated

Table 2. Homeostasis durations of the groups.

Bleeding

Stopped at

Second Minute

Bleeding

Stopped at

Fourth Minute

Unsuccessful

Control - 4 (40%) 6 (60%)

Ankaferd 4 (40%) 6 (60%) -

Chitosan 3 (30%) 6 (60%) 1 (10%)

Table 1. Weight and blood test results of the groups.

Normal Ranges for Rats Control Ankaferd Chitosan p

Weight (g) 200 206.80±9.02 208.50±5.74 206.90±7.47 0.853

Hemoglobin (mg/dL) 11-18 13.53±0.20 13.54±0.16 13.54±0.25 0.992

Hematocrit (%) 36-48 49.75±0.20 49.61±0.32 49.61±0.41 0.523

Platelets (103/µL) 500-1300 863.5±7.65 860.70±8.65 857.90±8.91 0.346

Blood urea nitrogen (mg/dL) 15-21 23.10±2.42 23.30±1.83 23.30±1.64 0.860

Creatinine (mg/dL) 0.2-0.8 0.33±0.57 0.32±0.05 0.31±0.04 0.752

Na (mmol/L) 143-156 136.60±2.01 135.90±1.52 135.90±1.52 0.576

K (mmol/L) 5.4-7 4.71±0.79 4.59±0.69 4.66±0.84 0.990

PT (s) 24.5-30.9 9.51±0.19 9.53±0.13 9.52±0.16 0.963

INR - 0.82±0.02 0.817±0.02 0.81±0.02 0.619

APTT (s) 13.0-19.2 15.45±0.19 15.39±0.22 15.37±0.23 0.688

AST (U/L) 45.7-80.8 45.30±1.64 46.70±2.00 45.30±2.63 0.890

ALT (U/L) 17.5-30.2 45.40±2.12 45.70±2.00 45.60±2.32 0.605

Ca (mg/dL) 3.2-8.5 9.31±0.34 9.15±0.36 9.14±0.33 0.473

APTT: Activated partial thromboplastin time, PT: prothrombin time, INR: international normalized ratio, K: potassium, Ca: calcium, Na: sodium AST: aspartate aminotransferase,

ALT: alanine aminotransferase.

50



hemostatic parameters and the early stages of healing potential

with Celox® and ABS on soft tissue in warfarin-treated rats [26].

As in our study, they found no differences in hemostasis time,

but they did find increased tissue factor values in the Celox®treated

group. Huri et al. also found no significant difference

between Celox® and ABS hemostasis time [27]. However, tissue

healing has been shown to be improved with ABS. Homeostatic

agents are mainly used to stop venous and small arterial

bleedings, but we used them for significant arterial bleedings

and showed their efficiency even in major bleeding.

Ersoy et al. showed that microporous polysaccharide

hemosphere shortens hemostasis time [3]. Hanks et al.

compared the homeostatic efficiencies of fibrin glue and

oxidized cellulose among patients who had undergone multiple

surgical operations; they reported a shorter homeostatic time in

the fibrin glue group, where the bleeding time was 1.6 min as

opposed to 3.3 min with oxidized cellulose [28]. In our study, in

the ABS group, the bleeding stopped in 40% of the rats in the

first 2 min and in the remaining rats in the first 4 min. In this

rat model with femoral bleeding, ABS was better than direct

compression to stop the bleeding in the first 4 min (p=0.011). In

60% of the control group, the bleeding did not stop in the first

4 min, but in the ABS group, the bleeding stopped in the first 4

min in all rats.

Topical homeostatic agents have additional advantages.

There are some studies reporting that they reduce secondary

complications in some interventions; acidic forms have

antibacterial and anticandidal effects and they accelerate

wound healing [28,29].

Conclusion

In conclusion, in the case of major bleeding, the main treatment

is surgical bleeding control, but outside of the hospital, we have

to use bleeding control procedures. Here we showed that ABS

and chitosan are better than direct pressure on the bleeding

region.

Study Limitations

The results of this study should be supported with larger studies.

Additionally, in our study, healthy rats were used. New studies

are needed to evaluate the results of already hypovolemic and

hypotensive subject groups in major artery bleeding.

Ethics

Ethics Committee Approval: Ankara Numune Education and

Research Hospital, 31.01.2011, protocol number: 2011/5,

Informed Consent: N/N.


Concept: Serkan Abacıoğlu, Kemal Aydın, Design: Serkan

Abacıoğlu, Data Collection or Processing: Fatih Büyükçam,

Bahattin Işık, Analysis or Interpretation: Fatih Büyükçam,

Literature Search: Ural Kaya, Muhammed Evvah Karakılıç,

Writing: Serkan Abacıoğlu, Ural Kaya, Fatih Büyükçam,

Muhammed Evvah Karakılıç.




included.

References

1. Committee on Injury Prevention and Control. Reducing the Burden of

Injury: Advancing Prevention and Treatment. Washington, DC, USA,

National Academy Press, 1999.

2. Brunett PH, Cameron PA. Trauma in adults. In: Tintinalli JE, Stapczynski

JS, Ma OJ, Cline DM, Cydulka RK, Meckler GD, (eds). Tintinalli’s Emergency

Medicine: A Comprehensive Study Guide. 17th ed. New York, NY, USA,

McGraw Hill, 2010.

3. Ersoy G, Kaynak MF, Yilmaz O, Rodoplu U, Maltepe F, Gokmen N. Hemostatic

effects of microporous polysaccharide hemosphere in a rat model with

severe femoral artery bleeding. Adv Ther 2007;24:485-492.

4. Ward KR, Tiba MH, Holbert WH, Blocher CR, Draucker GT, Proffitt EK, Bowlin

GL, Ivatury RR, Diegelmann RF. Comparison of a new hemostatic agent to

current combat hemostatic agents in a swine model of lethal extremity

arterial hemorrhage. J Trauma 2007;63:276-283.

5. Beyazit Y, Kurt M, Kekilli M, Goker H, Haznedaroglu IC. Evaluation of

hemostatic effects of Ankaferd as an alternative medicine. Altern Med Rev

2010;15:329-336.

6. Uçar Albayrak C, Caliskan U, Haznedaroglu IC, Goker H. Haemostatic actions

of the folkloric medicinal plant extract Ankaferd Blood Stopper. J Int Med

Res 2008;36:1447-1448.

7. Sonmez M, Baltacioğlu E, Sarac O, Erkut N. The use of Ankaferd blood

stopper in a patient with Glanzmann’s thrombasthenia with gingival

bleeding. Blood Coagul Fibrinolysis 2010;21:382-383.

8. Aydin S. Haemostatic actions of the folkloric medicinal plant extract

Ankaferd Blood Stopper. J Int Med Res 2009;37:279.

9. Teker AM, Korkut AY, Gedikli O, Kahya V. Prospective, controlled clinical

trial of Ankaferd Blood Stopper in children undergoing tonsillectomy. Int J

Pediatr Otorhinolaryngol 2009;73:1742-1745.

10. Meric Teker A, Korkut AY, Kahya V, Gedikli O. Prospective, randomized,

controlled clinical trial of Ankaferd Blood Stopper in patients with acute

anterior epistaxis. Eur Arch Otorhinolaryngol 2010;267:1377-1381.

11. Haznedaroglu BZ, Haznedaroglu IC, Walker SL, Bilgili H, Goker H, Kosar A,

Aktas A, Captug O, Kurt M, Ozdemir O, Kirazli S, Firat HC. Ultrastructural

and morphological analyses of the in vitro and in vivo hemostatic effects of

Ankaferd Blood Stopper. Clin Appl Thromb Hemost 2010;16:446-453.

12. Huri E, Haznedaroglu IC, Akgul T, Astarci M, Ustun H, Germiyanoulu C.

Biphasic effects of ankaferd blood stopper on renal tubular apoptosis in the

rat partial nephrectomy model representing distinct levels of hemorrhage.

Saudi Med J 2010;31:864-868.

13. Baykul T, Alanoglu EG, Kocer G. Use of Ankaferd Blood Stopper as a hemostatic

agent: a clinical experience. J Contemp Dent Pract 2010;11:E088-E094.

14. Ercetin S, Haznedaroglu IC, Kurt M, Onal IK, Aktas A, Kurt OK, Goker H,

Ozdemir O, Kirazli S, Firat HC. Safety and efficacy of Ankaferd Blood

Stopper in dental surgery. UHOD 2010;20:1-5.

15. Ergenoglu MU, Yerebakan H, Kucukaksu DS. A new practical alternative

for the control of sternal bleeding during cardiac surgery: Ankaferd Blood

Stopper. Heart Surg Forum 2010;13:379-380.

16. Haznedaroglu IC. Molecular basis of the pleiotropic effects of Ankaferd

Blood Stopper. IUBMB Life 2009;61:290.

51



17. Akalin C, Kuru S, Barlas AM, Kismet K, Kaptanoglu B, Demir A, Astarci HM,

Ustun H, Ertas E. Beneficial effects of Ankaferd Blood Stopper on dermal

wound healing: an experimental study. Int Wound J 2014;11:64-68.

18. Haznedaroglu BZ, Beyazit Y, Walker SL, Haznedaroglu IC. Pleiotropic cellular,

hemostatic, and biological actions of Ankaferd hemostat. Crit Rev Oncol

Hematol 2012;83:21-34.

19. Fischer TH, Connolly R, Thatte HS, Schwaitzberg SS. Comparison of structural

and hemostatic properties of the poly-N-acetyl glucosamine Syvek Patch

with products containing chitosan. Micrsosc Res Tech 2004;63:168-174.

20. Kim IY, Seo SJ, Moon HS, Yoo MK, Park IY, Kim BC, Cho CS. Chitosan and

its derivatives for tissue engineering applications. Biotechnol Adv

2008;26:1-21.

21. Dai T, Tegos GP, Burkatovskaya M, Castano AP, Hamblin MR. Chitosan

acetate bandage as a topical antimicrobial dressing for infected burns.

Antimicrob Agents Chemother 2009;53:393-400.

22. Dai T, Tanaka M, Huang YY, Hamblin MR. Chitosan preparations for wounds

and burns: antimicrobial and wound-healing effects. Expert Rev Anti Infect

Ther 2011;9:857-879.

23. Muzzarelli RA. Chitins and chitosans for the repair of wounded skin, nerve,

cartilage and bone. Carbohydrate Polymers 2009;76:167-182.

24. Muzzarelli RA. Chitins and chitosans as immunoadjuvants and nonallergenic

drug carriers. Mar Drugs 2010;8:292-312.

25. Kozen BG, Kircher SJ, Henao J, Godinez FS, Johnson AS. An alternative

hemostatic dressing: comparison of CELOX, HemCon, and QuikClot. Acad

Emerg Med 2008;15:74-81.

26. Aktop S, Emekli-Alturfan E, Ozer C, Gonul O, Garip H, Yarat A, Goker K.

Effects of Ankaferd Blood Stopper and Celox on the tissue factor activities

of warfarin-treated rats. Clin Appl Thromb Hemost 2014;20:16-21.

27. Huri E, Akgül KT, Yücel MÖ, Astarcı H, Üstün H, Germiyanoğlu RC. The

second step in vitro trial of Ankaferd® Bloodstopper®: comparison with

other hemostatic agents. Turk J Med Sci 2011;41:7-15.

28. Hanks JB, Kjaergard HK, Hollingsbee DA. A comparison of the haemostatic

effect of Vivostat patient-derived fibrin sealant with oxidised cellulose

(Surgicel) in multiple surgical procedures. Eur Surg Res 2003;35:439-444.

29. Tomizawa Y. Clinical benefits and risk analysis of topical hemostats: a

review. J Artif Organs 2005;8:137-142.

52

Brief REPORT

DOI: 10.4274/tjh.2014.0012


Antifungal Treatment in Stem Cell Transplantation Centers in

Turkey

Türkiye’deki Kemik İliği Transplantasyonu Merkezlerinde Antifungal Tedavi

Hamdi Akan, Erden Atilla


Abstract

Despite the development of various guidelines, the approach to

antifungal treatment in stem cell transplantation centers differs

according to country or even between centers. This led to the

development of another survey that aims to understand the antifungal

treatment policies of Turkish stem cell transplantation centers.

Although there has been an increasing trend towards the use of

diagnostic-based treatments in Turkey in the last few years, empirical

treatment is still the main approach. The practices of the stem cell

transplantation centers reflect the general trends and controversies in

this area, while there is a considerable use of antifungal combination

therapy.

Keywords: Antifungal treatment, Diagnosis, Stem cell transplantation

Öz

Çeşitli kılavuzlara rağmen, antifungal hastalıkların tedavisine yaklaşım

kök hücre nakil merkezlerinde ülkeden ülkeye, hatta aynı ülke

içerisinde farklı merkezlerde farklılık göstermektedir. Bu farklılıkları

belirlemek amacı ile ilk defa 2010 yılında Türkiye’deki kök hücre

nakli merkezlerinde profilaksi yaklaşımlarını anlamak üzere bir anket

düzenlemiştik. Bu anket, Türkiye’deki merkezlerde tedavi yaklaşımlarını

anlamamıza yol açacak yeni bir çalışma yapmamızı sağladı. Genel

olarak tanı-güdümlü yaklaşım giderek artma eğilimi gösterse de,

ampirik yaklaşım hala ilk seçenektir. Kök hücre nakli merkezlerindeki

yaklaşımlar genel eğilimlere ve tartışmalar uygun gözükse de, kombine

antifungal kullanımının yaygın olduğu görülmektedir.

Anahtar Sözcükler: Antifungal tedavi, Teşhis, Kemik iliği

transplantasyonu

Introduction

Despite the development of various guidelines [1,2,3],

approaches to antifungal treatment in stem cell transplantation

(SCT) centers differ according to country and even between

centers. This inspired the development of another survey aimed

at understanding the antifungal treatment policies of Turkish

SCT centers.


Out of 28 EBMT-registered SCT centers, 26 responded to the

survey (Figure 1). The questionnaire consisted of separate

sections defined to understand the basic treatment approach

in each center as empirical or diagnostic-driven, the use

of diagnostic tools to start or end a treatment, strategies in

empirical or diagnostic-driven treatment, and the use of

antifungal combinations.

Results

Center Characteristics

While 19 (73.1%) of these centers are adult SCT centers,

7 (26.9%) are pediatric SCT centers. While all centers (26)

are performing allogeneic transplants, 24 centers are also

performing autologous transplants. Among the 26 allogeneic

centers, 24 are performing non-myeloablative, 7 non-related,

and 6 cord blood cell transplants.

Treatment Approach

Four centers (16%) reported that they were only using empirical

antifungal treatment, while 56% of the centers reported that

they initially employ empirical treatment but that further

treatment decisions are based on diagnostic tools such as high

resolution computed tomography (HRCT) of the lungs and

Address for Correspondence/Yazışma Adresi: Hamdi AKAN, M.D.,


Phone : +90 312 595 73 42

E-mail : hamdiakan@gmail.com


Accepted/Kabul tarihi: March 31, 2014

53

Akan H and Atilla E: Antifungals in Stem Cell Transplantation


galactomannan (GM) (Figure 2). Twenty percent of the centers

reported that they always use a diagnostic-driven approach and

8% of the centers stated that they use an empirical approach

in selected cases.

Salvage Therapy

In non-responding patients, 70% of the centers stop the initial

antifungal treatment and switch to another class of antifungal.

Twenty-five percent of the centers reported that they add

another antifungal to the initial treatment.

Drug Selection

In empirical approaches, the first drug is amphotericin-B

(conventional in 6/21 centers, liposomal in 6/21 centers) in the

allogeneic setting (Figure 3). This is followed by voriconazole

(4/21) and caspofungin (2/21). This trend is similar in the

autologous setting, but voriconazole is less commonly used

in autologous transplants. Voriconazole is the main choice in

proven cases in allogeneic (23/25) and in autologous (21/23)

transplants.

When using antifungal combination therapy, 57% of the

centers add voriconazole to initial amphotericin-B treatment,

while 38% add caspofungin to initial amphotericin-B and 5%

use voriconazole with caspofungin (Figure 4).

End of Treatment

It was found that 33.3% of the centers continue the antifungal

treatment until the end of neutropenia in empirical treatment.

Other centers reported that they use both resolution of

neutropenia and other evidence such as the clinical condition,

diagnostic tools, presence of graft-versus-host disease (GVHD),

and pre-transplant fungal status to decide to stop the treatment.

In diagnostic-driven approaches, treatment mainly stops at 90

days (23.8%), after radiological improvement (19%), or after

resolution of neutropenia (14.3%).

Most of the centers continue oral antifungals, especially in

patients with partial radiological resolution and GVHD.

In centers treating their patients based on HRCT and GM

(diagnostic-driven treatment), the main drug of choice is

voriconazole (15/20), followed by amphotericin-B (5/20), in

the allogeneic setting. This trend is similar in other transplant

settings.

Figure 1. Distribution of stem cell transplantation centers

responding to the query.

Figure 3. The initial antifungal used for empirical treatment in

stem cell transplantation centers.

Figure 2. Approach to the treatment of invasive fungal disease in

stem cell transplantation centers.

Figure 4. The antifungal combinations used in centers giving

salvage treatment (25% of all centers) (Am-B: amphotericin-B,

CAS: caspofungin, Vori: voriconazole).

54


Akan H and Atilla E: Antifungals in Stem Cell Transplantation

Patient Selection

Diagnostic-driven treatment is mainly used in allogeneic settings

(19/26 in allogeneic transplants, 18/24 in nonmyeloablative

transplants), with a rate of 62.5% (15/24) in autologous settings.

Candida Treatment

Echinocandin is the first drug of choice in established Candida

infections at 17/25 centers in allogeneic and 17/23 centers in

autologous transplants, followed by amphotericin-B (5/25) in

allogeneic and fluconazole in autologous settings.

Further Treatment

In patients not responding to initial antifungal treatment, 70%

of the centers stop the initial antifungal and start a new one,

and 25% of the centers choose to use a combination antifungal

treatment.

Diagnostic Tools

HRCT is routine in 23/26 centers, GM is routine in 4/26 centers,

and beta-glucan and molecular diagnosis are routine in 4/26

centers.

General Approach

When asked about their view on empirical or diagnostic-driven

approaches in patients with prolonged fever and neutropenia,

46.2% responded in favor of empirical treatment and 11.5% in

favor of a diagnostic-driven approach, while 42.3% responded

that the choice should be made per patient and most of them

choose to use empirical treatment in high-risk patients (Figure 2).

Discussion and Conclusion

Although there has been an increasing trend towards the

use of diagnostic-based treatments in Turkey in the last few

years, empirical treatment is still the main approach. HRCT is

the major determinant of diagnostic-driven treatment, and

while amphotericin-B is the main drug in empirical treatment,

voriconazole is the main choice in diagnostic-driven treatment

and proven cases. Despite the guidelines, a large number of

centers are using antifungal drug combinations. Keeping in mind

that there is still controversy about the selection of empirical

therapy versus preemptive (diagnostic-driven) therapy, the

differences between the centers in this aspect is understandable.

The frequent use of antifungal combinations is interesting, but

especially in centers with inadequate diagnostic tools, this

approach is to be expected, especially when physicians are

faced with a fungal infection that may end up having dreadful

consequences.


Concept: Hamdi Akan, Design: Hamdi Akan, Data Collection or

Processing: Hamdi Akan, Erden Atilla, Analysis or Interpretation:

Hamdi Akan, Erden Atilla, Literature Search: Hamdi Akan, Erden

Atilla, Writing: Hamdi Akan, Erden Atilla.




included.

References

1. Akan H. Antifungal prophylaxis in stem cell transplantation centers in

Turkey. Turk J Hematol 2011;28:271-275.

2. European Society for Blood and Marrow Transplantation. ECIL 2011 Update

for Antifungal Therapy. Available at http://www.ebmt.org/contents/

resources/library/ecil/pages/ecil.aspx, accessed 1 September 2013.

3. Freifeld AG, Bow EJ, Sepkowitz KA, Boeckh MJ, Ito JI, Mullen CA, Raad II,

Rolston KV, Young JA, Wingard JR, Infectious Diseases Society of America.

Clinical practice guideline for the use of antimicrobial agents in neutropenic

patients with cancer: 2010 update by the infectious diseases society of

America. Clin Infect Dis 2011;52:56-93.

55

Brief REPORT

DOI: 10.4274/tjh.2014.0461


Hemoglobin H Disease in Turkey: Experience from Eight Centers

Türkiye’de Hemoglobin H Hastalığı: Sekiz Merkezin Deneyimi

Selma Ünal 1 , Gönül Oktay 2 , Can Acıpayam 3 , Gül İlhan 3 , Edip Gali 2 , Tiraje Celkan 4 , Ali Bay 5 , Barış Malbora 6 , Nejat Akar 7 , Yeşim Oymak 8 ,

Tayfur Toptaş 9

1Mersin University Faculty of Medicine, Department of Pediatric Hematology, Mersin, Turkey

2Antakya Hemoglobinopathy Diagnosis, Control, and Education Center, Antakya, Turkey

3Antakya Education and Research Hospital, Antakya, Turkey

4İstanbul University Cerrahpaşa Faculty of Medicine, Department of Pediatric Hematology, İstanbul, Turkey

5Gaziantep University Faculty of Medicine, Department of Pediatric Hematology, Gaziantep, Turkey

6Dr. Sami Ulus Education and Research Hospital, Ankara, Turkey

7TOBB University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey

8Behçet Uz Education and Research Hospital, İzmir, Turkey

9Van Education and Research Hospital, Van, Turkey

Abstract

The purpose of this study was to research the problem of hemoglobin

H (HbH) disease, to reveal the distribution patterns among different

health centers, and to emphasize the importance of this disease for

Turkey. A total of 273 patients were included from 8 hemoglobinopathy

centers. The Antakya Hemoglobinopathy Center reported 232 patients

and the remaining 7 centers reported 41 patients. PubMed was

also searched for published articles related to Turkish patients with

HbH disease, and we found 16 articles involving a total of 198 HbH

patients. Most of the patients were reported from Antakya; thus,

special attention should be paid to this region. This is a preliminary

study to investigate the extent of the problem of HbH disease and it

emphasizes the need for hematology associations or the Ministry of

Health to record all cases of HbH disease in Turkey.

Keywords: Thalassemia, Hemoglobinopathy, Hemoglobin H disease

Öz

Bu çalışmada, ülkemizdeki hemoglobin H (HbH) hastalığı sorununu

irdelemek, bölgelere göre dağılımını tespit etmek ve sonuç olarak

HbH hastalığının ülkemiz için önemini vurgulamak amaçlandı. Sekiz

hemoglobinopati merkezinden toplam 273 hasta çalışmaya dahil

edildi. Merkezler arasında en fazla bildirimi 232 hasta ile Antakya

Hemoglobinopati Merkezi sağladı. Kalan 7 merkezden toplam 41

HbH hastası bildirildi. Eş zamanlı olarak PubMed’den Türkiye’den

yayınlanan ya da Türk HbH hastalarının dahil edildiği makaleler

araştırıldı ve sonuçta 198 HbH hastasını içeren toplam 16 yayına

ulaşıldı. Bu çalışma, ülkemizde HbH hastalığı sorununu araştıran bir ön

çalışmadır. Çalışmamıza Antakya’dan oldukça fazla hasta bildiriminin

olması HbH hastalığı yönünden dikkatimizi bu bölgeye yöneltmemize

neden olacaktır. Sağlık Bakanlığı önderliğinde oluşturulacak kayıt

sistemleri ile tüm HbH’li hastalar kayıt altına alınmalı ve böylece HbH

hastalığının ülkemiz için önemi vurgulanmalıdır.

Anahtar Sözcükler: Talasemi, Hemoglobinopati, Hemoglobin H

hastalığı

Introduction

Hemoglobin H (HbH) disease occurs due to defects in 3 of the 4

alpha genes found in healthy people. HbH (--/-α) is compatible

with life and usually has a similar presentation to that of

thalassemia intermedia [1]. However, clinical signs vary among

patients, and while some patients may need intermittent or

frequent transfusions, others do not.

HbH disease is suspected in cases of unresponsiveness to iron

replacement therapy and findings of microcytic anemia in

complete blood counts and peripheral smears. In the early

decades of the disease, most patients do not need erythrocyte

transfusions. Diagnosis is established when 5%-30% HbH is

detected on Hb electrophoresis. Patients with HbH disease also

have 20%-40% Hb Barts in the evaluation of cord blood [2].

Brilliant cresyl staining can be used as a screening procedure

where the molecular diagnosis of HbH disease is not possible [3].

Address for Correspondence/Yazışma Adresi: Selma ÜNAL, M.D.,

Mersin University Faculty of Medicine, Department of

Pediatric Hematology, Mersin, Turkey

E-mail : unalselma@hotmail.com

Received/Geliş tarihi: December 01, 2014

Accepted/Kabul tarihi: May 12, 2015

56


Ünal S, et al. Hemoglobin H Disease in Turkey: Experience from Eight Centers

The prevalence of alpha thalassemia is 0.24% worldwide and

13,000 babies with HbH are born annually [4]. The overall

incidence rate of alpha thalassemia for Turkey is reported to

be 0.25%-4.1% [5,6,7]. However, according to different singlecenter

studies from the south of Turkey, the frequency of alpha

thalassemia ranges between 2.5% and 7.5% [4,5,6,7,8,9,10,11,12].

As the incidence of alpha thalassemia is high, the prevalence

of HbH disease may be assumed to be higher. However, due

to inadequate reporting of patients with HbH disease, the

exact spread and occurrence rates of HbH disease cannot be

determined. Thus, the patients who were reported from eight

different centers and those reported from previously published

studies related to Turkish cases of HbH disease were included in

the present study.


Hemoglobinopathy centers in Turkey were informed about and

invited to participate in this study. Eight centers accepted the

invitation and 273 patients were included in the study. Three of

these centers were university hospitals and the others were state

hospitals. The Antakya Hemoglobinopathy Diagnosis, Control,

and Education Center (AHDCEC) reported 232 patients. The

remaining 41 patients were reported from the other 7 centers.

All of the data collected from these centers were evaluated

retrospectively. Additionally, PubMed was searched for English

publications related to Turkish patients with HbH disease. As

publications in Turkish were difficult to locate and access, those

sources were not included.

Results

Of the 273 patients included in the study, 125 were female and

148 were male. Their ages ranged between 9 month and 78 years

(Table 1). Mean Hb level was 8.7 g/dL (range: 7.2-10.9), mean

red blood cell count was 5.16 (x1012/L) (range: 4.07-5.8), mean

MCV was 54.7 fL (range: 48-76.4), mean MCH value was 17.9 pg

(range: 15-23.9), mean MCHC value was 32.2 g/dL (range: 28.4-

34), mean RDW value was 26.7 (range: 14.6-28), mean HbA2

was 2% (range: 1.2%-2.8%), mean HbF was 2.9% (range: 0.2%-

3.3%), and mean HbH was 4.7% (range: 1.8%-17.9%).

The AHDCEC reported 232 patients with HbH disease who were

recorded in a data file reporting cases dating back to as far as

1993. In the evaluation of the patients from the AHDCEC, it was

observed that the diagnosis of HbH disease was based on clinical

findings, hemoglobin electrophoresis, and HbH detection, but

alpha gene mutation analysis was not performed for any of

these patients. The evaluation of data from 41 patients from the

centers other than the AHDCEC revealed that only one patient

reached adulthood with an age of 58. Premarital screening was

performed in only one family and one other family had a history

of hydrops fetalis. The centers contributing to the study and the

numbers of patients from these centers are shown in Table 1.

The PubMed search for publications on Turkish patients with

HbH disease yielded 16 articles with a total of 198 patients with

HbH disease (Table 2) [10,13,14,15,16,17,18,19,20,21,22,23,24,

25,26,27].

Discussion

Although reports about patients with beta thalassemia and

sickle cell anemia are available, insufficient data exist about

patients with HbH disease, which can be accompanied by

various complications and moderate or severe anemia that may

require transfusions.

A total of 273 patients from 8 centers were included in this study

and 232 of these patients were from Antakya, which is located in

the Mediterranean region and had high malaria incidence rates,

Table 1. Centers involved in the study and number of hemoglobin H patients.

Center

Reported

Number of

Patients

(n=273)

Antakya Hemoglobinopathy Diagnosis, Control, and Education Center 232 2-78 years 112 Female/

120 Male

Other centers:

- İstanbul University Cerrahpaşa Faculty of Medicine, Pediatric Hematology Department

(n=13)

- Gaziantep University Faculty of Medicine, Pediatric Hematology Department (n=9)

- Sami Ulus Education and Research Hospital (n=8)

- TOBB University Faculty of Medicine, Pediatric Hematology Department (n=6)

- Mersin University Faculty of Medicine, Pediatric Hematology Department (n=3)

- Behçet Uz Education and Research Hospital (n=1)

- Van Education and Research Hospital (n=1)

Total number of hemoglobin H patients 273

Age

41 9 months to 60

years

Sex

13 Female/

28 Male

57



Table 2. Articles associated with hemoglobin H disease in Turkey.

Reported Center Number of Patients References

Hacettepe University Faculty of Medicine 138 [14,15,16,18,19,22,23,26]

İstanbul University Faculty of Medicine 18 [13,17,20,27]

Çukurova University Faculty of Medicine 39 [20,21,25]

Ege University Faculty of Medicine 1 [24]

Mustafa Kemal University Faculty of Medicine 2 [10]

Total number of patients 198

probably due to Lake Amik, until recent years. It is known that

alpha thalassemia is common where malaria is endemic. It has

been observed that another reason why the majority of patients

with HbH disease are reported from this area is that the doctors

dealing with thalassemia have been working in the region for

many years, and thalassemia patients have been recorded since

1993 at the AHDCEC. The low reported number of patients from

cities within the same geographic region, such as Mersin and

other centers of population, may be due to the recent start of

recording patients with thalassemia and HbH disease.

We detected 198 patients with HbH disease when we searched

articles from PubMed related to Turkish patients with HbH disease

[10,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]. When the

results of our study were added to that number, we found 471

HbH cases to date from Turkey, and this number is fairly high

(Table 2). Since the origin of the patients was not recorded in

other published studies, no other region was pointed out like

Antakya where HbH disease was very frequent.

As the data used in this study were not obtained from every

center in Turkey, this does not accurately reflect the real number

of patients and data on HbH disease in Turkey. However, as many

of the HbH patients were reported from Antakya, the situation

of the disease in this particular region deserves attention first.

In addition, it is necessary to design studies using the data from

all centers in Turkey to determine the exact number of patients

with HbH disease.

Another important point is that cases of hydrops fetalis due to

alpha thalassemia are rarely reported in Turkey [28]. All cases

should be reported and families with HbH should be evaluated

for their history of nonimmunological hydrops fetalis. Prenatal

diagnosis should be offered to families who have history of

hydrops fetalis, in utero death, or abortion.

Conclusion

There are no exact data related to the prevalence of HbH disease

in Turkey. However, the 471 cases that are reported based on the

data from the literature and the eight centers included in this

study are noteworthy. Thalassemia is very common in Turkey,

and there are several centers to follow the disease. Recording

of HbH cases by these centers will illustrate the urgency of the

thalassemia problem in Turkey.

In this study, it was found that Antakya is the region where HbH

disease is most frequently encountered in Turkey. More studies

are required to understand the facts about alpha thalassemia-

HbH disease in Turkey.


Concept: Selma Ünal, Design: Selma Ünal, Data Collection or

Processing: Selma Ünal, Gönül Oktay, Can Acıpayam, Gül İlhan,

Edip Gali, Tiraje Celkan, Ali Bay, Barış Malbora, Nejat Akar, Yeşim

Oymak, Tayfur Toptaş, Analysis or Interpretation: Selma Ünal,

Literature Search: Selma Ünal, Writing: Selma Ünal.




included.

References

1. Chui DH, Fucharoen S, Chan V. Hemoglobin H disease: not necessarily a

benign disorder. Blood 2003;103:791-800.

2. Steinberg MH, Forget BG, Higgs DR, Weatherall DJ. Disorders of Hemoglobin.

2nd ed. Cambridge, Cambridge University, 2009.

3. Kulaç İ, Ünal Ş, Gümrük F. Brilliant cresyl blue staining for screening

hemoglobin H disease: reticulocyte smear. Turk J Hematol 2009;26:45.

4. Canatan D, Oğuz N, Güvendik İ, Yıldırım S. The incidence of alphathalassemia

in Antalya-Turkey. Turk J Haematol 2002;19:433-434.

5. Ozsoylu S, Malik SA. Incidence of alpha thalassemia in Turkey. Turk J Pediatr

1982;24:235-244.

6. Fei YJ, Kutlar F, Harris HF, Wilson MM, Milana A, Sciacca P, Schiliro G, Masala

B, Manca L, Altay C. A search for anomalies in the zeta, alpha, beta, and

gamma globin gene arrangements in normal black, Italian, Turkish, and

Spanish newborns. Hemoglobin 1989;13:45-65.

7. Arcasoy A. Türkiye’de Thalassemia Taşıyıcı Sıklığı. Ankara, Turkey: Ankara

Thalassemia Derneği, 1991 (in Turkish).

8. Kılınç Y. Hemoglobinopathies in Turkey. Turk J Hematol 2006;23:214-216.

9. Polat G, Yuregir GT, Aksoy K. Detection of deletional alpha thalassemia in

Cukurova. Ann Med Sci 1998;7:14-17.

10. Celik MM, Gunesacar R, Oktay G, Duran GG, Kaya H. Spectrum of

α-thalassemia mutations including first observation of (FIL) deletion in

Hatay Province, Turkey. Blood Cells Mol Dis 2013;51:27-30.

11. Aldemir Ö, İzmirli M. Hataydaki α-talasemi genotipleri ve α-talasemi

genotip frekansı. Abant Med J 2014;3:233-236 (in Turkish).

12. Kılınç Y, Kümi M, Gürgey A, Altay Ç. Adana bölgesinde doğan bebeklerde

kordon kanı çalısması ile alfa talasemi, G6PD enzim eksikliği ve HbS

sıklığının araştırılması. Doğa Tıp ve Ecz Der 1986;10:162 (in Turkish).

13. Aksoy M, Erdem S. Haemoglobin H disease study of an Eti-Turk family. Acta

Genet Stat Med 1968;18:12-22.

58



14. Ozsoylu S, Isik K. Haemoglobin H disease in a Turkish family. Scand J

Haematol 1973;10:54-58.

15. Orkin SH, Old J, Lazarus H, Altay C, Gurgey A, Weatherall DJ, Nathan DG. The

molecular basis of alpha-thalassemias: frequent occurrence of dysfunctional

alpha loci among non-Asians with Hb H disease. Cell 1979;17:33-42.

16. Altay C, Gurgey A, Tuncbilek E. Hematological evaluation of patients with

various combinations of alpha-thalassemia. Am J Hematol 1980;9:261-267.

17. Aksoy M, Kutlar A, Kutlar F, Harano T, Chen SS, Huisman TH. Hemoglobin

H disease in two Turkish females and one Iranian newborn. Hemoglobin

1985;9:373-384.

18. Kutlar F, Gonzalez-Redondo JM, Kutlar A, Gurgey A, Altay C, Efremov GD,

Kleman K, Huisman TH. The levels of zeta, gamma, and delta chains in

patients with Hb H disease. Hum Genet 1989;82:179-186.

19. Fei YJ, Oner R, Bozkurt G, Gu LH, Altay C, Gurgey A, Fattoum S, Baysal E,

Huisman TH. Hb H disease caused by a homozygosity for the AATAAA--

>AATAAG mutation in the polyadenylation site of the alpha 2-globin gene:

hematological observations. Acta Haematol 1992;88:82-85.

20. Yüregir GT, Aksoy K, Çürük MA, Dikmen N, Fei YJ, Baysal E, Huisman TH. Hb

H disease in a Turkish family resulting from the interaction of a deletional

α-thalassemia-1 and a newly discovered poly A mutation. Br J Haematol

1992;80:527-532.

21. Cürük MA, Dimovski AJ, Baysal E, Gu LH, Kutlar F, Molchanova TP, Webber

BB, Altay C, Gürgey A, Huisman TH. Hb Adana or α2(59)(E8)Gly-->Aspβ2,

a severely unstable α1-globin variant, observed in combination with

the -(α)20.5 KBα-thal-1 deletion in two Turkish patients. Am J Hematol

1993;44:270-275.

22. Oner C, Gürgey A, Oner R, Balkan H, Gümrük F, Baysal E, Altay C. The

molecular basis of Hb H disease in Turkey. Hemoglobin 1997;21:41-51.

23. Oner C, Oner R, Birben E, Balkan H, Gümrük F, Gürgey A, Altay C. HB H

disease with homozygosity for red cell G6PD deficiency in a Turkish female.

Hemoglobin 1998;22;157-160.

24. Durmaz AA, Akin H, Ekmekci AY, Onay H, Durmaz B, Cogulu O, Aydinok

Y, Ozkinay F. A severe α thalassemia case compound heterozygous for Hb

Adana in α1 gene and 20.5 kb double gene deletion. J Pediatr Hematol

Oncol 2009;31:592-594.

25. Cürük MA. Hb H (β4) disease in Cukurova, southern Turkey. Hemoglobin

2007;31:265-271.

26. Ünal Ş, Gümrük F. The hematological and molecular spectrum of alpha

thalassemias in Turkey: the Hacettepe experience. Turk J Hematol

2015;32:136-143.

27. Karakas Z, Koc B, Temurhan S, Elgun T, Karaman S, Asker G, Gencay G,

Timur Ç, Yıldırmak ZY, Celkan T, Devecioglu Ö, Aydın F. Evaluation of alpha

thalassemia mutations in cases with hypochromic microcytic anemia:

Istanbul perspective. Turk J Hematol 2015.

28. Gurgey A, Altay C, Beksaç MS, Bhattacharya R, Kutlar F, Huisman TH.

Hydrops fetalis due to homozygosity for alpha-thalassemia-1, -(alpha)-20.5

kb: the first observation in a Turkish family. Acta Haematol 1989;81:169-

171.

59

Brief REPORT

DOI: 10.4274/tjh.2015.0237


The Impact of Variant Philadelphia Chromosome Translocations

on the Clinical Course of Chronic Myeloid Leukemia

Kronik Myeloid Lösemide Varyant Philadelphia Translokasyonlarının Klinik Açıdan Etkisi

Damla Eyüpoğlu 1 , Süreyya Bozkurt 2 , İbrahim Haznedaroğlu 3 , Yahya Büyükaşık 3 , Deniz Güven 1

1Hacettepe University Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey

2Hacettepe University Faculty of Medicine Cancer Institute, Basic Oncology, Ankara, Turkey

3Hacettepe University Faculty of Medicine, Division of Hematology, Ankara, Turkey

Abstract

Chronic myeloid leukemia (CML) is genetically characterized by the

presence of the reciprocal translocation t(9;22) with the formation

of Philadelphia (Ph) chromosome. Sometimes, the Ph translocation is

generated by variant rearrangements. The prognostic impact of the

variant translocations is still controversial. Among the 180 patients

with Ph-positive CML who were treated in Hacettepe University

Faculty of Medicine Division of Hematology, variant translocations

were detected, and retrospectively clinical and prognostic features

were described. Also we performed a comprehensive literature review

on the prognosis of such variant cases before and after tyrosine

kinase inhibitor era. Five patients (2.7%) had variant Ph chromosomes,

involved in the rearrangements were chromosomes 2 (2 cases), 11, 14

and 15. Patients were treated with imatinib or dasatinib. All patients

reached a stable major molecular response suggesting a prognosis not

worse than standard translocation individuals. Our present data were

compatible with the data of previous studies indicating no difference

in the prognosis between standard and variant translocations in

tyrosine kinase inhibitors era of CML.

Keywords: Chronic myeloid leukemia, Variant Philadelphia, Tyrosine

kinase inhibitors, Prognosis

Öz

Kronik myeloid lösemi (KML) Philadelphia (Ph) kromozomu olarak

anılan t(9;22) resiprokal translokasyonuyla karakterize bir hastalıktır.

Bazen Ph kromozomu varyant rearranjmanlarla ortaya çıkabilir.

Varyant translokasyonların prognostik etkisi halen tartışmalıdır.

Hacettepe Üniversitesi Tıp Fakültesi Hematoloji Kliniği’nde tedavi

edilen 180 KML hastasından tanı anında varyant translokasyon

taşıyanlar tespit edildi, klinik ve prognostik özellikleri retrospektif

olarak incelendi. Ayrıca varyant olgularda tirozin kinaz inhibitörleri

dönemi öncesi ve sonrasındaki prognoz üzerine geniş bir literatür

taraması yapıldı. Hastalardan 5’i (%2,7) tanı anında 2. (2 olgu), 11., 14.

veya 15. kromozom rearranjmanlarını içeren varyant Ph taşımaktaydı.

Hastalar imatinib veya dasatinib ile tedavi edildi. Hastaların tümünde

stabil bir majör moleküler yanıt elde edilmesi standart translokasyona

göre daha kötü bir prognoza sahip olmadığını telkin etmektedir.

Mevcut verilerimiz, daha önce yapılmış, tirozin kinaz inhibitörleri

döneminde standart ve varyant translokasyonlar arasında prognoz

açısından farklılık belirtmeyen çalışmalarla uyumluluk göstermektedir.

Anahtar Sözcükler: Kronik myeloid lösemi, Varyant Philadelphia,

Tirozin kinaz inhibitörleri, Prognoz

Introduction

Chronic myeloid leukemia (CML) is a proliferative disorder of

hematopoietic pluripotent stem cells [1]. It presents with an

estimated incidence of 1/100,000 cases per year, which accounts

for 15%-20% of all leukemia cases [2]. CML is genetically

characterized by the presence of the reciprocal translocation

t(9;22) with the formation of the Philadelphia (Ph) chromosome

[3]. The BCR-ABL fusion gene encodes a constitutively active

protein tyrosine kinase and it is responsible for the leukemia

phenotype through the constitutive activation of multiple

signaling pathways [4]. The Ph chromosome is detected in

around 90% of CML patients, among whom 5%-10% may

have variant types [5]. Variant Ph chromosomes can present a

simple form (involving 22q11 and one additional breakpoint)

or a complex form (involving 22q11, 9q34, and at least one

additional breakpoint) [6].

The aim of this study is to assess the frequency and prognosis

of CML with variant Ph chromosomes. We also performed a

Address for Correspondence/Yazışma Adresi: Damla EYÜPOĞLU, M.D.,

Hacettepe University Faculty of Medicine, Department of Internal Medicine, Ankara, Turkey

Phone : +90 507 595 24 79

E-mail : damlakarakaplan@hotmail.com

Received/Geliş tarihi: July 14, 2015


60


Eyüpoğlu D, et al: The Impact of Variant Philadelphia Chromosome Translocations on the Clinical Course of Chronic Myeloid Leukemia

comprehensive literature review to understand the prognosis of

such cases before and after the tyrosine kinase inhibitor (TKI)

era.


Study Population

Between 2008 and 2014, 180 patients were diagnosed with

CML at our institution. The diagnosis of CML was established

on the basis of bone marrow examination and supported by

cytogenetic and molecular studies. Clinical, cytogenetic,

and molecular responses to TKIs were rated according to the

European Leukemia Net (ELN) 2013 guidelines [7].

Cytogenetic Studies

Conventional cytogenetic analysis was performed on

unstimulated bone marrow specimens after 24 h of culture.

Briefly, the cells were cultured and processed by conventional

methods. After trypsin-Giemsa banding (GTG-banding), 20

metaphases were analyzed and karyotypes were interpreted

according to the 2013 International System for Human

Cytogenetic Nomenclature [8].

Results

Among the 180 patients with Ph-positive CML, 5 had variant

Ph chromosomes. Rearrangements involving chromosomes

2 (2 cases), 11, 14, and 15 were detected. Four patients were

female, the median age was 60 (range: 49-68) years, and the

median white blood cell count was 64x103/µL (24-177x10 3 /

µL). In regard to cytogenetic characteristics, all of the variant

Ph translocations were reciprocal three-way translocations

that presented at diagnosis (Figure 1). One patient’s followup

data (case 2) were not available. The other four patients’

median follow-up time was 38.5 months (8-65 months), and

TKIs (imatinib, and dasatinib in the case of imatinib failure) were

used as therapeutic agents. The main clinical parameters and

cytogenetic responses are outlined in Table 1.

For evaluating the literature data on the impact of the variant

translocations on the prognosis and clinical features, we

performed an English literature review. For this review, the

PubMed (http://www.ncbi.nlm.nih.gov/pubmed) and Web of

Science (Web of Knowledge [v5.12], Thomson Reuters, http://

apps.webofknowledge.com/) databases were used. “CML AND

variant philadelphia” and “CML AND variant translocation” were

used as keywords. We analyzed the studies in which at least 4

cases were included and TKIs had been used as therapeutics. The

literature review was conducted in May 2015. The main criteria

of these studies are outlined in Table 2.

Discussion

In 2%-10% of cases, the Ph translocation is generated by variant

rearrangements, involving 9q34, 22q11, and one or several other

genomic regions [3]. In our study, 2.7% of our patients exhibited

variant Ph chromosomes, which corresponds to the lower

margin of the reported range [2,5,9]. Rearrangements involving

chromosomes 2 (2 cases), 11, 14, and 15 were detected in our

patients. The profile of the variant translocations was similar

with those of previous reports [2,10,11]. In our study, 2 out of

3 patients who had been followed for >12 months attained

complete cytogenetic response (CCyR) at 12 months. All of the

4 patients for whom follow-up data were available reached

major molecular response (MMR) and they were still in MMR at

the last follow-up. These data do not suggest worse prognosis

compared to our standard Ph patients, which has been reported

before [12].

The prognostic impact of the variant translocations was

reported in many studies. However, some authors have stated

that the involvement of additional oncogenes could be

associated with poorer prognosis [10,13,14], while the majority

of related studies have confirmed no difference in the prognosis

between standard and variant translocations [2,5,9,15,16,17].

The ELN recommendations do not provide any specific advice

for patients with variant translocations [7].

Johansson et al. [3] mentioned that the prognostic impact

of variant translocations and secondary abnormalities was

heterogeneous and most likely related to several parameters,

such as time of appearance, specific abnormalities, and

treatment modalities.

In the first such comprehensive study, El-Zimaity et al. [9]

investigated the characteristics and outcomes of 44 patients

with variant translocations among 721 CML patients treated

Figure 1. The karyotype of case 3; 46,XX t(9;11;22)(q34;p15;q11.2).

61



Table 1. Main clinical, hematological, and cytogenetic characteristics of the patients.

Hb

Case Sex/Age Karyotype

(g/

dl)

WBC

(10 3 /dL)

PLT

(10 3 /

dL)

Phase at

diagnosis

Sokal

score

Euro

score

IM

duration,

mo

Cytogenetic

response at

1 st year

Molecular

response

at 1 st year

Cause of

discontinuation

Current

response/

treatment

Survival,

mo

1 F/60

46,XX, t(2;9;22)

(q37;q34q11.2)

12.5 24 1548 CP Low Inter.

24 PCyR - Loss of PCyR

at 24 mo

MMR /

Dasatinib

Alive/65

2 M/60

46,XY, t(2;9;22)

(q11;q34;q11.2)

11.2 177.8 484 CP Low Low

n/a n/a n/a n/a n/a n/a

3 F/55

46,XX, der(9)

t(9;15;22)

(q34;?q;q11.2)/

der(15)

12.6 64 363 CP Low Low

50 CCyR MMR None MMR /IM Alive/50

4 F/68

46,XX, t(14;9;22)

(q31;q34;q11.2)

13.4 209.4 412 CP Inter. Low

28 CCyR MMR None MMR /IM Alive/28

5 F/49

46,XX, t(9;11;22)

(q34;p15;q11.2)

12.8 72.2 221 CP Low Low

8 - - None MMR /IM Alive/8

Hb: Hemoglobin, WBC: white blood cell count, PLT: platelet count, F: female, M: male, CP: chronic phase, Inter: intermediate, HU: hydroxyurea, IM: imatinib mesylate, mo: months, n/a: not available, PCyR: partial cytogenetic

response, MMR: major molecular response, CCyR: complete cytogenetic response.

a) IM dose: 400 mg/daily

b) Dasatinib dose: 100 mg/daily

c) Responses at 6 th month

62



Table 2. Summary of the studies describing the role of variant Philadelphia in chronic myeloid leukemia patients.

Study

Variant

Ph, n/(%)

Sex, Female/

Male, n (%)

Median

Age, Years

(range)

The Most

Frequent Ph

Chromosome

Variant Types in

the Study

Differences in Clinical and Laboratory

Presentation/Risk Scores

Therapies

Prognostic

Features

Johansson et al. [3], 2001 27/(5.9) 9/18 (33/67) 54 (12-78) ins (22;9), 3, 14 Heterogeneous features/ND ND Heterogeneous

Richebourg et al. [21], 2008 41 17/24 (41/59) ND 3, der(9), 15, 12 Heterogeneous features/ND IM ND

Costa et al. [22], 2006 7/(8.8) ND 38 (27-51) 1 and 11 ND/ND HU, IFN, IM ND

El-Zimaity et al. [9], 2004 44/(6.1) 20/24 (45.5/54.5) 48 (27-75)

11, 7, 19, 10, 3, 12

Higher frequency of accelerated phase/Similar IM Similar

Valencia et al. [15], 2009 5/(6) 3/2 (60/40) 59 (50-71) 3, 5, 7, 8, 10 No significant difference/ND IM Similar

Fabarius et al. [16], 2011 69/(6) 31/38 (45/55) 54 (16-88) 1, 3, 2, 12, 5, 17

No significant difference/Similarb IM, IFN,

cytarabine

Similar

Koshiyama et al. [2], 2013 8/(8.6) 3/5 (37.5/62.5) 38 (13-75)

X, 3, 4, 5, 6, 7, 11,

14, 17, 19

No significant difference/Similara IM Similar

Marzocchi et al. [5], 2011 30/(5.4) 11/19 (37/63) 52 (33-84) 17, 1, 11, 12, 15 No significant difference/Similar a,b IM Similar

Hsiao et al. [17], 2011 6/(7.2) ND ND 3, 4, 9, 12, 14, 15

Compared with cytotoxic therapy, IM showed a

higher response/Similara,b

Cytotoxic

agents, IM

Similar

Stagno et al. [10], 2010 10/(6.5) 1/9 (10/90) 57 (29-74) 8 and 17

Higher amount of BCR-ABL/ND

IM, NIL Poor

Gorusu et al. [13], 2007 22 ND ND 1, 11, 17 Increased activity of genomic rearrangement/ND HU, IFN, IM Poor

Lee et al. [14], 2012 9/(3.2) 6/3 (67/33)

51

(32-70)

1 Overall survival and failure-free survival were

IM Poor

inferior/Worsea

ND: Not described, HU: hydroxyurea, IM: imatinib, IFN: interferon therapy, NIL: nilotinib, Ph: Philadelphia.

a) Sokal risk score

b) Euro risk score

63



with imatinib. The only significant difference in clinical

characteristics was a higher frequency of accelerated phase in

those with variant translocations (56% vs. 38%).

In a large retrospective study, Fabarius et al. [16] mentioned

that there was no significant difference in the median time of

CCyR (0.95 and 1.01 years), the median time to MMR (1.58 and

1.40 years), the 5-year progression-free survival (81% and 90%),

and the 5-year overall survival (87% and 92%). In the study

of Marzocchi et al. [5], no significant differences in complete

hematological response (93% and 98%), CCyR at 12 months

(70% and 78%), or MMR at 12 months (57% and 59%) were

observed between both groups in terms of the initial therapy

with imatinib mesylate.

Hsiao et al. [17] compared the clinical features of CML patients

with standard and variant translocations. Apart from the other

studies, they not only included TKIs as therapeutic agents but

also investigated clinical outcomes of the cytotoxic protocols. It

was stated that there was no significant difference in sex, age,

complete blood counts, disease status, and survival between

variant and classical Ph groups.

On the other hand, several studies pointed out the poor

prognosis of variant translocations. Lee et al. [14] stated that

variant Ph at diagnosis was associated with lower event-free

survival (EFS) (p=0.02) and failure-free survival (p=0.008).

Stagno et al. [10] identified that the median amount of BCR-

ABL at diagnosis was significantly higher in the variant Ph group.

After 18 months of imatinib (8 patients) or nilotinib (2 patients)

treatment, 8 patients achieved suboptimal response or failed,

while 7 patients had a cytogenetic or a molecular suboptimal

response. As a result, the authors stated that complex variant

translocations are associated with genomic instability and

a more aggressive form of CML. Gorusu et al. [13] confirmed

that deletions of the ABL1 or BCL locus were more prevalent

in variant translocation CML cases and indicated statistically

worse therapeutic responses (p<0.04) and outcomes.

The impact of the variant translocations on Sokal and Euro

scores was also found to differ in several studies. Some studies

mentioned that there was no significant difference between

patients with variant and standard Ph translocations regarding

Sokal and Euro scores [2,5,9,16]. On the other hand, it was

reported that intermediate Sokal risk score is more frequent

in patients with variant translocations and that patients with

intermediate Sokal risk had lower EFS (p=0.047) in another

study [14].

Conclusion

Between 2% and 10% of patients with CML may have variant

translocations [5]. Although it is generally accepted that the

clinical, prognostic, and hematological features of CML cases

with variant translocations are not distinct from those with the

typical t(9;22) translocation [3,18], controversies were found in

respect to the prognostic meaning of variant Ph chromosomes

[19,20]. Although our patient number was relatively limited, our

data were coherent with the studies mentioning no difference

in the prognosis between standard and variant translocations in

the TKI era.


Concept: Yahya Büyükaşık, Design: Yahya Büyükaşık, İbrahim C.

Haznedaroğlu, Data Collection or Processing: Damla Eyüpoğlu,

Yahya Büyükaşık, Analysis or Interpretation: Süreyya Bozkurt,

Damla Eyüpoğlu, Literature Search: Süreyya Bozkurt, Deniz

Güven, Writing: Damla Eyüpoğlu.




included.

References

1. Abramson S, Miller RG, Phillips RA. The identification in adult bone marrow

of pluripotent and restricted stem cells of the myeloid and lymphoid

systems. J Exp Med 1977;145:1567-1579.

2. Koshiyama DB, Capra ME, Paskulin GA, Rosa RF, Oliveira CA, Vanelli T,

Fogliatto LM, Zen PR. Cytogenetic response to imatinib treatment in

Southern Brazilian patients with chronic myelogenous leukemia and

variant Philadelphia chromosome. Ann Hematol 2013;92:185-189.

3. Johansson B, Fioretos T, Mitelman F. Cytogenetic and molecular genetic

evolution of chronic myeloid leukemia. Acta Haematol 2001;107:76-94.

4. Quintás-Cardama A, Cortes J. Molecular biology of bcr-abl1-positive

chronic myeloid leukemia. Blood 2009;113:1619-1630.

5. Marzocchi G, Castagnetti F, Luatti S, Baldazzi C, Stacchini M, Gugliotta

G, Amabile M, Specchia G, Sessarego M, Giussani U, Valori L, Discepoli G,

Montaldi A, Santoro A, Bonaldi L, Giudici G, Cianciulli AM, Giacobbi F,

Palandri F, Pane F, Saglio G, Martinelli G, Baccarani M, Rosti G, Testoni N;

Gruppo Italiano Malattie E Matologiche dell’Adulto (GIMEMA) Working

Party on Chronic Myeloid Leukemia. Variant Philadelphia translocations:

molecular-cytogenetic characterization and prognostic influence on

frontline imatinib therapy, a GIMEMA Working Party on CML analysis. Blood

2011;117:6793-6800.

6. Huret J. Complex translocations, simple variant translocations and Phnegative

cases in chronic myelogenous leukaemia. Hum Genet 1990;85:565-

568.

7. Baccarani M, Deininger MW, Rosti G, Hochhaus A, Soverini S, Apperley

JF, Cervantes F, Clark RE, Cortes JE, Guilhot F, Hjorth-Hansen H, Hughes

TP, Kantarjian HM, Kim DW, Larson RA, Lipton JH, Mahon FX, Martinelli G,

Mayer J, Müller MC, Niederwieser D, Pane F, Radich JP, Rousselot P, Saglio

G, Saußele S, Schiffer C, Silver R, Simonsson B, Steegmann JL, Goldman

JM, Hehlmann R. European Leukemia Net recommendations for the

management of chronic myeloid leukemia: 2013. Blood 2013;122:872-884.

8. Shaffer LG, McGowan-Jordan J, Schmid M. ISCN 2013: An International

System for Human Cytogenetic Nomenclature (2013). Basel, Switzerland,

Karger Medical and Scientific Publishers, 2013.

9. El-Zimaity MM, Kantarjian H, Talpaz M, O’Brien S, Giles F, Garcia-Manero

G, Verstovsek S, Thomas D, Ferrajoli A, Hayes K, Nebiyou Bekele B, Zhou X,

Rios MB, Glassman AB, Cortes JE. Results of imatinib mesylate therapy in

chronic myelogenous leukaemia with variant Philadelphia chromosome. Br

J Haematol 2004;125:187-195.

64



10. Stagno F, Vigneri P, Del Fabro V, Stella S, Cupri A, Massimino M, Consoli

C, Tambè L, Consoli ML, Antolino A, Di Raimondo F. Influence of complex

variant chromosomal translocations in chronic myeloid leukemia patients

treated with tyrosine kinase inhibitors. Acta Oncol 2010;49:506-508.

11. Lin HY, Rocher LL, McQuillan MA, Schmaltz S, Palella TD, Fox IH. Cyclosporineinduced

hyperuricemia and gout. N Engl J Med 1989;321:287-292.

12. Uz B, Buyukasik Y, Atay H, Kelkitli E, Turgut M, Bektas O, Eliacik E, Isik

A, Aksu S, Goker H, Sayinalp N, Ozcebe OI, Haznedaroglu IC. EUTOS CML

prognostic scoring system predicts ELN-based ‘event-free survival’ better

than Euro/Hasford and Sokal systems in CML patients receiving front-line

imatinib mesylate. Hematology 2013;18:247-252.

13. Gorusu M, Benn P, Li Z, Fang M. On the genesis and prognosis of variant

translocations in chronic myeloid leukemia. Cancer Genet Cytogenet

2007;173:97-106.

14. Lee SE, Choi SY, Bang JH, Kim SH, Jang EJ, Byeun JY, Park JE, Jeon HR, Oh YJ,

Kim M, Kim DW. The long-term clinical implications of clonal chromosomal

abnormalities in newly diagnosed chronic phase chronic myeloid leukemia

patients treated with imatinib mesylate. Cancer Genet 2012;205:563-571.

15. Valencia A, Cervera J, Such E, Barragán E, Bolufer P, Fuster O, Collado R,

Martínez J, Sanz MA. Complex variant t (9; 22) chromosome translocations

in five cases of chronic myeloid leukemia. Adv Hematol 2009;2009:187125.

16. Fabarius A, Leitner A, Hochhaus A, Müller MC, Hanfstein B, Haferlach C,

Göhring G, Schlegelberger B, Jotterand M, Reiter A, Jung-Munkwitz S,

Proetel U, Schwaab J, Hofmann WK, Schubert J, Einsele H, Ho AD, Falge

C, Kanz L, Neubauer A, Kneba M, Stegelmann F, Pfreundschuh M, Waller

CF, Spiekermann K, Baerlocher GM, Lauseker M, Pfirrmann M, Hasford J,

Saussele S, Hehlmann R; Schweizerische Arbeitsgemeinschaft für Klinische

Krebsforschung (SAKK) and the German CML Study Group. Impact of

additional cytogenetic aberrations at diagnosis on prognosis of CML: longterm

observation of 1151 patients from the randomized CML Study IV.

Blood 2011;118:6760-6768.

17. Hsiao HH, Liu YC, Tsai HJ, Hsu JF, Yang WC, Chang CS, Lin SF. Additional

chromosome abnormalities in chronic myeloid leukemia. Kaohsiung J Med

Sci 2011;27:49-54.

18. Al-Achkar W, Wafa A, Liehr T. A new t(9;11;20;22)(q34;p11.2;q11.21;q11)

in a Philadelphia-positive chronic myeloid leukemia case. Oncol Lett

2013;5:605-608.

19. Zhao Y, Wu G, Wu K, Wu K, Liu L, Cao W, Yu X, Luo Y, Shi J, Tan Y, Huang

H. Simultaneous occurrence of variant Philadelphia translocations and

ABL mutations in two patients with chronic myeloid leukemia. Leuk Res

2009;33:e85-e87.

20. Chauffaille Mde L, Bandeira AC, da Silva ASG. Diversity of breakpoints of

variant Philadelphia chromosomes in chronic myeloid leukemia in Brazilian

patients. Rev Bras Hematol Hemoter 2015;37:17-20.

21. Richebourg S, Eclache V, Perot C, Portnoi MF, Van den Akker J, Terré C,

Maareck O, Soenen V, Viguié F, Laï JL, Andrieux J, Corm S, Roche-Lestienne

C; Fi-LMC Group. Mechanisms of genesis of variant translocation in chronic

myeloid leukemia are not correlated with ABL1 or BCR deletion status or

response to imatinib therapy. Cancer Genet Cytogenet 2008;182:95-102.

22. Costa D, Carrió A, Madrigal I, Arias A, Valera A, Colomer D, Aguilar JL, Teixido

M, Camós M, Cervantes F, Campo E. Studies of complex Ph translocations in

cases with chronic myelogenous leukemia and one with acute lymphoblastic

leukemia. Cancer Genet Cytogenet 2006;166:89-93.

65

CASE REPORT

DOI: 10.4274/tjh.2015.0027


Secondary Neoplasms in Children with Hodgkin’s Lymphoma

Receiving C-MOPP and Radiotherapy: Presentation of Four Cases

C-MOPP ve Radyoterapi Alan Hodgkin Lenfomalı Çocuklarda İkincil Neoplaziler: Dört Olgu Sunumu

Sevgi Gözdaşoğlu1, Ali Pamir1, Emel Ünal1, İsmail Haluk Gökçora2, Ömer Uluoğlu3, Koray Ceyhan4, Haluk Deda5, Erdoğan Işıkman6,

Gülsan Yavuz1, Nurdan Taçyıldız1, Ayhan Çavdar1

1Ankara University Faculty of Medicine, Department of Pediatric Hematology and Oncology, Ankara, Turkey

2Ankara University Faculty of Medicine, Department of Pediatric Surgery, Ankara, Turkey

3Gazi University Faculty of Medicine, Department of Pathology, Ankara, Turkey

4Ankara University Faculty of Medicine, Department of Pathology, Ankara, Turkey

5TOBB University of Economics and Technology Hospital, Clinic of Neurosurgery, Ankara, Turkey

6Ankara University Faculty of Medicine, Department of Radiotherapy, Ankara, Turkey

Abstract

Patients who survive Hodgkin lymphoma (HL) are at increased risk of

secondary neoplasms (SNs). A wide variety of SNs have been reported,

including leukemias, non-Hodgkin’s lymphomas, and solid tumors,

specifically breast and thyroid cancers. Herein we report subsequent

neoplasms in four patients with HL receiving chemoradiotherapy. It

is interesting that three SNs, fibrosarcoma, thyroid carcinoma, and

retrobulbar meningioma, were observed in the radiation area in one

of our patients. A hypopharyngeal epithelioid malignant peripheral

nerve sheath tumor as an unusual secondary malignant neoplasm

developed in another patient, while a benign thyroid nodule and

invasive ductal breast carcinoma were observed at different times in

the female patient. Follicular adenoma of the thyroid gland developed

in one of our patients.

Keywords: Secondary neoplasms, Chemoradiotherapy, Hodgkin’s

lymphoma

Öz

Yaşayan Hodgkin lenfomalı olgularda ikincil neoplazilerin gelişme riski

yüksektir. Lösemiler, Hodgkin-dışı malign lenfomalar ve solid tümörler

özellikle meme ve tiroid kanserlerini içeren çok çeşitli ikincil malign

neoplaziler raporlanmıştır. Burada kemoradyoterapi alan Hodgkin

lenfomalı dört olguda gelişen ikincil neoplaziler sunulmuştur. İlginç

olarak hastalarımızdan birinde ışın alanında üç ikincil neoplazm;

fibrosarkom, tiroid karsinom ve retrobulber meningiom, diğer bir

hastamızda sıradışı ikincil malign neoplazm olarak hipofaringeal

epitelyal malign periferik sinir kılıfı tümörü gelişirken kız hastada

farklı zamanlarda benign tiroid nodülü ve invaziv duktal meme kanseri

saptandı. Bir hastamızda ise, tiroidde folliküler adenoma gelişti.

Anahtar Sözcükler: İkincil neoplaziler, Kemoradyoterapi, Hodgkin

lenfoması

Introduction

Developments in chemoradiotherapy have enabled most

patients with Hodgkin’s lymphoma (HL) to be cured. However,

the long-term effects of the treatment include an increased risk

of secondary neoplasms (SNs). SNs are defined as histologically

distinct neoplasms developing at least 2 months after the

completion of treatment for the primary malignancy [1]. SNs may

be benign or malignant in characteristics. The occurrence of SNs

following HL has now been recognized as a major problem. Among

long-term survivors who received C-MOPP (cyclophosphamide,

vincristine, procarbazine, and prednisone) plus radiotherapy (RT),

SNs developed only in 4 out of 28 patients. In 3 respective cases,

follicular adenoma of the thyroid gland, invasive ductal breast

carcinoma, and hypopharyngeal epithelioid malignant peripheral

nerve sheath tumor (EMPNST) occurred individually. In another

patient, 3 neoplasms, fibrosarcoma, papillary thyroid cancer, and

retrobulbar meningioma, were observed subsequently. Herein

we report these cases.

Case Presentations

Case 1

A 9-year-old male patient was diagnosed with clinical stage I HL

with the mixed cellularity (MC) histopathological subtype and

Address for Correspondence/Yazışma Adresi: Sevgi GÖZDAŞOĞLU, M.D.,

Kemer Sk. No: 21/78 Büyükesat, Ankara, Turkey

E-mail : sgozdasoglu@hotmail.com


Accepted/Kabul tarihi: July 02, 2015

66


Gözdaşoğlu S, et al: Secondary Neoplasms in Children with Lymphoma

had received local RT of 40 Gy to the neck region. He relapsed 3.5

years later with clinical stage IV disease and was given 6 cycles

of C-MOPP and 4 cycles of maintenance C-MOPP, for a total

of 10 cycles. A fibrosarcoma developed in the radiation area 8

years following the initial treatment (Figure 1), then followed by

papillary thyroid carcinoma diagnosed at 16 years and a rightsided

retrobulbar meningioma at 30 years later consecutively.

The patient was treated according to his neoplasms and he is

alive at the present time.

Case 2

A 15-year-old male patient was diagnosed with clinical stage

I HL with lymphocytic predominance (LP) and received 3

cycles of C-MOPP and 40 Gy RT to the neck region. A thyroid

nodule developed 27 years after the initial treatment. Surgical

excision was performed and the histopathological diagnosis was

follicular adenoma of the thyroid gland. The patient is presently

alive with no symptoms.

Case 3

A 10-year-old female patient was diagnosed with clinical stage

II HL of the nodular sclerosis (NS) type and received nitrogen

mustard, vinblastine, and local RT (40 Gy) to the neck region.

She developed a mediastinal relapse 4 years after the initial

treatment and was given cyclophosphamide (CTX), vincristine

(VCR), and adriamycin (ADM) (four cycles) and C-MOPP (two

cycles), and mediastinal RT at a dose of 37 Gy. She further

developed a benign thyroid nodule 14 years later, which was

excised. An invasive ductal carcinoma appeared in her left

breast 30 years after the initial chemoradiotherapy. After the

diagnosis of the breast carcinoma, the patient did not return

and was lost to follow-up.

Case 4

A 13-year-old male patient was diagnosed with clinical stage

III HL with MC histopathology and he received 40 Gy RT to the

neck region and C-MOPP plus maintenance C-MOPP (a total

of 10 cycles). A swan-like neck developed 12 years after the

treatment (Figure 2). An EMPNST (Figure 3) developed 30 years

after the initial treatment. Although the patient received four

cycles of iphosphamide and ADM combination chemotherapy,

he died with progression of his malignant disease 6 months

after diagnosis.

lymphomas, and solid tumors, specifically breast and thyroid

cancers. Breast cancer was the most common solid tumor with

an estimated actuarial incidence of 35% in women by 40 years

of age [4]. In one article, these SNs occurred from 3 months

to 21 years after the diagnosis of HL, with leukemias having

a median latent period of 5.5 years and solid tumors 9.5 years

from diagnosis [5].

Age at treatment has a major effect on the risk of SNs after

therapy for HL. A cohort group of 5519 patients with HL treated

during 1963-1993 was evaluated and followed for SNs, and it

was found that 322 SNs occurred. Relative risks of solid cancers

and of leukemia increased significantly with younger age at first

treatment [6].

On the other hand, in addition to chemoradiotherapy,

genetic predisposing factors such as Li-Fraumeni syndrome,

neurofibromatosis, and genetic retinoblastoma further enhance

the potential for developing SNs. Genetic susceptibility may

play an aggravating role [7].

Thirty-nine children with previously untreated HL were treated

with MOPP and RT between 1970 and 1984 in our department.

The median age was 10 years (range: 3 to 15 years); 29 were

males and 10 were females. The majority of the patients were

at stage III-IV with a predominance of the MC histopathological

subtype. Twenty-four patients received C-MOPP combination

chemotherapy (10-12 cycles), whereas 14 patients were given

sandwich therapy of 3 C-MOPP plus EF RT 40 Gy plus 3 C-MOPP,

and one case was treated with CTX, VCR, and ADM (4 cycles)

and C-MOPP (2 cycles) plus 38 Gy mediastinal RT. Eleven of

the 39 patients could not be followed, while 28 patients had

a complete evaluation with a median follow-up period of 234

months. Among long-term survivors, SNs occurred only in 4

out of 28 patients: follicular adenoma of the thyroid gland,

invasive ductal breast carcinoma, and hypopharyngeal EMPNST,

and, in one case, three neoplasms, fibrosarcoma, papillary

thyroid cancer, and retrobulbar meningioma, were observed

Discussion

The incidence of SNs has been extensively investigated in

patients treated for HL. The observed number of SNs was 3.8

times that expected among patients treated with MOPP only, 3.2

times that expected among those treated with extended field

or total nodal irradiation only, and 23.0 times that expected

among those treated with MOPP and extended field or total

nodal irradiation [2]. Approximately 25% of the mortality after

treatment for HL is believed to be due to SNs [3]. A wide variety

of SNs have been reported, including leukemias, non-Hodgkin’s

Figure 1. Histopathologic section of fibrosarcoma showing

fascicles of spindle-shaped cells (H&E stain, 20 x ).

67



subsequently. All of these SNs developed in the radiation areas.

The patients’ details are given in Table 1.

Radiation-related solid SNs account for 80% of all SNs and

demonstrate a strong relationship with RT. The risk of these solid

tumors increases with the total dose of radiation, exposure at a

younger age, and longer follow-up after RT [1].

Radiation-induced fibrosarcoma occurred at random intervals

from 3 to 38 years after irradiation, usually after high dosages

[8]. Fibrosarcoma developed 8 years after the initial treatment

in the radiation area in Case 1. Fibrosarcoma was observed as

the first SN in our experience with HL [9].

Figure 2. Appearance of swan-like neck.

In our patients, thyroid abnormalities occurred in 50% of the

patients who received 40 Gy to the neck region. All patients

without irradiation to the neck region showed normal thyroid

function [10]. Radiation therapy at a young age is the major risk

factor for the development of secondary thyroid cancers. The risk

has been reported to be 18-fold that of the general population

[1]. In Case 1, papillary thyroid cancer developed 16 years after

40 Gy radiation to the neck. Total thyroidectomy was performed

and hormonal replacement treatment was given. In addition to

these two malignancies, a right-sided retrobulbar meningioma

developed 30 years after the initial RT. It is interesting that these

3 SNs, fibrosarcoma, papillary thyroid cancer, and meningioma,

were observed consecutively in the radiation areas of the patient

and all 3 tumors were completely resected.

Benign thyroid lesions including follicular adenoma were also

reported [11]. Follicular adenoma developed 27 years after the

initial treatment in Case 2.

Best et al. identified two variants at chromosome 6q21 associated

with decreased basal PRDM1 expression and impaired induction

of PRDM1 by radiation exposure [12]. PRDM1 encodes a zinc

finger transcriptional repressor involved in a variety of cellular

processes including proliferation, differentiation, and apoptosis.

Loss of heterozygosity at chromosome 6q was found to be

significantly more common in breast cancers following RT for

HL than in sporadic breast cancers (42% vs. 10%) [12].

Figure 3. Epithelioid malignant peripheral nerve sheath tumor

cells with May Grunwald-Giemsa and diaminobenzidine stainings.

a) Binucleated tumor cells and cytoplasmic perinuclear small

vacuoles (May Grunwald-Giemsa stain, 200 x ). b) Diffuse nuclear

and cytoplasmic S-100 protein positivity (Diaminobenzidine stain,

40 x ).

The risk of breast cancer is high among women treated with

RT for childhood HL. Excess risk has been reported in female

survivors treated with high-dose, extended-volume radiation at

age 30 years or younger. In patients treated with chest RT before

16 years of age, the cumulative incidence approaches 20% by

age 45 years. Radiation-associated breast cancer has been

reported to have more adverse clinicopathological features [1].

Our patient (case 3) was treated with 40 Gy to the neck region

at the first admission and 38 Gy to the mediastinum for relapse

in addition to chemotherapy.

68



Table 1. Characteristics of the patients with secondary neoplasias.

No Age/Sex Histopathological

Type

Clinical

Stage

1 9/Male MC I Local RT 40 Gy

Treatment

3.5 years after relapse

Latency

Period

(Years)

Second Neoplasm

IV MOPP (10 cycles) 8

16

30

Fibrosarcoma

Papillary thyroid carcinoma

Retrobulbar meningioma

2 15/Male LP I 3 MOPP+Local RT 40 Gy 27 Follicular adenoma of thyroid gland

3 10/Female NS II Nitrogen mustard+VB+Local RT 40 Gy

4 years after RT, mediastinal relapse

IV CTX+VCR+ADM (4 cycles)+MOPP (2

cycles)+Mediastinal RT 38 Gy

14

30

Benign thyroid nodule

Invasive ductal breast carcinoma

4 13/Male MC III Local RT 40 Gy+MOPP (10 cycles) 28 Hypopharyngeal epithelioid

malignant peripheral nerve sheath

tumor

Case 4 was treated with 40 Gy RT to the neck region and a

total of 10 cycles of C-MOPP. A swan-like neck developed

in the patient 12 years after the initial treatment and a

hypopharyngeal EMPNST developed in the irradiated area 30

years after RT [13]. The epithelioid variant is an unusual form of

malignant peripheral nerve sheath tumors with poor prognosis

and represents approximately 5% of malignant peripheral nerve

sheath tumors [14]. Adamson et al. reported two patients with

malignant peripheral nerve sheath tumors of the spine after RT

for HL, and despite prompt surgical resection in the patients,

the tumors exhibited aggressive behavior [15]. EMPNST is

uncommon but an important fatal complication of RT.

There is increasing evidence that RT doses used in the past were

higher than necessary. Children receiving RT have an increased

risk of developing serious complications such as pulmonary or

cardiac toxicity and other cancers later in life. Hence, the new

concept is the use of involved side RT and involved node RT. The

goal of this new concept is to reduce both treatment volume

and treatment dose while maintaining efficacy and minimizing

acute and late sequelae [16,17].

Conclusion

All children who have received these treatments remain at risk

and continued surveillance is warranted. Treatment alterations

have potentially decreased the future appearance of SNs.

Monitoring for the detection of late effects in adult survivors

of childhood cancer necessitates good collaboration between

pediatric and adult oncology units.

Ethics

Informed Consent: It was taken.


Concept: Sevgi Gözdaşoğlu, Design: Ali Pamir, Sevgi Gözdaşoğlu,

Data Collection or Processing: Ali Pamir, Emel Ünal, Sevgi

Gözdaşoğlu, Analysis or Interpretation: Sevgi Gözdaşoğlu,

Literature Search: Sevgi Gözdaşoğlu, Writing: Sevgi Gözdaşoğlu.


of interest, including specific financial interest, relationships, and/

or affiliations relevant to the subject matter or materials included.

References

1. National Cancer Institute. Late Effects of Treatment for Childhood Cancer

(PDQ®). Second Cancers. Rockville, MD, USA, National Cancer Institute,

2012. Available online at http://www.cancer.gov/types/childhood-cancers/

late-effects-pdq#section/_36.

2. Green DM. Long-Term Complications of Therapy for Cancer in Childhood and

Adolescence. Baltimore, MD, USA, The Johns Hopkins University Press, 1989.

3. Wolden SL, Lamborn KR, Cleary SF, Tate DJ, Donaldson SS. Second cancers

following pediatric Hodgkin’s disease. J Clin Oncol 1998;16:536-544.

4. Bhatia S, Robison LL, Oberlin O, Greenberg M, Bunin G, Fossati-Bellani F,

Meadows AT. Breast cancer and other second neoplasms after childhood

Hodgkin’s disease. N Engl J Med 1996;334:745-751.

5. Meadows AT, Baum E, Fossati-Bellani F, Green D, Jenkin RD, Marsden B,

Nesbit M, Newton W, Oberlin O, Sallan SG, Siegel S, Strong LC, Voute PA.

Second malignant neoplasms in children: an update from the Late Effects

Study Group. J Clin Oncol 1985;3:532-538.

6. Swerdlow AJ, Barber JA, Hudson GV, Cunningham D, Gupta RK, Hancock BW,

Horwich A, Lister TA, Linch DC. Risk of second malignancy after Hodgkin’s

disease in a collaborative British cohort: the relation to age at treatment. J

Clin Oncol 2000;18:498-509.

7. Boice JD Jr. Cancer following irradiation in childhood and adolescence. Med

Pediatr Oncol Suppl 1996;1:29-34.

69



8. O’Neil MB Jr, Cocke W, Mason D, Hurley EJ. Radiation induced soft-tissue

fibrosarcoma: surgical therapy and salvage. Ann Thorac Surg 1982;33:624-628.

9. Gözdasoglu S, Cavdar AO, Babacan E. Late effects (including 2nd malignancy)

in Turkish children with Hodgkin’s disease. In: SIOP XVIII Annual Meeting.

Belgrade, Yugoslavia, Abstract Book, 1986:183-185.

10. Gözdasoglu S, Cavdar AO, Babacan E, Mengübas K, Yavuz G, Unal E, Pamir A,

Ocal G, Gökcora I. Late effects of chemoradiotherapy in pediatric Hodgkin’s

disease. J Chemother 1995;7:463-466.

11. Acharya S, Sarafoglou K, LaQuaglia M, Lindsley S, Gerald W, Wollner N, Tan

C, Sklar C. Thyroid neoplasms after therapeutic radiation for malignancies

during childhood or adolescence. Cancer 2003;97:2997-2403.

12. Best T, Li D, Skol AD, Kirchhoff T, Jackson SA, Yasui Y, Bhatia S, Strong LC,

Domchek SM, Nathanson KL, Olopade OI, Huang RS, Mack TM, Conti DV,

Offit K, Cozen W, Robison LL, Onel K. Variants at 6q21 implicate PRDM1

in the etiology of therapy-induced second malignancies after Hodgkin

lymphoma. Nat Med 2011;17:941-943.

13. Gözdaşoğlu S, Ceyhan K, Üstüner E, Akyar S, Doğan M, İçli F. Hypopharyngeal

epithelioid malignant schwannoma following treatment of Hodgkin’s

lymphoma. Int J Case Report Med 2015;2015:275309.

14. Reis Filho JS, Pope LZ, Balderrama CM, Fillus-Neto J, Schmitt FC. Epithelioid

malignant peripheral nerve sheath tumour: case report and review of the

previously published cases. Cytopathology 2002;13:54-63.

15. Adamson DC, Cummings TJ, Friedman AH. Malignant peripheral nerve

sheath tumor of the spine after radiation therapy for Hodgkin’s lymphoma.

Clin Neuropathol 2004;23:245-255.

16. Yeoh KW, Mikhaeel NG. Role of radiotherapy in modern treatment of

Hodgkin’s lymphoma. Adv Hematol 2011;2011:258797.

17. Specht L, Yahalom J, Illidge T, Berthelsen AK, Constine LS, Eich HT, Girinsky

T, Hoppe RT, Mauch P, Mikhaeel NG, Ng A; ILROG. Modern radiation therapy

for Hodgkin lymphoma: field and dose guidelines from the international

lymphoma radiation oncology group. Int J Radiat Oncol Biol Phys

2014;89:854-862.

70

LETTERS TO EDITOR


First Observation of Hemoglobin G-Waimanalo and Hemoglobin

Fontainebleau Cases in the Turkish Population

Türk Toplumunda Gözlenen ilk Hemoglobin G-Waimanalo ve Hemoglobin Fontainebleau Olguları

Duran Canatan 1,2 , Türker Bilgen 3 , Vildan Çiftçi 1 , Gülsüm Yazıcı 1 , Serpil Delibaş 2 , İbrahim Keser 4

1Antalya Genetic Diagnostic Center, Antalya, Turkey

2Hemoglobinopathy Diagnostic Center of Mediterranean Blood Diseases Foundation, Antalya, Turkey

3Namık Kemal University Research and Application Center for Scientific and Technological Investigations, Tekirdağ, Turkey

4Akdeniz University Faculty of Medicine, Department of Biology and Genetics, Antalya, Turkey

To the Editor,

Deletional alpha thalassemia mutations can be detected by

various methods such as reverse dot blot, gap-polymerase

chain reaction (gap-PCR), and multiplex ligation-dependent

probe amplification. Point mutations leading to abnormal

hemoglobins (Hb) are also observed in common populations.

When a point mutation is suspected, resequencing of the alpha

genes has become a routine procedure [1]. Hb G-Waimanalo

is a silent mutation characterized by a substitution to aspartic

acid from asparagine at codon 64 [A64(A2), Asp>Asn] [2]. Hb

Fontainebleau is a slightly unstable mutation characterized by

a substitution to alanine from proline at codon 21 [A21(A2),

Ala>Pro] [3]. To date, they have not been reported from Turkey

[4]. Here we present two cases with abnormal Hbs.

Case 1: NB, a 33-year-old female, was admitted to the

hemoglobinopathy diagnostic center for premarital thalassemia

testing. Her complete blood count (CBC) was found to be

normal with 24.2% abnormal bands in high-performance

liquid chromatography (HPLC) (Table 1). A blood sample was

studied further at the genetic diagnostic center. Following DNA

extraction with a commercial kit (Roche, Mannheim, Germany)

and amplification of the whole beta globin gene by standard

protocols of PCR and DNA sequencing (Applied Biosystems,

USA), mutation was not found in the beta globin gene. Sequence

analyses of alpha genes A1 and A2 were performed and an

abnormal Hb in the HBA2: c.193G>A change was detected. This

change in the HbA2 gene was at codon 64 GAC>AAC (Asp>Asn),

known as Hb G-Waimanalo.

Case 2: ND, a 37-year-old, female, was also admitted to the

hemoglobinopathy diagnostic center for premarital testing. She had

normocytic anemia in CBC and abnormal bands detected at 16.2%

in HPLC. This band result was found to be lower because it may have

been fragmented with results of slightly unstable mutation (Table 1).

Her blood was studied by the same method at the genetic diagnostic

center. Mutation was not found in the beta globin gene. The HbA2 and

HbA2 genes were then selectively amplified by standard protocols of

PCR. DNA sequencing revealed a G to C change at nucleotide position

21 in the HbA2 gene. This mutation at codon 21 GCT>CCT (Ala>Pro)

in the HbA2 gene is known as Hb Fontainebleau.

Hb G-Waimanalo is an abnormal Hb and asymptomatic. It was

reported in association with alpha and/or beta thalassemia [5,6].

There were no hematological findings in our case; the beta gene

was found to be normal. Hb G-Waimanalo was identified in five

cases in a study in China [7].

Hb Fontainebleau was described as a silent mutation for the

first time in a family of Italian origin [8]. Two cases with mild

microcytosis were reported in New Zealand [3]. Our patient had

normocytic anemia based on CBC and lower abnormal bands

by reason of a slightly unstable mutation in HPLC. Beta gene

analysis was normal. So far, a total of 22 cases including 1

homozygous case without clinical findings and 11 heterozygous

cases have been reported from premarital screening in the

United Arab Emirates [9].

In conclusion, abnormal bands, especially in HPLC, should be

investigated with sequence analysis to corroborate alpha and/or

beta globin gene mutations.

Table 1. The results of complete blood count and high-performance liquid chromatography in the presented cases.

Case Hb (g/dL) Hct (%) RBC (10 12 /L) MCV (fL) MCH (pg) HbA1 (%) HbA2 (%) HbF (%) Abnormal bands (%)

Case 1 13.3 39.4 4.31 89.1 34.6 73.5 1.6 0.3 24.6

Case 2 11.1 37.8 4.66 83.1 24.4 72.0 1.2 0.6 16.2

Hb: Hemoglobin, Hct: hematocrit, RBC: red blood cell, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin.

71

LETTERS TO EDITOR


Keywords: Abnormal hemoglobins, Hemoglobin G-Waimanalo,

Hemoglobin Fontainebleau

Anahtar Sözcükler: Anormal hemoglobinler, Hemoglobin

G-Waimanalo, Hemoglobin Fontainebleau


Concept: Duran Canatan, Design: Duran Canatan, Data

Collection or Processing: Serpil Delibaş, Gülsüm Yazıcı, Vildan

Çiftçi, Analysis or Interpretation: Türker Bilgen, İbrahim Keser,

Gülsüm Yazıcı, Vildan Çiftçi, Literature Search: Duran Canatan,

Türker Bilgen, Writing: Duran Canatan.




included.

References

1. Harteveld CL, Higgs DR. Alpha-thalassaemia. Orphanet J Rare Dis 2010;5:13.

2. Blackwell RQ, Jim RT, Tan TG, Weng MI, Liu CS, Wang CL. Hemoglobin G

Waimanalo: alpha-64 Asp leads to Asn. Biochim Biophys Acta 1973;322:27-33.

3. Brennan SO, Chan T, Ryken S, Ruskova A. A second case of Hb Fontainebleau

[alpha21(B2)Ala-->Pro] in an individual with microcytosis. Hemoglobin

2009;33:258-261.

4. Akar N. An updated review of abnormal hemoglobins in the Turkish

population. Turk J Hematol 2014;31:97-98.

5. Lin M, Wu JR, Yang LY, Chen H, Wang PP, Wang Q, Zheng L. Hb G-Waimanalo:

occurrence in combination with alpha-thalassemia-1 Southeast Asian

deletion. Blood Cells Mol Dis 2009;42:36-37.

6. Tan TG, Jim RT, Blackwell RQ. Hemoglobin G Waimanalo beta thalassemia.

Hawaii Med J 1978;37:235-239.

7. Lin M, Wang Q, Zheng L, Huang Y, Lin F, Lin CP, Yang LY. Prevalence and

molecular characterization of abnormal hemoglobin in eastern Guangdong

of southern China. Clin Genet 2012;81:165-171.

8. Wajcman H, Blouquit Y, Gombaud-Saintonge G, Riou J, Galacteros F.Hb

Fontainebleau [alpha 21(B2)Ala----pro], a new silent mutant hemoglobin.

Hemoglobin 1989;13:421-428.

9. Turner A, Sasse J, Varadi A. Hb Fontainebleau (HBA2: c.64G > C) in the

United Arab Emirates. Hemoglobin 2014;38:216-220.

Address for Correspondence/Yazışma Adresi: Duran CANATAN, M.D.,

Antalya Genetic Diagnostic

Center, Antalya, Turkey

E-mail : durancanatan@gmail.com

Received/Geliş tarihi: August 18, 2015


DOI: 10.4274/tjh.2015.0299

Serum Lipids in Turkish Patients with β-Thalassemia Major and

β-Thalassemia Minor

Türk β-Talasemi Majör ve β-Talasemi Minör Hastalarının Serum Lipidleri

Yasemin Işık Balcı 1 , Şule Ünal 2 , Fatma Gümrük 3

1Pamukkale University Faculty of Medicine, Department of Pediatric Hematology, Denizli, Turkey

2Hacettepe University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey

3Hacettepe University Faculty of Medicine, Department of Radiology, Ankara, Turkey

To the Editor,

It is well-known that β-thalassemia is associated with changes

in plasma lipids and lipoproteins [1,2,3]. To our knowledge, no

data are available on lipid profiles in Turkish β-thalassemia

major (TM) and β-thalassemia trait (TT) patients together. The

aim of this study was to evaluate lipid profiles in two groups of

patients with β-TM and β-TT and to compare them with healthy

controls. The study included a total of 311 subjects. Group 1

included 131 β-TM patients (mean age: 16.3±7.58 years). Group

2 included 68 β-TT patients (mean age: 7.25±4.43 years). Group

3 consisted of 112 age- and sex-matched healthy controls (mean

age: 9±4.7 years). Serum ferritin level was 2487±1103 (range:

661-5745) ng/mL in Group 1. In comparing the correlation

between ferritin and lipid parameters, while a significantly

negative relationship was detected between ferritin and highdensity

lipoprotein cholesterol (HDL-C) (p=0.000, r=-0.602), a

significantly positive relationship was detected between ferritin

and triglyceride (TG) levels (p=0.02) in TM patients. Serum lipid

profiles of the 3 groups are shown in Table 1.

Previous studies have shown total serum cholesterol, HDL-C,

lower low-density lipoprotein cholesterol (LDL-C), and higher

TG in β-TM patients compared to healthy controls [1,2,3]. In

our study, we found lower serum total cholesterol, lower

HDL-C, LDL-C, and higher TG in β-TM patients compared to

healthy controls. The pathophysiology of hypocholesterolemia

in thalassemia remains obscure, although several mechanisms

have been proposed; plasma dilution due to anemia, increased

cholesterol requirement associated with erythroid hyperplasia,

macrophage system activation with cytokine release, and

increased cholesterol uptake by the reticuloendothelial system

[4,5]. Previous studies reported different variations in lipid

profiles of β-TT patients [6,7]. In our study, we demonstrated

72


LETTERS TO EDITOR

Table 1. Lipid profiles and their significance in patients with β-thalassemia major, patients with β-thalassemia trait, and

controls.

Group 1 Group 2 Group 3 p-values

β-TM (n=131) β-TT (n=68) Control (n=112) Groups 1-2 Groups 1-3 Groups 2-3

T-Chol 118.5±30.6 145.6±27.6 154.3±31.7 0.00 0.00 NS

LDL-C 59.1±27.6 82.5±24.9 89.6±26.1 0.00 0.00 NS

HDL-C 34.4±11.2 45.7±12.2 45.5±11.1 0.00 0.00 NS

TG 121.8±50.8 82.9±34.6 97.8±52.4 0.00 0.00 NS

T-Chol: Total cholesterol, LDL-C: low-density lipoprotein cholesterol, HDL-C: high-density lipoprotein cholesterol, TG: triglyceride, β-TM: β-thalassemia major, β-TT: β-thalassemia

trait, NS: non-significant.

similar lipid profiles in β-TT patients and healthy controls. Based

on statistical insignificance, we considered that the effects

of lipid profile on the development of atherosclerotic vessel

disease were similar in both β-TT patients and the healthy

control group. Serum iron and iron stores, expressed as elevated

ferritin levels, have been implicated in coronary artery disease.

Iron overload depletes the antioxidant and HDL-C levels. Lower

HDL-C level is an important risk factor for development of

coronary heart diseases [8]. We found significant relationships

of serum ferritin levels with TG and HDL-C in β-TM patients.

These results indicate that β-TM patients who need life-long

red blood cell transfusions should receive chelation therapy not

only for iron overload-induced congestive heart failure but also

in order to prevent cardiovascular diseases resulting from lipid

profile alterations.

In conclusion, lipid profiles of β-TM patients differed from

those of β-TT patients and healthy controls. The present study

demonstrates that lower levels of HDL-C in β-TM should be a

reason for concern for better evaluation of the cardiovascular

risk factors in β-TM. In order to reduce the effects of lipid

metabolism on cardiovascular disorders, an effective chelating

therapy is essential in TM patients.

Keywords: Thalassemia major, Thalassemia minor, Serum lipids

Anahtar Sözcükler: Talasemi majör, Talasemi minör, Serum

lipidleri


Concept: Yasemin Işık Balcı, Design: Yasemin Işık Balcı, Data

Collection or Processing: Yasemin Işık Balcı, Şule Ünal, Fatma

Gümrük, Analysis or Interpretation: Yasemin Işık Balcı, Şule

Ünal, Literature Search: Yasemin Işık Balcı, Şule Ünal, Fatma

Gümrük, Writing: Yasemin Işık Balcı.

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

interest, including specific financial interests, relationships, and/


References

1. Maioli M, Vigna GB, Tonolo G, Brizzi P, Ciccarese M, Donega P, Maioli M,

Fellin R. Plasma lipoprotein composition, apolipoprotein (a) concentration

and isoforms in β-thalassemia. Atherosclerosis 1997:131;127-133.

2. Mansi KM, Aburjai TA. Lipid profile in Jordanian children with β-thalassemia

major. Int J Hematol 2008;18:93-98.

3. Nasr MR, Abdelmaskoud AM, Abd El-Aal KS, Mabrouk NA, Ismael WM.

Plasma lipid profile and lipid peroxidation in beta-thalassemic children. J

Clin Lipidol 2008;2:405-409.

4. Papanastasiou DA, Siorokou T, Haliotis FA. β-Thalassaemia and factors

affecting the metabolism of lipids and lipoproteins. Haematologia (Budap)

1996;27:143-153.

5. Hashemieh M, Javadzadeh M, Sihirkavand A, Sheibani K. Lipid profile in

minor thalassemic patients: a historical cohort study. Bangladesh Med Res

Counc Bull 2011;37:24-27.

6. Namazi MR. Minor thalassemia as a protective factor against cerebrovascular

accidents. Med Hypotheses 2002;59:361-362.

7. Maioli M, Pettinato S, Cherchi GM, Giraudi D, Pacifico A, Pupita G, Tidore MG.

Plasma lipids in β-thalassemia minor. Atherosclerosis 1989;75:245-248.

8. Brizzi P, Isaja T, D’Agata A, Malaguarnera L, Malaguarnera M, Musumeci

S. Oxidized LDL antibodies (OLAB) in patients with β-thalassemia major. J

Atheroscler Thromb 2002;9:139-144.

Address for Correspondence/Yazışma Adresi: Yasemin IŞIK BALCI, M.D.,

Pamukkale University Faculty of Medicine, Department of Pediatric Hematology, Denizli, Turkey

Phone : +90 532 547 71 79

E-mail : dryibalci@gmail.com



DOI: 10.4274/tjh.2015.0168

73

LETTERS TO EDITOR


Extranodal Natural Killer/T-Cell Lymphoma: A Rare Nasal-Type

Case

Ekstranodal Natural Killer/T-Hücreli Lenfoma: Nadir Bir Nasal Tip Olgusu

Esra Sarıbacak Can 1 , Harika Okutan 1 , Murat Albayrak 1 , Ünsal Han 2

1Dışkapı Yıldırım Beyazıt Research and Training Hospital, Clinic of Hematology, Ankara, Turkey

2Dışkapı Yıldırım Beyazıt Research and Training Hospital, Clinic of Pathology, Ankara, Turkey

To the Editor,

Nasal type extranodal natural killer (NK) NK-cell/T-cell

lymphoma (NKTCL) is a rare extranodal lymphoma of NK-cell

or T-cell origin that most commonly affects immunocompetent

middle-aged men of Asian or Native American descent [1]. The

pathogenesis is not understood completely, but it is related

in part to infection of the tumor cells with Epstein-Barr virus

(EBV) [2]. Around 6-7% of all non-Hodgkin’s lymphoma (NHL)

in Southeast Asia accounts for NKTCL. However, the incidence of

NHL is lower in the United States at 1.5% [3,4].

Disease within the nasal cavity has a better prognosis. Radiation

therapy alone can be curative. Over 60% of patients with stage

1 disease remain in long-term remission following treatment

with radiation therapy with or without chemotherapy [5].

Nasal disease may be cured with radiotherapy at a rate of 85%.

However, the relapse rate is high at 25%. Therefore, it is highly

crucial for this aggressive disease to be diagnosed and treated

at an early stage [6,7].

In our case, a 29-year-old female complained of nasal

obstruction and had a necrotizing mass in the upper middle

concha at the right nasal cavity; biopsy was planned. CD56 was

strongly positive (Figure 1), CD8 was positive in a few scattered

cells, and CD4 was positive in the majority of infiltrating T

cells in the sample of necrotic tissue pieces. TIA-1 and perforin

were commonly positive. Granzyme was commonly strongly

cytoplasmic-positive. Epstein-Barr encoding region (EBER) in situ

hybridization analysis was done with a probe cocktail containing

EBV. Early RNA transcript showed that NKTCL compatibility

existed with commonly strongly nuclear-positivity in EBER

infiltrating cells. Positron emission tomography-computed

tomography (PET-CT) revealed pathologically heterogeneous

soft tissue mucosal thickening, pushing the nasal septum

slightly to the left and hypertrophy of the right ethmoid cells in

the upper middle concha level of the nasal cavity, with increased

metabolic uptake (SUV max : 3.18). The patient was diagnosed

with stage 1E based on PET-CT evaluation and received a total

of 38 Gy external radiotherapy at 200 cGy daily. No involvement

was detected month after radiotherapy and complete response

was considered to have occurred after 1 year.

NKTCL of the palate and sinuses has been reported in many

cases. However, the incidence of NKTCL is much lower in the

United States. Nasal obstruction, bleeding, pain, or local

swelling are usually observed and ulcerative, destructive lesions

within extranodal sites can be produced. Often it is associated

with EBV. Immunophenotypically, the tumor cells express CD2,

CD3, and CD56. The cells can lack CD56 and express CD8+ T-cell

antigens in some cases [7]. The course of NKTCL, nasal type, is

aggressive where a 5-year overall survival ranges from 25% to

50% [8].

Symptoms of nasal type NKTCL can include nasal discharge,

nasal obstruction and other nonspecific sinonasal symptoms.

However, sore throat and dysphagia, also known as symptoms of

nasal type NKTCL, are frequently missed and treated as viral and

bacterial pharyngitis, which leads to late diagnosis. Therefore,

morbidity and mortality are increased. NK/T-cell lymphoma,

nasal type, is rarely observed in Turkey and early diagnosis of

the disease is of vital importance.

Figure 1. Strong staining of diffuse cytoplasmic natural killer cells

with CD56 (CD56, IHC, 200x).

74


LETTERS TO EDITOR

Keywords: Extranodal natural killer/T-cell lymphoma, Non-

Hodgkin lymphoma

Anahtar Sözcükler: Ekstranodal natural killer/T-hücreli

lenfoma, Hodgkin dışı lenfoma


Concept: Esra Sarıbacak Can, Design: Esra Sarıbacak Can,

Data Collection or Processing: Esra Sarıbacak Can, Analysis or

Interpretation: Esra Sarıbacak Can, Harika Okutan, Literature

Search: Esra Sarıbacak Can, Murat Albayrak, Writing: Esra

Sarıbacak Can, Ünsal Han.


interest, including specific financial interests, relationships, and/or

affiliations relevant to the subject matter or materials included.

References

1. Roschewski M, Wilson WH. EBV-associated lymphomas in adults. Best Pract

Res Clin Haematol 2012;25:75-89.

2. Niedobitek G. Epstein-Barr virus infection in the pathogenesis of

nasopharyngeal carcinoma. Mol Pathol 2000;53:248-254.

3. Al-Hakeem DA, Fedele S, Carlos R, Porter S. Extranodal NK/T-cell lymphoma,

nasal type. Oral Oncol 2007;43:4-14.

4. Jaccard A, Hermine O. Extranodal natural killer/T-cell lymphoma: advances

in the management. Curr Opin Oncol 2011;23:429-435.

5. Liu QF, Wang WH, Wang SL, Liu YP, Huang WT, Lu N, Zhou LQ, Ouyang H,

Jin J, Li YX. Immunophenotypic and clinical differences between the nasal

and extranasal subtypes of upper aerodigestive tract natural killer/T-cell

lymphoma. Int J Radiat Oncol Biol Phys 2014;88:806-813.

6. Liang R. Diagnosis and management of primary nasal lymphoma of T-cell or

NK-cell origin. Clin Lymphoma 2000;1:33-38.

7. Hasserjian RP, Harris NL. NK-cell lymphomas and leukemias: a spectrum

of tumors with variable manifestations and immunophenotype. Am J Clin

Pathol 2007;127:860-868.

8. Kwong YL. Natural killer-cell malignancies: diagnosis and treatment.

Leukemia 2005;19:2186-2194.

Address for Correspondence/Yazışma Adresi: Esra SARIBACAK CAN, M.D.,

Dışkapı Yıldırım Beyazıt Research and Training Hospital,

Clinic of Hematology, Ankara, Turkey

E-mail : esracanercan@hotmail.com



DOI: 10.4274/tjh.2015.0169

T-Cell Lymphoma Presenting with Auricular and Parotid Gland

Involvement

Aurikula ve Parotis Bezi Tutulumu ile Seyreden T-Hücreli Lenfoma

Birgül Öneç 1 , Alper Koç 2 , Elif Nisa Ünlü 3 , İlhan Ünlü 4 , Hüseyin Yaman 4 , Durdu Mehmet Köş 2

1Düzce University Faculty of Medicine, Department of Hematology, Düzce, Turkey

2Düzce University Faculty of Medicine, Department of Internal Medicine, Düzce, Turkey

3Düzce University Faculty of Medicine, Department of Radiology, Düzce, Turkey

4Düzce University Faculty of Medicine, Department of Otorhinolaryngology, Düzce, Turkey

To the Editor,

The external auditory canal is an unusual presenting site for

lymphomas, with only a few case reports in the literature [1,2].

Malignant lymphomas arising from the salivary glands are also

uncommon, accounting for approximately 5% of extranodal

lymphomas, and the majority of them are of B-cell lineage.

Primary salivary gland T-cell lymphomas are extremely rare

[3,4,5].

A 63-year-old man was admitted with swelling of the left

side of his face and left auricle. Considered as an infection, it

was empirically treated with systemic and topical antibiotics.

Examination by an otorhinolaryngologist revealed a suppurative

lesion that consisted of ulcerated areas and granulation tissue in

the external auditory canal and preauricular region in addition to

edema (Figure 1A). A lobulated mass lesion of 47x39 mm arising

from the left parotid and extending to the left auditory canal

was detected in computed tomography (CT) with accompanying

lymph nodes in the left subauricular and cervical localization.

Fine-needle biopsy was nondiagnostic and incisional biopsy

found only severely active chronic inflammation. Finally, deep

excisional biopsy revealed CD3-, CD5-, and CD30-positive and

S100-negative lymphoid cells diffusely infiltrating the dermis.

The case was considered as stage 2 peripheral T-cell lymphoma

not otherwise specified peripheral T-cell lymphoma-not

otherwise specified (PTCL-NOS) and CHOP (cyclophosphamide,

75

LETTERS TO EDITOR


for proper assignment of lineage of salivary gland lymphomas

[5]. Reactive lymphoid infiltrate located in the periphery of the

lymphoma may contribute to the delay of diagnosis and larger

biopsy samples are needed.

Keywords: Parotid gland, T-Cell lymphoma, Auricula

Anahtar Sözcükler: Parotis bezi, T-Hücreli lenfoma, Aurikula

Figure 1. A) A suppurative lesion that consisted of ulcerated areas

and granulation tissue was observed in the external auditory canal

and preauricular region. B) Significant regression of lesion after

4 cycles of CHOP (cyclophosphamide, doxorubicin, vincristine,

prednisone) treatment.

doxorubicin, vincristine, prednisone) protocol was started.

Despite clinical improvement in the lesions (Figure 1B), CT

revealed progression after the fourth cycle. The second-line

treatment is ongoing with DHAP (dexamethasone, high-dose

ara-C, cisplatin) at the 8th month of follow-up.

The auricula, external auditory canal, and parotid glands

are unusual locations for T-cell lymphoma. Presentations of

lymphomas are indistinguishable from other swellings of the

auricle or parotid gland and therefore a high index of suspicion

should be maintained in patients who present with presumptive

cutaneous infections that do not respond to antibiotic therapy

in these locations. A suppurative auricular lesion suggests

an infectious disease rather than a lymphoma, but primary

cutaneous lymphomas and cutaneous manifestations of

lymphomas must be kept in mind. Early excisional biopsy

may prevent excessive waste of time with unnecessary

antibiotherapies. Parotid lymphomas are most likely to be

B-cell non-Hodgkin lymphoma, but non-B-cell lymphomas

have a more aggressive course in all salivary gland lymphomas.

Both B-cell and T-cell lymphomas share many morphological

similarities; therefore, immunohistochemical analysis is required


Concept: Birgül Öneç, Design: Birgül Öneç, Alper Koç, Data

Collection or Processing: Birgül Öneç, Alper Koç, Analysis or

Interpretation: Birgül Öneç, Durdu Mehmet Köş, Elif Nisa Ünlü,

Literature Search: Birgül Öneç, Hüseyin Yaman, İlhan Ünlü,

Writing: Birgül Öneç, Alper Koç.




included.

References

1. Marçal N, Campelos S, Dias L, Gonçalves M, Pereira G, Godinho T. Primary

cutaneous CD30-positive anaplastic large-cell lymphoma of the external

auditory canal. Ear Nose Throat J 2012;91:10-12.

2. González Delgado A, Argudo Marco F, Sánchez Martínez N, Sprekelsen

Gassó C. T cell non-Hodgkin’s lymphoma of the external auditory canal.

Acta Otorrinolaringol Esp 2008;59:200-201.

3. Yanagawa N, Osakabe M, Furuse H, Maeda K, Tamura G. Peripheral T-cell

lymphoma arising from an intraglandular lymph node in the parotid gland:

a case report and literature review. Pathol Int 2012;62:60-64.

4. Metikurke SH, Krishnappa R, Ramachar SM, Arora I. Primary malignant

lymphoma of the parotid gland. J Cancer Res Ther 2012;8:641-643.

5. Chan JK, Tsang WY, Hui PK, Ng CS, Sin VC, Khan SM, Siu LL. T- and T/

natural killer-cell lymphomas of the salivary gland: a clinicopathologic,

immunohistochemical and molecular study of six cases. Hum Pathol

1997;28:238-245.

Address for Correspondence/Yazışma Adresi: Alper KOÇ, M.D.,

Düzce University Faculty of Medicine, Department of Internal Medicine,

Düzce, Turkey

E-mail : alperkoc44@hotmail.com



DOI: 10.4274/tjh.2015.0217

76


LETTERS TO EDITOR

Immune Thrombocytopenia Resolved by Eltrombopag in a Carrier

of Glucose-6-Phosphate Dehydrogenase Deficiency

Glukoz-6-Fosfat Dehidrogenaz Eksikliği Taşıyıcısında Eltrombopag Yanıtlı İmmün Trombositopeni

Laura Scaramucci, Pasquale Niscola, Massimiliano Palombi, Andrea Tendas, Marco Giovannini, Paolo de Fabritiis

Sant’Eugenio Hospital, Clinic of Hematology, Rome, Italy

To the Editor,

Eltrombopag, a thrombopoietin mimetic peptide, may provide

excellent clinical efficacy in steroid-refractory patients with

immune thrombocytopenic purpura (ITP) [1,2]. Eltrombopag

is generally well tolerated. However, its use in the particular

setting of glucose-6-phosphate dehydrogenase (G6PD) and

history of acute hemolytic anemia (AHA) has not been reported

so far. A 51-year-old female was diagnosed as having ITP in

September 2014. She was not taking any medication and her

past history was negative, apart from having been diagnosed

a carrier (heterozygous) of G6PD deficiency (Mediterranean

variant) after a familial screening by molecular and biochemical

methods. She presented with only slightly reduced (about 50%)

enzyme level, belonging to World Health Organization-defined

class 3 [3,4]. In the following years, the patient experienced some

episodes of AHA, which were managed at outside institutions;

in particular, a severe episode of AHA, probably triggered by

urinary infection and antibiotics [5], had complicated her

second and last delivery. The hemolytic episodes were selflimiting

and resolved without sequelae. No other causes of

hemolysis were documented. When the case came to our

attention, a diagnosis of ITP was made; hemolytic parameters

were normal, although the G6PD enzyme concentration was

not measured. Oral prednisone (1 mg/kg) was given with only

a transient benefit. The patient was then a candidate for

elective splenectomy. However, given her extremely low platelet

count, she was started in October 2014 on eltrombopag at 50

mg/day as a bridge to splenectomy. Given that, to the best of

our knowledge, the use of this drug has never been reported

in the particular setting of G6PD deficiency, the patient was

constantly monitored. A prompt platelet increase (178x109/L)

was observed 1 week after the start of treatment. After she

achieved the target platelet count, the dose of eltrombopag was

tapered to the lowest effective dose. The patient’s response was

stabile while she remained on a dose of eltrombopag between

25 and 50 mg/day without any adverse events; in particular, no

variations of hemolytic parameters were observed. As of today,

after 6 months of continuous eltrombopag administration, the

patient has constantly maintained the target platelet counts

and she is awaiting elective splenectomy. According to our

knowledge, we report for the first time the evidence regarding

the safe use of this thrombomimetic agonist, which provided

an excellent treatment outcome without any adverse effects,

in a steroid-refractory adult ITP patient at risk of drug-induced

AHA as a G6PD-deficient heterozygous carrier. G6PD deficiency

is an X-linked, hereditary genetic defect [2,3,4] for which

heterozygous women, who are usually asymptomatic, have a

mosaicism of normal and G6PD-deficient erythrocytes. Given

the possible decreased amount of G6PD enzyme, although

exceptional and only under particularly stressing conditions,

such as urinary tract infections and/or the use of certain

antibiotics such as nitrofurantoin, AHA may occur [5]. Although

our report, being limited to a single patient, is purely anecdotal,

considering the high prevalence of G6PD deficiency and the

increasing use of thrombomimetic drugs, further collection of

such cases would be very useful to determine the complication

risks associated in this setting with the use of thrombopoietin

agonists.

Keywords: Idiopathic thrombocytopenic purpura, Glucose-6-

phosphate dehydrogenase deficiency, Thrombopoietin mimetic

peptide, TMP mimetic peptide

Anahtar Sözcükler: İdiyopatik immün trombositopeni, Glukoz-

6-fosfat dehidrogenaz eksikliği, Trombopoetin uyarıcı peptit,

TPO uyarıcı peptit


Concept: Laura Scaramucci, Pasquale Niscola, Massimiliano

Palombi, Andrea Tendas, Marco Giovannini, Paolo de Fabritiis,

Design: Laura Scaramucci, Pasquale Niscola, Massimiliano

Palombi, Andrea Tendas, Marco Giovannini, Paolo de Fabritiis,

Data Collection or Processing: Laura Scaramucci, Marco

Giovannini, Analysis or Interpretation: Laura Scaramucci,

Pasquale Niscola, Marco Giovannini, Literature Search: Pasquale

Niscola, Massimiliano Palombi, Andrea Tendas, Writing: Laura

Scaramucci, Pasquale Niscola, Final Approval: Paolo de Fabritiis.




included.

77

LETTERS TO EDITOR


References

1. Tripathi AK, Shukla A, Mishra S, Yadav YS, Yadav DK. Eltrombopag therapy

in newly diagnosed steroid non-responsive ITP patients. Int J Hematol

2014;99:413-417.

2. Luzzatto L, Seneca E. G6PD deficiency: a classic example of pharmacogenetics

with on-going clinical implications. Br J Haematol 2014;164:469-480.

3. No authors listed. Glucose-6-phosphate dehydrogenase deficiency. WHO

Working Group. Bull World Health Organ 1989;67:601-611.

4. Cappellini MD, Fiorelli G. Glucose-6-phosphate dehydrogenase deficiency.

Lancet 2008;371:64-74.

5. van de Mheen L, Smits SM, Terpstra WE, Leyte A, Bekedam DJ, van den

Akker ES. Haemolytic anaemia after nitrofurantoin treatment in a pregnant

woman with G6PD deficiency. BMJ Case Rep 2014:2014.

Address for Correspondence/Yazışma Adresi: Pasquale NISCOLA, M.D.,

Sant’Eugenio Hospital, Clinic of Hematology, Rome, Italy

Phone: +39 065 100 31 41

E-mail : pniscola@gmail.com



DOI: 10.4274/tjh.2015.0181

Wernicke’s Encephalopathy in an Acute Myeloid Leukemia

Patient: A Case Study

Bir Akut Myeloid Lösemi Hastasında Wernicke Ensefalopatisi: Bir Olgu Sunumu

Muhammet Maden 1 , Gülsüm Pamuk 1 , Yahya Çelik 2 , Ercüment Ünlü 3

1Trakya University Faculty of Medicine, Department of Hematology, Edirne, Turkey

2Trakya University Faculty of Medicine, Department of Neurology, Edirne, Turkey

3Trakya University Faculty of Medicine, Department of Radiodiagnostics, Edirne, Turkey

To the Editor,

Wernicke’s encephalopathy (WE) is a life-threatening disease

with acute onset, resulting from thiamine deficiency. Causes

are alcohol intake, malnutrition, gastric bypass surgery,

human immunodeficiency virus infection, total parenteral

nutrition (TPN), chronic dialysis, and cancer [1]. WE may cause

neurological symptoms such as nystagmus, ophthalmoplegia,

ataxia, confusion, convulsions, delirium, coma, and acute

psychoses [2].

An 18-year-old female patient with refractory acute myeloid

leukemia (AML) was given FLAG-IDA (fludarabine, cytosine

arabinoside, idarubicin) chemotherapy protocol. As she

developed typhlitis, oral intake was stopped; broad-spectrum

antibiotics and TPN without any vitamin supplementation

were started. She developed a fixed look to a point, chin and

upper extremity spasms, and urinary incontinence on the 38th

day of chemotherapy. Neurological examination showed nonlateralization,

bilateral light reflexes were +/+, verbal stimuli

were negative, and the reflex response to painful stimuli was

positive. Laboratory results showed hemoglobin of 7.2 g/dL,

leukocytes of 3380/mm3, neutrophils of 2890/mm 3 , platelets of

48,000/mm3, and normal blood biochemistry. The level of serum

thiamine could not be measured, because the laboratory did not

have the capabilities to measure it. Brain diffusion MRI showed

increased signal intensity in the medial thalami (Figure 1). The

patient was diagnosed with WE. She was given 1500 mg/day

thiamine i.v. for 3 days and 250 mg/day thiamine i.v. for another

5 days. On the fourth day of thiamine infusion, her general

condition began to improve and she started giving one-word

responses to verbal stimuli. Her convulsions disappeared and she

started to form short sentences and walk without assistance;

she was discharged on the 30 th day of thiamine replacement

therapy. Currently, 2 years have passed since the WE and the

patient is in complete hematologic remission.

Figure 1. Coronal T2-weighted image shows symmetrical, weak,

and limited increased signal intensity in the medial thalami and

in the third ventricle-facing surface.

78


LETTERS TO EDITOR

In cancer patients, WE may develop because of certain

chemotherapeutic agents (especially doxifluridine, ifosfamide, and

5-fluorouracil) [3], malnutrition, and thiamine depletion due to

fast-growing tumor cells or gastrointestinal bypass surgery [2]. In

the literature, there are a few cases of WE in AML. The diagnosis of

WE in cancer patients is difficult because there are many causes of

similar symptoms, such as confusion-causing hypoxia, infections,

electrolyte imbalance, opioid medications, chemotherapy, brain and

meningeal metastases, and delirium [4]. To make a definite diagnosis

of WE, it should be clinically suspected. WE may be verified with the

measurement of thiamine concentration in blood or erythrocyte

transketolase activity; however, these tests are not widely available

[3]. Magnetic resonance imaging (MRI) has 93% specificity and

53% sensitivity to verify the diagnosis [2]. Bilateral dorsomedial

thalamus, tectal plaque, and periaqueductal gray matter signal

abnormalities are observed as classical in MRI [5]. WE should be

treated empirically with 500 mg of thiamine 3 times per day for 2-3

days. If there is no response, supplementation may be discontinued

after 2-3 days. In case an effective response is observed, 250 mg of

thiamine should be continued daily until clinical improvement [2].

Thiamine supplementation should definitely be added to TPN

or the diets of hematologic malignancy patients with poor

oral nutrition. When symptoms such as ataxia, confusion, or

ophthalmoplegia appear in these patients, brain MRI should

be immediately performed and thiamine infusion should be

initiated immediately in the case of strong suspicion.

Keywords: Thiamine, Wernicke’s encephalopathy, Acute myeloid

leukemia

Anahtar Sözcükler: Tiamin, Wernicke ensefalopatisi, Akut

myeloid lösemi


Concept: Muhammet Maden, Gülsüm Pamuk, Design:

Muhammet Maden, Gülsüm Pamuk, Data Collection or

Processing: Muhammet Maden, Ercüment Ünlü, Yahya Çelik,

Analysis or Interpretation: Muhammet Maden, Gülsüm Pamuk,

Ercüment Ünlü, Yahya Çelik, Literature Search: Muhammet

Maden, Gülsüm Pamuk, Writing: Muhammet Maden.




included.

References

1. Papila B, Yildiz O, Tural D, Delil S, Hasiloglu ZI, Ayan F, Papila C. Wernicke’s

encephalopathy in colon cancer. Case Rep Oncol 2010;3:362-367.

2. Sechi G, Serra A. Wernicke’s encephalopathy: new clinical settings and

recent advances in diagnosis and management. Lancet Neurol 2007;6:442-

455.

3. Basu TK, Dickerson JW. The thiamin status of early cancer patients with

particular reference to those with breast and bronchial carcinomas.

Oncology 1976;33:250-252.

4. Kuo SH, Debnam JM, Fuller GN, de Groot J. Wernicke’s encephalopathy:

an underrecognized and reversible cause of confusional state in cancer

patients. Oncology 2009;76:10-18.

5. Zuccoli G, Siddiqui N, Bailey A, Bartoletti SC. Neuroimaging findings in pediatric

Wernicke encephalopathy: a review. Neuroradiology 2010;52:523-529.

Address for Correspondence/Yazışma Adresi: Muhammet MADEN, M.D.,

Trakya University Faculty of Medicine, Department of Hematology,

Edirne, Turkey

E-mail : drmaden@yandex.com

Received/Geliş tarihi: June 24, 2015


DOI: 10.4274/tjh.2015.0249

Kaleidoscopic Views in the Bone Marrow: Oxalate Crystals in a

Patient Presenting with Bicytopenia

Kemik İliğinde Oksalat Kristalleri Birikimine Bağlı Gelişen Bisitopeni

Yelda Dere 1 , Simge Erbil 2 , Murat Sezak 2 , Başak Doğanavşargil 2 , Mümtaz Yılmaz 3 , Nazan Özsan 2 , Mine Hekimgil 2

1Sıtkı Koçman University Faculty of Medicine, Department of Pathology, Muğla, Turkey

2Ege University Faculty of Medicine, Department of Pathology, İzmir, Turkey

3Ege University Faculty of Medicine, Department of Nephrology, İzmir, Turkey

To the Editor,

Our patient is a 24-year-old female who was admitted to the

nephrology clinic of our hospital with fatigue, weakness, and

swelling of the feet. From her medical history, we learned that

she had two operations for nephrolithiasis at the ages of 9 and

12, and she underwent renal transplantation in 2013, but she

was still on hemodialysis. Laboratory tests showed bicytopenia

(anemia and leukopenia) with hemoglobin of 8.2 g/dL and white

blood cell count of 3800/mm3, and she underwent a bone marrow

(BM) biopsy. Microscopically, an almost complete suppression of

79

LETTERS TO EDITOR


hematopoietic cells with the replacement of BM cells by foreignbody

reactive fibrous tissue and numerous birefringent crystalline

materials were detected (Figures 1A and 1B). The crystals formed

rosettes with needle-like radial extensions surrounded by foreign

body-type giant cells. Under polarized light, the crystals formed

multicolored rosettes (Figure 1C). In BM aspirates envelope-like

crystals were found in the background of a few myeloid cells

with normal morphology (Figure 1D). Based on histopathological

examination integrated with clinical results, a diagnosis of

hypocellular BM associated with crystal deposition concordant

with oxalate crystals was made. Because of the absence of genetic

tests performed to date, the patient was referred to the genetics

department, and after genetic studies the diagnosis of primary

hyperoxaluria was confirmed.

Pancytopenia associated with BM infiltration of different

deposits is a rare condition mostly associated with amyloidosis

or the accumulation of iron. One of the rarest deposits in the

BM is oxalate crystals due to hyperoxaluria [1,2,3]. Primary

hyperoxaluria, a genetic disorder due to mutation in the alanine

glyoxylate aminotransferase gene, located on chromosome

2q37.3 and resulting in the conversion of glyoxylate to

oxalate, is characterized by increased production of oxalic acid

because of the specific liver enzyme deficiency and generally

presents with renal stones, renal or liver failure, and oxalosis

[4]. Calcium oxalate may even be deposited into various tissues

such as those of the retina, peripheral nerves, arterial media,

and heart [4,5]. The medical history of nephrolithiasis at early

ages, characteristic appearance of birefringent crystals forming

rosettes in the BM, and the envelope-like forms in the BM

aspirates seen in our case supported the diagnosis of primary

hyperoxaluria, which is best confirmed by genetic studies and

treated with liver transplantation because of the location of the

abnormal enzymes in the hepatocytes.

Keywords: Oxalosis, Hyperoxaluria, Bone marrow

Anahtar Sözcükler: Oksalozis, Hiperoksalüri, Kemik iliği


Concept: Yelda Dere, Mine Hekimgil, Design: Simge Erbil,

Yelda Dere, Data Collection or Processing: Mümtaz Yılmaz,

Figure 1. A, B: Characteristic appearance of oxalate crystals in the

bone marrow, H&E, 100x. C: Colorful rosette-like crystal under

polarized light, H&E, 200 x . D: Colorful envelope-like crystal in the

bone marrow aspirate, Giemsa stain, 100 x .

Başak Doğanavşargil, Murat Sezak, Nazan Özsan, Analysis or

Interpretation: Mine Hekimgil, Yelda Dere, Simge Erbil, Literature

Search: Başak Doğanavşargil, Murat Sezak, Simge Erbil, Writing:

Yelda Dere, Mine Hekimgil, Simge Erbil.


interest, including specific financial interests, relationships, and/or

affiliations relevant to the subject matter or materials included.

References

1. Walter MJ, Dang CV. Pancytopenia secondary to oxalosis in a 23-year-old

woman. Blood 1998;91:4394.

2. Halil O, Farringdon K. Oxalosis: an unusual cause of leucoerythroblastic

anaemia. Br J Haematol 2003;122:2.

3. Taşlı F, Özkök G, Ok ES, Soyer N, Mollamehmetoğlu H, Vardar E. Massive bone

marrow involvement in an end stage renal failure case with erythropoietinresistant

anemia and primary hyperoxaluria. Ren Fail 2013;35:1167-1169.

4. Hoppe B, Beck BB, Milliner DS. The primary hyperoxalurias. Kidney Int

2009;75:1264-1271.

5. Doganavsargil B, Akil I, Sen S, Mir S, Basdemir G. Autopsy findings of a case

with oxalosis. Pediatr Dev Pathol 2009;12:229-232.

Address for Correspondence/Yazışma Adresi: Yelda DERE, M.D.,

Sıtkı Koçman University Faculty of Medicine, Department of Pathology, Muğla, Turkey

Phone : +90 505 465 31 98

E-mail : yeldamorgul@gmail.com



DOI: 10.4274/tjh.2015.0256

80


LETTERS TO EDITOR

An Unexpected Innocent Complication Associated with Azacitidine

Treatment of Myelodysplastic Syndrome: Erythema Annulare

Centrifugum

Miyelodisplastik Sendrom Tedavisi Seyrinde Azasitidin İlişkili Beklenmedik Masum

Komplikasyon: Eritem Annulae Santrifuj

Esra Turan Erkek, Sevgi Kalayoğlu Beşışık

İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine, Division of Hematology, İstanbul, Turkey

To the Editor,

Skin lesions accompanying hematological malignancies can

be formed due to either direct tumor infiltration of the skin

or indirect effects. Indirectly developing lesions may be a

component of paraneoplastic syndrome. Erythema annulare

centrifugum (EAC) is considered to be a hypersensitivity

reaction developed against various antigens associated with

infections, drugs, and endocrine diseases. EAC, rarely seen in

neoplastic diseases, has been reported in lymphoma, leukemia,

histiocytosis, and prostate cancer. Here we report EAC in a

patient using a hypomethylating agent, azacitidine.

A 69-year-old female patient was admitted to our polyclinic

with weakness and ecchymosis in her legs existing for 3

months. She was considered as having refractory anemia with

excess blasts-2 according to myelodysplastic syndrome (MDS)

classification [1]. Because there was only hyperdiploidy in

conventional cytogenetic examination, she was classified in

group intermediate-2 of the International Prognostic Scoring

System. She had a history of radical mastectomy and adjuvant

chemoradiotherapy for breast cancer 3 years ago. She said that

variously sized round and oval erythematous, itching, painless

lesions had formed in the abdominal region on the 4 th day of

azacitidine usage (75 mg/m 2 /day, 7 days, s.c.) (Figure 1 and 2).

There were no concomitant complaints or physical examination

findings except fatigue. After azacitidine was stopped, a skin

biopsy was taken. In the biopsy, mild perivascular inflammatory

infiltration accompanying vascular ectasia in the papillary dermis

was detected. The possibility of paraneoplastic syndrome was

excluded due to the disappearance of all lesions by 1 week after

cessation of treatment. During the second course of azacitidine,

the lesions reoccurred on the second day. Subsequently to the

second course, the patient died of sepsis, which developed after

pneumonia.

EAC was first defined by Darier in 1916, and it was classified

into categories of superficial and deep forms by Ackerman

in 1978 [2]. In the deep form, the lesions are hard and are

usually seen together with desquamation without itching. The

superficial form is characterized by itchy lesions with uncertain

borders and desquamation. EAC formation is associated

with trauma, ectoparasites, tuberculin test, PUVA therapy

(photochemotherapy), viral infections, and diabetes. There

are publications reporting that EAC may be associated with

Hodgkin’s lymphoma rarely and lung, colon, cervix, prostate,

stomach, and ovarian cancers even more rarely [3,4]. Lesions

are often observed on the trunk, proximal portions of the

limbs, and the buttocks. Today EAC is defined as a characteristic

hypersensitivity reaction that can be triggered by many different

antigens and disappears within 1-2 weeks.

Skin lesions, whose most common forms are Sweet syndrome and

myeloid sarcoma, are rarely observed in MDS [5]. Azacitidine, a

Figure 1. Erythema annulare centrifugum rashes formed during

treatment.

Figure 2. Skin lesions’ regression after treatment interruption.

81

LETTERS TO EDITOR


nucleoside analogue, is one of the low-density treatment options

in MDS. Azacitidine usage may cause cutaneous reactions such

as urticaria, skin dryness, nodules, localized hematoma at the

injection area, rash, granuloma, swelling, pigmentation changes,

and induration.

This case was presented because no AEC development during

azacitidine use in MDS had been reported previously.

Keywords: Erythema annulare centrifugum, Azacitidine,

Myelodysplastic syndrome

Anahtar Sözcükler: Eritem annuler santrifuj, Azasitidin,

Miyelodisplastik sendrom


Concept: Sevgi Kalayoğlu Beşışık, Design: Sevgi Kalayoğlu

Beşışık, Data Collection or Processing: Esra Turan Erkek, Sevgi

Kalayoğlu Beşışık, Analysis or Interpretation: Esra Turan Erkek,

Sevgi Kalayoğlu Beşışık, Literature Search: Esra Turan Erkek,

Sevgi Kalayoğlu Beşışık, Writing: Esra Turan Erkek.




included.

References

1. Vardiman JW, Harris NL, Brunning RD. The World Health Organization

(WHO) classification of the myeloid neoplasms. Blood 2002;100:2292-2302.

2. Weyers W, Diaz-Cascajo C, Weyers I. Erythema annulare centrifugum:

results of a clinicopathologic study of 73 patients. Am J Dermatopathol

2003;25:451-462.

3. Ural AU, Ozcan A, Avcu F, Kaptan K, Taştan B, Beyan C, Yalçin A. Erythema

annulare centrifugum as the presenting sign of CD 30 positive anaplastic

large cell lymphoma-association with disease activity. Haematologia

(Budap) 2001;31:81-84.

4. Carlesimo M, Fidanza L, Mari E, Pranteda G, CacchiC, Veggia B, Cox MC,

Camplone G. Erythema annularecentrifugum associated with mantle B-cell

non-Hodgkin’s lymphoma. Acta Derm Venereol 2009;89:319-320.

5. Soppi E, Nousiainen T, Seppa A, Lahtinen R. Acute febrile neutrophilic

dermatosis (Sweet’s syndrome) in association with myelodysplastic

syndromes: a report of three cases and a review of the literature. Br J

Haematol 1989;73:43-47.

Address for Correspondence/Yazışma Adresi: Esra TURAN ERKEK, M.D.,

İstanbul University İstanbul Faculty of Medicine, Department of Internal Medicine,

Division of Hematology, İstanbul, Turkey

E-mail : dresraturan@gmail.com



DOI: 10.4274/tjh.2015.0268

82


LETTERS TO EDITOR

Thrombotic Microangiopathic Hemolytic Anemia without Evidence

of Hemolytic Uremic Syndrome

Hemolitik Üremik Sendrom Bulguları Olmayan Trombotik Mikroanjiyopatik Hemolitik Anemi

Şinasi Özsoylu

Retired Professor of Pediatrics, Hematology and Hepatology, Honorary Fellow of American Academy of Pediatrics,

Honorary Member of American Pediatric Society

To the Editor,

In a recent issue of this journal Dr. Oymak and her colleagues

presented a clinically and genetically well-studied 5-year-old

boy who was seen with severe microangiopathic hemolytic

anemia without laboratory findings of renal involvement

despite complement factor H gene mutations [1].

Because of Yeneral’s extensive review [2] on atypical hemolytic

uremic syndrome (aHUS) published recently in the Turkish

Journal of Hematology, I brought it to readers’ attention that

more recently some authors do not use ‘aHUS’, which was

historically used to distinguish heterogeneous uncharacterized

syndromes from Shiga toxin-related HUS, since the term lacks

both specificity and suggested causes [3].

Though in our patient with thrombotic thrombocytopenic

purpura renal involvement was documented at the beginning

but not in the last two recurrences, neither serum nor urinary

findings indicated kidney involvement [4].

Although the discussions of Dr. Oymak et al. are well taken,

the term ‘microangiopathic hemolytic anemia’ is covering the

syndrome to a large extent as suggested by George and Nester [5].

Keywords: Microangiopathy, Kidney functions, Hemolytic

anemia

Anahtar Sözcükler: Mikroanjiopati, Böbrek fonksiyonları,

Hemolitik anemi

Conflict of Interest: The author of this paper has no conflicts of

interest, including specific financial interests, relationships, and/


References

1. Oymak Y, Karapınar TH, Ay Y, Özcan E, Müminoğlu N, Aydın Köker S, Töret

E, Berdeli A, Serdaroğlu E, Vergin C. Thrombotic microangiopathy with

complement factor H gene mutations unassociated with atypical hemolytic

uremic syndrome. Turk J Hematol 2015;32:275-276.

2. Yenerel MN. Atypical hemolytic uremic syndrome: differential diagnosis

from TTP/HUS and management. Turk J Hematol 2014;31:216-225.

3. Özsoylu Ş. About microangiopathic hemolytic anemia. Turk J Hematol

2015;32:92.

4. Özsoylu Ş. A case of acquired thrombotic thrombocytopenic purpura: three

recurrences in 25 years. Turk J Hematol 2015;32:279-280.

5. George JN, Nester CM. Syndromes of thrombotic microangiopathy. N Engl J

Med 2014;371:654-666.

Address for Correspondence/Yazışma Adresi: Şinasi ÖZSOYLU, M.D.,

Retired Professor of Pediatrics, Hematology and Hepatology, Honorary Fellow of

American Academy of Pediatrics, Honorary Member of American Pediatric Society

E-mail : sinasiozsoylu@hotmail.com

Received/Geliş tarihi: August 19, 2015


DOI: 10.4274/tjh.2015.0301

83

IMAGES IN HEMATOLOGY

DOI: 10.4274/tjh.2015.0046


Radiologic Image of a Child with Leukemia Who Developed Sepsis

and Fulminant Thrombosis during Induction Therapy

İndüksiyon Tedavisi Sırasında Sepsis ve Fulminan Tromboz Gelişen Lösemili Bir Çocuğun

Radyolojik Görüntüsü

Eda Ataseven 1 , Ömer Özden 2 , Şebnem Yılmaz Bengoa 1 , Handan Güleryüz 3 , Murat Duman 4 , Hale Ören 1

1Dokuz Eylül University Faculty of Medicine, Department of Pediatric Hematology, İzmir, Turkey

2Dokuz Eylül University Faculty of Medicine, Department of Pediatric Intensive Care, İzmir, Turkey

3Dokuz Eylül University Faculty of Medicine, Department of Pediatric Radiology, İzmir, Turkey

4Dokuz Eylül University Faculty of Medicine, Department of Pediatric Emergency, İzmir, Turkey

Figure 1. Thorax and abdominal computed tomography of the patient demonstrating bilateral areas of consolidation in the lung

parenchyma and multiple infarcts in the left lower lobe of the lungs, in the liver, in the spleen, in the left upper lobe of the left and

middle zone of the right kidney, and in some parts of the intestines (arrows).

Address for Correspondence/Yazışma Adresi: Hale ÖREN, M.D.,

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

Phone : +90 232 412 61 41

E-mail : hale.oren@deu.edu.tr


Accepted/Kabul tarihi: February 02, 2015

84


Ataseven E, et al: Sepsis and Thrombosis in a Child with Acute Lymphoblastic Leukemia

In a 5-year-old girl with acute lymphoblastic leukemia

(ALL), febrile neutropenia occurred in the induction phase of

chemotherapy. She was not using a central venous catheter.

Despite empiric antibiotics, she developed tachypnea, bilateral

rales, and disseminated intravascular coagulation (DIC). Viral,

bacterial, and fungal investigations were unremarkable.

Thorax and abdominal computed tomography showed bilateral

consolidation areas in the lungs and multiple infarcts in the left

lower lobe of the lungs, the liver, the spleen, the kidneys, and the

intestines (Figure 1). Heparin infusion was started. No inherited

prothrombotic defect could be shown; antiphospholipid

antibodies were negative. She died of pulmonary failure.

Sepsis secondary to an unknown pathogen is the most

common cause of mortality and the overall risk of symptomatic

thrombosis is 5.2% in ALL [1,2,3]. Despite a high incidence

of central nervous system and upper venous system events,

widespread thromboembolism seems to be rare [3,4]. Our patient

had multiple acquired risk factors, such as leukemia, concurrent

administration of Escherichia coli asparaginase and prednisone,

infection, and DIC. After administration of anticoagulant

therapy, patients usually show improvement, but in our patient

we could not reduce the occlusive events. This case is a good

reminder for hematologists that the onset of neutropenic fever

may be very aggressive and thrombotic events may occur rapidly

and may be fulminant in children with ALL.

Keywords: Acute leukemia, Sepsis, Thrombosis

Anahtar Sözcükler: Akut lösemi, Sepsis, Tromboz


Concept: Eda Ataseven, Hale Ören, Design: Hale Ören, Data

Collection or Processing: Eda Ataseven, Handan Güleryüz, Murat

Duman, Ömer Özden, Analysis or Interpretation: Hale Ören,

Şebnem Yılmaz Bengoa, Literature Search: Eda Ataseven, Hale

Ören, Writing: Eda Ataseven.


of interest, including specific financial interests, relationships, and/


References

1. Kulkarni KP, Marwaha RK. Mortality pattern in childhood acute

lymphoblastic leukemia with sepsis as a major barrier. J Pediatr Hematol

Oncol 2012;34:264-265.

2. Lund B, Åsberg A, Heyman M, Kanerva J, Harila-Saari A, Hasle H, Söderhäll

S, Jónsson ÓG, Lydersen S, Schmiegelow K; Nordic Society of Paediatric

Haematology and Oncology. Risk factors for treatment related mortality

in childhood acute lymphoblastic leukaemia. Pediatr Blood Cancer

2011;56:551-559.

3. Caruso V, Iacoviello L, Di Castelnuovo A, Storti S, Mariani G, de Gaetano

G, Donati MB. Thrombotic complications in childhood acute lymphoblastic

leukemia: a meta-analysis of 17 prospective studies comprising 1752

pediatric patients. Blood 2006;108:2216-2222.

4. Nowak-Göttl U, Kenet G, Mitchell LG. Thrombosis in childhood acute

lymphoblastic leukaemia: epidemiology, aetiology, diagnosis, prevention

and treatment. Best Pract Res Clin Haematol 2009;22:103-114.

85

IMAGES IN HEMATOLOGY

DOI: 10.4274/tjh.2015.0054


Hereditary Elliptocytosis with Pyropoikilocytosis

Piropoikilositozlu Herediter Eliptositoz

Turan Bayhan, Şule Ünal, Fatma Gümrük

Hacettepe University Faculty of Medicine, Department of Pediatric Hematology, Ankara, Turkey

Figure 1. The peripheral blood smear of the patient: prominent elliptocytic erythrocytes, in addition to microcytic erythrocytes (arrows)

and fragmented erythrocytes (circles).

Address for Correspondence/Yazışma Adresi: Turan BAYHAN, M.D.,

Hacettepe University Faculty of Medicine,

Department of Pediatric Hematology, Ankara, Turkey

E-mail : turanbayhan@yahoo.com


Accepted/Kabul tarihi: March 09, 2015

86


Bayhan T, et al: Hereditary Elliptocytosis with Pyropoikilocytosis

A 17-day-old boy was admitted because of jaundice and

anemia. He was born weighing 2900 g subsequent to a term

gestation as the fourth child of first-degree cousin parents. The

previous history revealed the administration of phototherapy for

4 days starting from the first day of life. Complete blood count

revealed hemoglobin (Hb) of 6.9 g/dL, hematocrit of 19.8%,

mean corpuscular volume (MCV) of 87.5 fL, red cell distribution

width (RDW) of 37%, white blood cell count of 11.4x10 9 /L,

and platelet count of 263x10 9 /L. Corrected reticulocyte count

was 5.3%. Peripheral blood smear revealed polychromasia and

pyropoikilocytosis. Direct antibody test was negative. Erythrocyte

glucose-6-phosphate dehydrogenase, pyruvate kinase, and

pyrimidine 5’ nucleotidase levels were normal. An erythrocyte

transfusion was administered with a diagnosis of non-immune

hemolytic anemia and the patient was discharged at the 26 th day

of life with initiation of folic acid. During his outpatient followup,

he required erythrocyte transfusions 2 more times and the

last transfusion was performed when he was 3 months old. At

a visit 3 months after the last transfusion, his blood count was

as follows: Hb of 9.5 g/dL, hematocrit of 28.2%, MCV of 68.2 fL,

and RDW of 30.5%. Erythrocyte osmotic fragility was found to

be normal and Hb electrophoresis revealed Hb F of 6.6% and Hb

A2 of 1.7%. Upon physical examination he had mild jaundice and

no splenomegaly. The parents’ blood counts were within normal

ranges. Peripheral blood smear revealed prominent elliptocytes

and occasional microcytic and fragmented erythrocytes with

poikilocytosis (Figure 1). The clinical findings and laboratory

results were diagnostic for the hereditary pyropoikilocytosis (HPP)

type of hereditary elliptocytosis (HE), but in vitro fragmentation

testing was not performed.

HE is a common hemolytic red cell membrane disease with

variant clinical presentations [1]. Common mutations that cause

HE are found in the α-spectrin, β-spectrin, and protein 4.1 genes

[2]. The majority of patients with HE are asymptomatic, but HPP

is a severe form of HE that presents with hemolytic anemia and

jaundice during the infantile period. Erythrocyte morphology

in HPP resembles that of blood smears in thermal burns with

poikilocytes, red blood cell fragments, microspherocytes, and

elliptocytes [3]. Low MCV (25 to 75 fL) due to fragmented red

blood cells is characteristic and osmotic fragility is commonly

normal [1,3].

Keywords: Anemia, Elliptocytosis, Pyropoikilocytosis

Anahtar Sözcükler: Anemi, Eliptositoz, Piropoikilositoz


Concept: Turan Bayhan, Design: Turan Bayhan, Şule Ünal, Data

Collection or Processing: Şule Ünal, Analysis or Interpretation:

Fatma Gümrük, Literature Search: Şule Ünal, Fatma Gümrük,

Writing: Turan Bayhan.




included.

References

1. Da Costa L, Galimand J, Fenneteau O, Mohandas N. Hereditary spherocytosis,

elliptocytosis, and other red cell membrane disorders. Blood Rev

2013;27:167-178.

2. Gallagher PG. Hereditary elliptocytosis: spectrin and protein 4.1R. Semin

Hematol 2004;41:142-164.

3. King MJ, Zanella A. Hereditary red cell membrane disorders and laboratory

diagnostic testing. Int J Lab Hematol 2013;35:237-243.

87

v33i1

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?