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RESEARCH ARTICLEDOI: 10.4274/tjh.2016.0504Turk J Hematol 2018;35:54-60Incomplete Antibodies May Reduce ABO Cross-MatchIncompatibility: A Pilot Studyİnkomplet Antikorlar ABO Çapraz Karşılaştırma Uyumsuzluğunu Azaltabilirler:Bir Başlangıç ÇalışmasıMehmet Özen 1 , Soner Yılmaz 2 , Tülin Özkan 3 , Yeşim Özer 4 , Aliye Aysel Pekel 5 , Asuman Sunguroğlu 3 , Günhan Gürman 6 ,Önder Arslan 61Ufuk University Faculty of Medicine, Department of Hematology, Ankara, Turkey2University of Health Sciences, Gülhane Training and Research Hospital, Blood Bank Unit, Ankara, Turkey3Ankara University Faculty of Medicine, Department of Medical Biology, Ankara, Turkey4Ankara University Faculty of Medicine, Unit of Blood Bank, Ankara, Turkey5University of Health Sciences, Gülhane Training and Research Hospital, Clinic of Immunology and Allergy Diseases, Ankara, Turkey6Ankara University Faculty of Medicine, Department of Hematology, Ankara, TurkeyAbstractObjective: Any erythrocyte transfusion among humans having type A orB blood groups is impossible due to antibodies causing fatal transfusioncomplications. A cross-match test is performed to prevent immunetransfusion complications before transfusion. Our hypothesis is that thefragment antibody (Fab) part of the antibody (incomplete antibody) maybe used to prevent an immune stimulus related to the complete antibody.Therefore, we designed a pilot study to evaluate the effectiveness of theseincomplete antibodies using cross-match tests.Materials and Methods: Pepsin enzyme and staphylococcal protein Acolumns were used to cut anti-A and anti-B monoclonal antibodies andpurify their Fab (2) fragments, respectively. An Rh-positive erythrocytesuspension with purified anti-A Fab (2) solution and B Rh-positiveerythrocyte suspension with purified anti-B Fab (2) solution werecombined correspondingly. Cross-match tests were performed by tube andgel centrifugation methods. The agglutination levels due to the anti-A andanti-B Fab (2) antibodies and their effects on the agglutination normallyobserved with complete antibodies were then measured.Results: No agglutination for the purified incomplete anti-A Fab (2) withA Rh+ erythrocyte and anti-B Fab (2) with B Rh+ erythrocyte combinationswas observed in the tube cross-match tests. These agglutination levels were1+ in two wells in the gel centrifugation cross-match tests. Fab (2)-treatederythrocytes were also resistant to the agglutination that normally occurswith complete antibodies.Conclusion: We determined that the Fab (2) fragments of antibodies maynot only be used to obtain a mild or negative reaction when comparedto complete antibodies, but they might also be used for decreasing ABOincompatibility. Incomplete antibodies might be a therapeutic option inautoimmune hemolytic anemia and they may also be used in solid organor hematopoietic stem cell transplantation. Therefore, we have planned anin vivo study to prove these in vitro findings.Keywords: Transfusion medicine, Red blood cells, Complications, Humoralimmune responseÖzAmaç: Tip A ve B kan grubuna sahip insanlar arasında herhangi bir eritrositnakli öldürücü transfüzyon komplikasyonlarına neden olan antikorlarnedeniyle imkansızdır. İmmün transfüzyon komplikasyonlarını önlemek içintransfüzyondan önce çapraz karşılaştırma testi yapılır. Hipotezimiz kompletantikorla ilişkili bağışıklık yanıtını önlemekte antikorun fragman antikor(Fab) parçasının (inkomplet antikor) kullanılabileceğidir. Bu inkompletantikorların etkinliğini değerlendirmek için de çapraz karşılaştırmatestlerini kullanarak bir başlangıç çalışması tasarladık.Gereç ve Yöntemler: Anti-A ve anti-B monoklonal antikorlarını kesmek vesaflaştırmak için sırasıyla pepsin enzimi ve stafilokokal protein A kolonlarıkullanıldı. A Rh pozitif eritrosit süspansiyonu ile saflaştırılmış anti-A Fab(2) solüsyonu ve B Rh pozitif eritrosit süspansiyonu ile saflaştırılmış anti-BFab (2) solüsyonu sırasıyla birleştirildi. Çapraz karşılaştırma testleri tüp vejel santrifügasyon yöntemleri kullanılarak çalışıldı. Sonrasında anti-A veanti-B Fab (2) antikorlara bağlı aglütinasyon düzeyi ve bunların kompletantikorlarla normalde gözlenen aglütinasyon üzerine etkileri ölçüldü.Bulgular: Tüp yöntemi ile yapılan çapraz karşılaştırma testinde saflaştırılmışinkomplet anti-A Fab (2) ile A Rh pozitif eritrosit ve anti-B Fab (2) ile B Rh pozitiferitrosit kombinasyonlarında aglütinasyon gözlenmedi. Jel santrifügasyonyöntemi ile yapılan çarpraz karşılaştırma testinde bu aglütinasyon düzeyleriher iki kuyucukta da 1 pozitifti. Fab (2) ile muamele edilen eritrositler kompletantikorla normalde oluşan aglütinasyona da dirençliydiler.Sonuç: Antikorların Fab (2) fragmanlarının sadece komplet antikorlarakıyasla daha hafif veya negatif reaksiyonu elde etmekte değil, aynı zamandaABO uyumsuzluğunu azaltmakta da kullanılabileceğini değerlendirdik.İnkomplet antikorlar otoimmün hemolitik anemide bir tedavi seçeneğiolabileceği gibi aynı zamanda solid organ veya hematopoeitik kök hücrenaklinde kullanılabilir. Bu nedenle in vitro bulguları doğrulamak için in vivobir çalışma planladık.Anahtar Sözcükler: Transfüzyon tıbbı, Alyuvarlar, Komplikasyonlar,Hümöral bağışıklık yanıtı©Copyright 2018 by Turkish Society of HematologyTurkish Journal of Hematology, Published by Galenos Publishing HouseAddress for Correspondence/Yazışma Adresi: Mehmet ÖZEN, M.D.,Ufuk University Faculty of Medicine, Department of Hematology, Ankara, TurkeyPhone : +90 536 275 00 74E-mail : kanbilimci@gmail.com ORCID-ID: orcid.org/0000-0002-0910-9307Received/Geliş tarihi: December 30, 2016Accepted/Kabul tarihi: May 22, 201754
Turk J Hematol 2018;35:54-60Özen M, et al: ABO Blood Group and Fab AntibodiesIntroductionThere are many blood groups used for the human population,including ABO, Rh, Kidd, Kell, Duffy, MNS, and Lewis. The ABOsystem is the most important of all blood groups in transfusionpractice due to the reciprocal antibodies [1]. These antibodiesconsistently and predictably present in the sera of normal peoplewhose erythrocytes lack the corresponding antigen(s) [2]. Theseantibodies may cause immediate lysis of donor red blood cells(RBCs) during ABO-incompatible transfusion and initiate fatalhemolytic transfusion reactions [1].Typing and screening are the first steps of pretransfusioncompatibility tests. These tests are used to define the patient’sABO group and Rh type and to detect expected and unexpectedantibodies in the patient’s serum. The cross-match is the finalstep of pretransfusion testing [3]. In this test, donor cellsare combined with the patient’s serum and checked foragglutination, which would signify incompatible blood [4]. Thisprocess, also known as major cross-matching, serves as the lastsafeguard to ensure a safe transfusion [4,5,6].Antibodies are also essential for humoral immunity [7].Many antibodies have been shown to be primarily relatedto autoimmune diseases and such diseases are referred to asantibody-related autoimmune diseases [7,8,9]. Many of thesediseases may disappear in the absence of certain antibodies [9].All antibodies have two fragments. The antigen-binding fragment(Fab) binds to an antigen, and the crystallizable fragment (Fc)stimulates the immune system by activating the complement[10]. Additionally, macrophages or lymphocytes detect the Fcfragment of antibodies [11,12]. Therefore, the Fab fragmentdetects antigens and the Fc fragment stimulates the immunesystem. An antibody with the Fc part removed, in which onlythe Fab fragment exists, may be called an incomplete antibody.Papain or pepsin enzymes can be used in the fragmentation ofantibodies and can produce Fab or Fab (2) fragments of theantibodies, respectively [13]. The effectiveness levels of Faband Fab (2) fragments of an antibody are similar and theyare interchangeable [14]. Our hypothesis is that incompleteantibodies may be used to prevent an immune stimulus. Wedesigned a pilot study to examine the effectiveness of theseincomplete antibodies in incompatible cross-matches dueto ABO antibodies and we are presenting it here. Local ethicscommittee approval was obtained for this study.Materials and MethodsAnti-A and anti-B monoclonal antibodies (Eryclone, VernaIndustrial Estate, Verna, India) were used for this study. First thepepsin enzyme was used to cut these monoclonal antibodiesand staphylococcal protein A columns were used to purify theirFab (2) fragments. The Pierce F(ab’)2 Preparation Kit (ThermoFisher Scientific, Rockford, IL, USA) was used to produce theFab (2) fragments from complete antibodies. This process wasconducted according to the manufacturer’s instructions.After obtaining purified Fab (2)s, we began the second part ofthe study. During purification of Fab (2)s, the volume of theproducts changed. The ratios of the complete monoclonalantibodies to the standard erythrocyte solution for anoptimal cross-match test were calculated according to themanufacturer’s instructions. We used these ratios for the anti-Aor -B Fab (2) to the A or B Rh-positive erythrocyte solutions forthe cross-match tests, respectively.After calculation, an anti-IgG cross-match card (Ortho-ClinicalDiagnostics, High Wycombe, UK) was used for the compatibilitytests. We combined 10 µL of A Rh-positive 5% erythrocytesuspension with 150 µL of purified anti-A Fab (2) solution in thesame well to conduct a cross-match test in order to prove thatthe erythrocytes were covered with anti-A Fab (2). We also used150 µL of complete anti-A antibodies for the positive controland 150 µL of phosphate-buffered saline (PBS) for the negativecontrol. We incubated all cards at 37 °C for 10 min and thencentrifuged them for 5 min. The negative controls lacked thecomplete and incomplete antibodies. We repeated the sameprocess with complete and incomplete anti-B antibodies and theB Rh-positive erythrocyte suspension. In addition, we repeatedthese tests using Across Gel ® Anti-Human Globulin IgG+C3dcross-match cards (Dia Pro, İstanbul, Turkey). We also evaluatedagglutination levels when complete and incomplete antibodieswere put in the same well at the same time, noting the amountsfor A and B erythrocyte suspensions. We conducted an antibodytitration test and repeated this last test with several ratios(32/1, 8/1, 4/1, 1/1, 1/4, and 1/16) for complete to incompleteantibodies when used simultaneously. Finally, we evaluated thereactions in all wells.We also performed a tube test to confirm the results of thecard tests and to show whether incomplete antibodies inhibitednormal agglutination with complete antibodies or not. First,we treated A Rh+ erythrocytes with anti-A Fab (2) and B Rh+erythrocytes with anti-B Fab (2) in two separate tubes. We thenadded complete anti-A and anti-B antibodies to the respectivetubes and mixed them. As a positive control, A Rh+ erythrocyteswere treated only with complete anti-A antibodies and B Rh+erythrocytes were treated only with complete anti-B antibodiesin a tube without adding incomplete fragments. Consequently,there were no incomplete antibodies in positive control tubes.We then evaluated the agglutination levels in the tubes.In addition, we performed a flow cytometric analysis to provethe results of all these tests. The B Rh+ erythrocyte samplewas transferred to a tube containing K 3EDTA and that tube’scontents were divided into four tubes. We mixed each tube with55
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