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Positive Mixed Lymphocyte Culture Test Result Due To Hla –dp Mismatch

Authors:
Tulay Kilicaslan Ayna at Izmir Kâtip Celebi University, Turkey
Tulay Kilicaslan Ayna
  • Izmir Kâtip Celebi University, Turkey
Hilmi tozkır at Namık Kemal University
Hilmi tozkır
Hayriye Senturk Ciftci at Istanbul University
Hayriye Senturk Ciftci
Hakan Gurkan at Trakya University
Hakan Gurkan
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Abstract

Graft Versus Host Disease (GVHD) remains a major cause of morbidity and mortality in allogeneic stem cell transplantation (SCT). The MLC assay has been generally accepted as a standard test for determining HLA-D region compatibility. In this study, the MLC test has been carried out on the related recipient-donor couple who were prepared for allogeneic SCT and were found to be matched for HLA-A, -B, -Cw, DR, DO but not for -DP. The result of the MLC test was positive. We observed that HLA-DP mismatch was responsible for the increased proliferation values in MLC test.
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224
HLA-DP Uyumsuzluğuna Bağlı Mikst Lenfosit
Kültür Testindeki Pozitifl ik
1İstanbul Üniversitesi İstanbul Tıp Fakültesi, Tıbbi Biyoloji Anabilim Dalı, İstanbul, Türkiye
2Trakya Üniversitesi Tıp Fakültesi, Tıbbi Biyoloji Anabilim Dalı, Edirne, Türkiye
3Trakya Üniversitesi Tıp Fakültesi, İç Hastalıkları Anabilim Dalı, Hematoloji Bilim Dalı, Edirne, Türkiye
Positive Mixed lymphocyte Culture Test Result Due to HLA-DP Mismatch
Tülay Kılıçaslan Ayna1, Hilmi Tozkır2, Hayriye Şentürk Çiftçi1, Hakan Gürkan2, Emre Tekgündüz3, Çetin Algüneş2,
Mehmet Gürtekin1, Mahmut Çarin1
Case Report
ÖZET
Graft Versus Host Hastalığı (GVHH) allojeneik kök hücre nakli (KHN) sey-
rinde halen önemli bir morbidite ve mortalite nedenidir. Mikst lenfosit
kültür (MLC-Karışık Lenfosit Kültür) testi, genellikle HLA- D bölge uyumu-
nu saptayan standart bir test olarak kabul edilir. Bu çalışmada allojeneik
KHN’ne hazırlanan ve HLA-A, -B, -Cw, -DR, -DQ lokusları uyumlu ve -DP
lokusu uyumsuz olan akraba hasta -verici çiftine MLC testi yapılmıştır. Test
sonucu pozitif olarak saptanmış ve HLA-DP farklılığının MLC testindeki
proliferasyon değerlerindeki artışlardan sorumlu olduğu gözlemlenmiştir.
Anahtar Sözcükler: Mikst lenfosit kültürü, HLA-DP, kök hücre nakli
Geliş tarihi: 16.06.2009 Kabul tarihi: 18.08.2009
ABSTRACT
Graft Versus Host Disease (GVHD) remains a major cause of morbidity and
mortality in allogeneic stem cell transplantation (SCT). The MLC assay has
been generally accepted as a standard test for determining HLA-D region
compatibility. In this study, the MLC test has been carried out on the re-
lated recipient-donor couple who were prepared for allogeneic SCT and
were found to be matched for HLA-A, -B, -Cw, DR, DQ but not for -DP.
The result of the MLC test was positive. We observed that HLA-DP mis-
match was responsible for the increased proliferation values in MLC test.
Key Words: Mixed lymphocyte culture, HLA-DP, stem cell transplantation
Received: 16.06.2009 Accepted: 18.08.2009
1960’lı yıllardan beri KHN birçok hematolojik hastalığın
tedavisinde artan sıklıkta kullanılmaktadır (1). KHN sırasında
transfüze edilen kök hücreler otolog veya allojenik (İnsan Lö-
kosit Antijenleri-Human Leukocytes Antigen-HLA) uyumlu kar-
deşler, akraba olmayan vericiler ve kordon kanı) kaynaklardan
elde edilmektedir. Bu kaynaklardan en çok kullanılan ve çoğu
endikasyonda en başarılı olanı HLA tam uyumlu kardeşlerden
yapılanıdır (1, 2). GVHH graftta bulunan immünokompetan T
lenfositlerinin konağın dokularına karşı reaktivite kazanma-
sı sonucu oluşur. KHN’deki başarının artmasında, yeni tedavi
yöntemlerinin geliştirilmesi yanında hasta ve vericinin HLA uy-
gunluklarını saptamak amacıyla yapılan çeşitli moleküler testler
de önemli yer tutmaktadır (1, 3). HLA (sınıf I ve sınıf II) anti-
jenlerini belirlemede başlangıçta serolojik yöntemler kullanı-
lırken, teknolojik gelişmeler neticesinde günümüzde molekü-
ler yöntemler tercih edilmektedir (4). MLC ise, hasta ve verici
arasındaki özellikle HLA-sınıf II (HLA-D) antijenlerinin uygun-
suzluğunda hücresel bağışık yanıtın ölçülmesinde kullanılan
bir test yöntemidir. Genetik açıdan birbirinden farklı kişilerin
lenfositleri in vitro karşılaştırıldığında, lenfosit yüzeyinde ifade
edilen HLA-D grubu antijenleri diğer lenfosit grubunda DNA
sentezini uyarmaktadır. DNA sentezindeki artış, hasta ile ve-
rici arasındaki uyuşmazlığın derecesi ile orantılıdır (5). HLA-D
bölgesi DR, DQ ve DP, olmak üzere 3 önemli alt bölgeye ay-
rılmaktadır. Sınıf II molekülleri α ve β polipeptit zincirlerinin
birlikte oluşturduğu moleküllerdir. DR bölgesi sadece bir α
zincir genini kapsar (DRA). Buna karşılık bazıları fonksiyonel
olan (DRB1, 3, 4 ve 5), bazıları fonksiyonel olmayan (DRB2,
6, 7, 8, 9) 9 β zincir geni vardır (DRB). Fonksiyonel olan DRB1
genlerinin her biri farklı β zincirini kodlar. DQ bölgesinde
hem α, hem de β zincirleri polimorfi ktir. DP bölgesinde ise,
DP α zinciri düşük polimorfi zm gösterirken, DP β zinciri bü-
yük oranda polimorfi ktir (6). Çalışmamızda, Polymerase Chain
Reaction (Polimeraz Zincir Reaksiyonu- PCR)-Sequence Spe-
cifi c Primers (Diziye Özgü Primerler-SSP) yöntemi ile DP allel
uyumsuzluğu MLC pozitifl iği tespit ettiğimiz bir akraba hasta-
verici çifti sunulmaktadır.
Olgu Sunumu
Kronik Lenfositik Lösemi (KLL) nedeniyle tedavi edilmek-
te olan 52 yaşında bir erkek hasta ile 49 yaşında olan erkek
kardeşinin HLA uyumu araştırılmıştır. Hasta ve vericinin HLA
doku tiplemesi Trakya Üniversitesi Tıp Fakültesi Tıbbi Biyoloji
A.D.’nda belirlenmiştir. Her birinden EDTA’lı tüpe 5 cc perife-
rik venöz kan alınarak QIAMP DNA Blood Mini Kit (QİAGEN,
Cat. No:51104) ile DNA izolasyonları yapılmıştır. HLA- A, -B,
-Cw, -DRB, -DQB ve -DPB lokuslarının genotiplendirmesinde
düşük rezolüsyonlu Olerup SSP A,B, Cw, DR, DQ, DP SSP
Combi Tray, Lot. No.: V99) kiti kullanılmıştır. Amplifi kasyon-
Address for Correspondence: Dr. Tülay Kılıçaslan Ayna, İstanbul Üniversitesi İstanbul Tıp Fakültesi, Tıbbi Biyoloji Anabilim Dalı, İstanbul, Türkiye
Phone: +90 212 414 20 00 E-mail: tulayayna@gmail.com
Balkan Med J 2011; 28: 224-226 • DOI: 10.5174/tutfd.2009.02852.1
© Trakya University Faculty of Medicine
da GeneAmp PCR Sistem 9700 (Applied Biosystems, Foster
City, California) kullanılarak; 1 siklus 94°C’de 2 dak., 10 sik-
lus 94°C’de 10 sn., 65°C’de 1 dak., 20 siklus 94°C’de 10 sn.,
61°C’de 50 sn., 72°C’de 30 sn. ve (+) 4°C’de sonsuz PCR prog-
ramı uygulanmıştır. Elde edilen PCR ürünlerine %0.5 TBE ile
%2 agaroz jelde,160 volt, 450 amp., 10 dakika elektroforez
uygulanmıştır. Daha sonra UV. transilluminatör yardımı ile aga-
roz jel değerlendirilerek amplifi kasyon gerçekleşen kuyucuklar
belirlenmiş ve Score bilgisayar yazılım programında değerlen-
dirilmiştir. Hastanın HLA allelleri; A*02, 03, B*27, 35, Cw*02,
07, DRB1*01, 16, DQB1*05, 05 ve DPB1*0201, 0401 olarak
saptanırken, vericinin HLA allellerinin A*02, 03, B*27, 35,
Cw*02, 07, DRB1*01, 16, DQB1*05, 05 ve DPB1*0401, 0402
olduğu belirlenmiştir.
Hasta ve verici çiftine İstanbul Üniversitesi İstanbul Tıp Fa-
kültesi, Tıbbi Biyoloji Anabilim Dalı’nda MLC testi uygulanmış-
tır. Test için, hasta ve vericiden lityum heparinli tüpe 8 cc peri-
ferik venöz kan alınarak lenfositler Ficoll-hypaque (Pharmacia,
Piscataway, NJ) gradient tekniği ile izole edilmiştir. Elde edilen
lenfositler RPMI 1640 (glutaminli, hepesli, Sigma-50 u/mlpe-
nisilin ve 50 μg/ml streptomisin) ile üç kez yıkandıktan sonra
hücreler RPMI 1640 ve insan serumu (9:1) içeren medyumda
1×106 olacak şekilde sayılmıştır. Hasta hücreleri 5000 rad’da
(cGy) ışınlanmıştır. Hücreler 96 kuyulu plaklarda 96 saat 37°C
ve %5 CO2’li ortamda inkübe edildikten sonra, her bir kuyuya
3H Thymidine koyulmuş ve 16 -18 saat sonra tüm hücreler top-
lanmıştır. Kültürün sonlanmasından sonra likit sintilasyon saya-
cında sayım yapılmıştır. Tek yönlü MLC test sonucunda iki ayrı
değerlendirme, Stimülasyon indeksi (SI) ve Göreceli Cevap
İndeksi (Relative Response Index - RRI) hesaplanmıştır (For-
mül 1, Formül 2). MLC testinde SI değerinin ≤1 olması ve RRI
değerinin negatif olması vericinin hasta için uygun olduğunu
belirtmektedir. Tek yönlü MLC testi hasta hücre sayısı yetersiz
olduğundan verici yönünde çalışılmıştır. SI: 2.66 ve RRI değer-
leri + (2.2) ile + (4.4) arasında bir dağılım göstermiştir.
Formül 1:
SI= VH*/VV
Formül 2:
RRI = (VH*-VV /VK*-VV) ×100
*: ışınlanmış hücreler
VH*:Verici hücreleri ile hastanın ışınlanmış hücreleri
VV: Sadece verici hücreleri
VK*: Verici hücreleri ile ışınlanmış kontrol (hasta ve verici
ile akrabalığı olmayan sağlıklı kişilerden elde edilen hücreler)
hücreleri
Tartışma
Günümüzde çeşitli hematolojik neoplaziler, bazı kalıtsal
hastalıklar ve kemik iliği yetersizliği sendromları KHN ile tedavi
edilmektedir. Hasta ve verici arasındaki HLA uyumu graft red-
di, GVHH hastalığı ve yetersiz immun yapılanma ile ilişkilidir.
Serolojik HLA tiplemesi, hasta ve vericilerinin HLA karşılaştır-
masını saptamada yetersizdir. Serolojik sonuçlar hasta ve verici
uyumunun allel düzeyinde karşılaştırılmasına imkan vermez. Bu
sebeple, HLA polimorfi zmini saptamak için moleküler biyoloji
tekniklerinin kullanımına izin veren daha ileri metodlara ihtiyaç
vardır. PCR SSP metodu ile diziye özgü sentetik oligonukleo-
tidler kullanılarak hasta verici çiftleri arasındaki uyum araştırıl-
maktadır. MLC ise, sınıf II molekülleri (HLA-DR, DQ ve DP) ara-
sındaki farklılığı saptayan bir metoddur (7). Hasta- alıcı çiftleri
arasındaki HLA-DRB1 uyumsuzlukları MLC testlerinde prolife-
ratif cevapların en önemli nedenidir. Ancak, DRB1 uyumlu olsa
bile, hasta ve verici arasında DRB3, DQ ve DP uyumsuzlukları
da MLC testinde proliferasyona sebep olabilir (9). Özellikle ak-
raba olmayan vericilerden yapılan KHN’lerinde HLA-C, E, DP
gibi bölgelerindeki uyumsuzluklar da, KHN sonrası GVHH ris-
kinin artmasına katkıda bulunabilmektedir. Bu durum akraba
olmayan KHN’lerinde akut GVHH ve rejeksiyon ihtimalinin ne-
den daha yüksek olduğunu açıklayabilir (9-11). Bizim vakamız-
da, KLL tanılı hasta ve verici kardeşinin PCR-SSP yöntemi ile
HLA-A,-B,-C,-DR,-DQ ve -DP lokusları çalışılmış, hasta ve verici
arasında sadece DP uyumsuzluğu saptanmıştır. MLC testinde
ise hasta-verici hücrelerinin proliferasyonlarındaki artış nedeni
ile test sonucu pozitif olarak değerlendirilmiştir. Böylece, HLA-
DP uyumsuzluğu MLC testi ile de doğrulanmıştır.
Olerup ve ark.’ları, akraba olan ve olmayan, DP uyumlu
olan ve olmayan çiftlere MLC testi yaptıklarında, DP uyumsuz-
luğu olan 3 (%100) akraba çiftin hepsinin MLC sonucunun po-
zitif olduğunu görmüşlerdir. Akraba olmayan HLA-DP uyumsuz
bireylerden elde edilen hücrelerle uygulanan MLC testinde
ise %9.8 oranında pozitifl ik saptanmıştır. Araştırmacılar MLC
testini değerlendirme de, RRI sonuçlarını esas almışlardır. RRI
değeri %8 veya üzerinde ise sonuç pozitif olarak kabul edilmiş-
tir. Akraba olmayan grupta MLC testi yapılan vakalarda pozitif
sonuçların az olmasının sebebini de kabul edilen yüksek RRI
değerlerinden kaynaklanmış olabileceğini belirtmişlerdir. Ayrı-
ca bu çalışmada iki DP uyumsuzluğu olan hasta verici çiftlerin-
de, tek DP uyumsuzluğuna göre RRI değerlerinde daha fazla
artış olduğu görülmüştür (8). Vakamızdaki DP uyumsuz akraba
çiftin MLC sonucunun pozitif bulunması Olerup ve ark.’larının
akraba çiftlerde gözlemledikleri uyumsuz DP ile MLC pozitifl iği
ilişkisi açısından uyumludur.
Penzes ve grubu yaptıkları çalışmaya, KHN için akraba
uygun vericisi olmayan 28 Macar hasta ve bu hastalara sero-
lojik olarak HLA-A, -B, -DR uyumlu 61 (26 hasta için birden
fazla potansiyel verici var) akraba olmayan vericiyi dahil et-
mişlerdir. Çiftlerin sadece %11.5’inde moleküler yöntemlerle
HLA uyumu saptanmıştır. Bu çiftlere MLC testi yapıldığında,
daima (-) MLC sonucu saptanırken, HLA-DPB1*0201-0301 ve
DPB1*0301-0401 allel kombinasyonunun ise, tüm vakalarda
reaktif olduğunu bulmuşlardır. Sonuç olarak, araştırmacılar
MLC testinde bazı DP allel kombinasyonlarının düşük immu-
jenite gösterebileceğini, bazı kombinasyonların ise, pozitif
sonuçlar ortaya çıkarabileceğini belirtmişlerdir. Bu sonuçların-
da, KHN için en uygun vericinin seçilmesine yardım edeceğini
açıklamışlardır (12).
Vakamızda hastanın HLA-DP’si DPB1*0201-0401 vericinin
HLA-DP’si ise, DPB1*0401-0402‘dir. MLC testi sonucunda,
DPB1*0201-0402 allel kombinasyonunun da reaktif olduğunu
belirledik. Penzes ve ark.’larının çalışması ile vakamızı değer-
lendirdiğimizde, HLA-DPB1*0201-0301 ve DPB1*0301-0401
kombinasyonlarına ilave olarak DPB1*0201-0402 kombinasyo-
nunun da MLC testinde pozitif sonuç ortaya çıkarabileceğini
225
Balkan Med J
2011; 28: 224-6
Ayna ve ark.
HLA-DP Uyumsuzluğunda Lenfosit Kültür Testi
saptadık. Ancak DPB1*0201-0402 allel kombinasyonu daima
yüksek immun cevaba sahiptir diyebilmek için test sayısını ar-
tırmaya ihtiyaç vardır.
Sonuç olarak, moleküler yöntemlerle HLA bölgesi allel dü-
zeyinde belirlenmektedir. MLC testi ile de, HLA-DR, DQ anti-
jenlerinin yanı sıra DP antijen farklılıklarının da saptanması ile,
HLA sınıf II antijenlerinin uygunluğu doğrulanmaktadır. HLA
uyumlu kardeşler aynı haplotiplere sahip olduklarından dolayı
genellikle allel uyumsuzluğu beklenmez. Bu çiftteki DP uyum-
suzluğu crossing over esnasında gelişmiş olmalıdır. Ayrıca yap-
tığımız çalışma doku tiplendirmesi yaparken her lokusun ayrı
önemi olduğunu ve KHN öncesinde DP lokusların araştırılma-
sının önemini vurgulamaktır. Bu değerlendirmenin, nakil için
uygun vericinin seçilmesine katkıda bulunacağı ve nakil sonrası
GVHH riskini de azaltacağı düşüncesindeyiz.
Çıkar Çatışması
Yazarlar herhangi bir çıkar çatışması bildirmemişlerdir.
Kaynaklar
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sus host disease in HLA-identical sibling donors. Egypt J Immu-
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JJ, Faras AJ, McGlave PB and Rich SS.Frequent HLA class I and
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ResearchGate has not been able to resolve any citations for this publication.
View
The predictive value of HLA-DR oligotyping for MLC response
Article
  • Jul 1992
Comparison of HLA proteins between a patient and potential unrelated marrow donors is difficult because many similar, but not identical, HLA proteins are expressed in the human population. A reliable and practical method to detect these subtle differences is provided by oligotyping, a new technique that identifies polymorphic sequences in the genes encoding the HLA proteins. Oligotyping was used to compare polymorphic HLA-DR sequences in 286 pairs of samples from patients and potential unrelated donors who were serologically matched for HLA-DR specificities. Oligotyping detected HLA-DR differences in 53% of these pairs and all mismatched pairs were reactive in primary mixed lymphocyte cultures. Where HLA-DR disparity was not detected by oligotyping, 37% of the pairs were nonreactive in MLC. The remaining 63% often contained an allele associated with the HLA-DRw11 serological specificity. In the absence of HLA-DRB1*11, oligotyping was predictive of MLC reactivity for samples with HLA-DR2, -DR4, and DRw52. In clinical settings, the ability to predict MLC reactivity on the basis of precise HLA typing provides an alternative to MLC. Further, the relationship between specific polymorphic sequences and reactivity in MLC may lead to more fundamental insights into the mechanisms involved in alloreactive responses.
View
Selection of unrelated donors for bone marrow transplantation is improved by HLA class II genotyping with oligonucleotide hybridization
Article
  • Sep 1991
As the demand for donors for bone marrow transplantation increases, the use of HLA-matched, genetically unrelated donors represents a promising strategy. It is well documented that the clinical outcome of bone marrow transplantation is directly dependent on optimal matching for HLA class I and class II specificities. Molecular studies have revealed the existence of a much larger number of HLA class II alleles than was anticipated, many of which cannot be recognized by routine serological typing. Currently this "hidden" polymorphism represents a major limitation to the generalized use of unrelated donors for bone marrow transplantation. It has recently become possible, however, to identify HLA allelic polymorphism directly at the DNA level by hybridization with sequence-specific oligonucleotide probes ("HLA oligotyping") after amplification of DNA by polymerase chain reaction. In this study, we have investigated whether donor-recipient pairs that are fully matched for HLA by serology are truly HLA-DR, -DQ, and -DP identical and to what extent class II differences influence the primary mixed lymphocyte culture. We show that HLA oligotyping, performed on 50 pairs of HLA class I and II serologically matched individuals, can indeed reveal phenotypically relevant allelic differences at either DRB or DQB loci in 56% of these pairs and can therefore improve HLA class II typing and the choice of bone marrow donors quite significantly. Oligotyping for DRB/DQB/DPB polymorphism also allows prediction of a positive mixed lymphocyte culture, as established in 31 donor/recipient combinations, and even detection of polymorphic differences that were not revealed by this test. This approach is well suited for accurate HLA typing of large pools of bone marrow donors and was successfully applied to select fully matched donors for bone marrow transplantation.
View
HLA-DP incompatibilities induce significant proliferation in primary mixed lymphocyte cultures in HLA-A, -B, -DR and -DQ compatible individuals: Implications for allogeneic bone marrow transplantation
Article
  • Dec 1990
The major part of the proliferative response in primary mixed lymphocyte cultures (MLC) is caused by HLA-DRB1 incompatibilities. In DRB1-matched pairs the proliferation induced by HLA-DRB3, -DQ and -DP mismatches may be unmasked. In most previous studies the influence of HLA-DP incompatibilities in primary MLC has been investigated in homozygous typing cells representing only a few Dw specificities. We were interested in determining the stimulatory capacity of isolated HLA-DP mismatches, ascertained by RFLP analysis, in primary MLC in HLA-A, -B, -DR and -DQ compatible, unrelated heterozygous individuals of many different Dw specificities. Thirty-eight MLCs performed with cells from related pairs and 67 with cells from unrelated pairs were evaluated. All but nine of the MLCs were analyzed in both directions, giving a total of 201 investigated reactions. The relative responses (RR) in the three MLCs performed between DP incompatible, related pairs were all positive (RR greater than or equal to 8%). Eighty of 82 MLCs performed with cells from DP incompatible, unrelated individuals were positive, whereas 37 of 46 MLCs between DP compatible, unrelated pairs were negative (RR less than 8%) (p less than 10(-10)). The magnitude of the RR was influenced by the number of DP mismatches. Thus, the mean RR was approximately twice as high in MLCs in which responder and stimulator cells differed by two DP antigens (mean RR 60.5%) compared with reactions with only one DP mismatch (mean RR 35.4%) (p less than 10(-3)). RFLP-defined HLA-DP incompatibilities predict a positive primary MLC in HLA-A, -B, -DR and -DQ matched individuals with a high degree of accuracy (98%).(ABSTRACT TRUNCATED AT 250 WORDS)
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Marrow transplantation from HLA matched unrelated donors for treatment of hematologic malignancies
Article
  • Mar 1991
Less than 40% of the patients who could benefit from marrow transplantation have an HLA-matched relative who can serve as a donor. For this reason, several centers have explored marrow transplantation from other categories of donors. This retrospective study analyzes the results of marrow transplantation for 52 patients receiving grafts from HLA-A,B,DR,Dw-phenotypically matched, MLC-compatible, unrelated volunteer donors compared to a disease, disease-stage, and age-matched cohort of 104 patients transplanted from HLA-genotypically identical sibling donors. The patients transplanted from unrelated donors had an increased incidence of grade II-IV acute graft-versus-host disease compared to patients transplanted from related donors (79% vs. 36%, P much less than 0.001). However, the probability of relapse-free survival appears similar in the two groups (P = 0.39 over all, with estimates of 41% vs. 46% at 1 year). We conclude from this preliminary data that marrow transplantation from HLA-matched unrelated donors should be considered in most, if not all, circumstances where transplantation from an HLA-matched sibling would be indicated if such a donor were available.
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Marrow transplantation for hematologic malignancies: A brief review of current status and future prospects
Article
  • Aug 1988
  • SEMIN HEMATOL
The ability to transplant bone marrow makes it possible to treat patients with hematologic malignancies with doses of systemic radiotherapy or chemotherapy that would ordinarily result in fatal myelosuppression. With this approach, some otherwise incurable patients can be treated successfully. For most hematologic malignancies (acute nonlymphocytic leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, and malignant lymphoma), if transplantation is delayed until patients have end-stage disease (drug-resistant relapse) or blast crisis, approximately 10 to 20% of patients can be saved. If transplantation is carried out earlier in the course of disease, the outcome improves considerably. Some of the major limitations of marrow transplantation are the need for an appropriate source of marrow, the severe myelosuppression seen in the immediate posttransplant period, the complications of graft-v-host disease (GVHD), and disease recurrence. The use of autologous marrow and the formation of a national donor registry may make transplantation more widely applicable. The availability of growth factors can hasten hematopoietic recovery, making the immediate posttransplant period safer. New immunosuppressive regimens and T-cell depletion of donor marrow can diminish the impact of GVHD. The need for better preparative regimens persists and has emerged as the major requirement for continued progress in marrow transplantation.
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Role of the mixed lymphocyte culture (MLC) reaction in marrow donor selection: Matching for transplants from related haploidentical donors
Article
  • Sep 1994
The utility of the MLC assay as a test of HLA-D region matching and predictor of acute graft-versus-host disease (GvHD) was evaluated in 157 patients receiving marrow grafts from HLA-A, B identical related haploidentical donors. All donors and recipients were tested by HLA-DR serology, by Dw phenotyping with homozygous typing cells (HTC) and by standard MLC. Ninety-nine of the donor-recipient pairs were mismatched for a serologically defined HLA-DR antigen while 109 pairs were mismatched for the HLA-DR region by HTC typing. Donor antirecipient relative responses (RR) in MLC, corresponding to the GvHD vector in marrow transplantation, ranged from -4% to 100%, with a median of 25%. A comparison of reactivity in MLC with presence or absence of matching by Dw phenotyping, however, showed a significant overlap in the distribution of RRs from HLA-Dw matched versus Dw mismatched pairs, suggesting that the MLC was not a reliable predictor of HLA-Dw matching. Using an optimally-defined cutoff of 3% RR, the MLC was correlated with risk of developing clinically significant grades II-IV acute GvHD (p = 0.03) but not with risk of developing severe grades III-IV GvHD (p = 0.18). In contrast, matching by Dw phenotype was a significant predictor of GvHD, with Dw-compatible transplant recipients less likely to develop either grades II-IV (p = 0.004) or III-IV (p = 0.036) GvHD than Dw-incompatible transplant recipients. Overall, these results underscore the difficulty in using the MLC to measure HLA-D region compatibility and predict the risk of severe graft-versus-host disease among patients receiving related haploidentical marrow grafts. HLA-D (HTC) typing results correlate primarily with DRB compatibility, and with the advent of DRB1 allele matching by sequence-specific oligonucleotide probes (SSOP) or by direct sequencing, the precision in donor matching achievable with these methods is far greater than with either HLA-D typing or direct MLC testing.
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Frequent HLA class I and DP sequence mismatches in serologically (HLA-A, HLA-B, HLA-DR) and molecularly (HLA-DRB1, HLA-DQA1, HLA-DQB1) HLA-identical unrelated bone marrow transplant pairs
Article
  • Feb 1994
The rates of graft-versus-host disease (GVHD) and rejection are significantly higher among recipients of unrelated donor marrow (BM) than in recipients of marrow from HLA-identical siblings, even when donors and recipients are mixed lymphocyte culture (MLC) compatible and serologically and Dw identical. It has been hypothesized that phenotypically silent HLA class I and DP sequence mismatches might be associated with these differences, but little is known about their incidence. We have sequenced the HLA-A, HLA-B, HLA-C, HLA-DPA1, and HLA-DPB1 genes expressed by 12 unrelated marrow transplant pairs, 11 of whom were molecularly matched at DRB, DQA1, and DQB1 loci. Nine of these pairs were also HLA-A and HLA-B matched by serology. Six of these nine "HLA-identical" pairs were HLA-A (2 of 6), HLA-B (1 of 6), and HLA-C (6 of 6) mismatched at the sequence level. The mismatched class I alleles of all these pairs had strikingly different sequence motifs in the six specificity pockets of their antigen recognition site, and in five pairs they also had sequence differences at positions implicated in T-cell receptor (TCR) binding. Two of the three pairs who were serologically mismatched for one HLA-A or HLA-B antigen were also sequence mismatched at HLA-C. Finally, 10 of 11 pairs tested expressed different DP sequences. These data indicate that HLA class I, especially HLA-C, and DP sequence mismatches are frequent among unrelated subjects defined as HLA identical by current typing methods. We speculate that these sequence differences may explain, at least in part, the higher incidence of acute GVHD and rejection in unrelated BM transplantation as opposed to transplantation between HLA-identical siblings. Because of their high frequency, the role of HLA-A, HLA-B, HLA-C, and HLA-DP mismatches in transplantation outcome is now amenable to direct study.
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Influence of HLA-DPB1 mismatches on MLR responses: the role of high resolution HLA class II typing and MLC in unrelated donor selection for BMT
Article
  • Jan 1999
  • BONE MARROW TRANSPL
Twenty eight Hungarian patients lacking a compatible related donor and their 61 HLA-A,B,DR serologically identical potential unrelated donors (selected from BMDW) were investigated in this study. Out of the 61 donor-recipient pairs only 7 (11,5%) proved to be HLA-identical at DNA level. Thirty one pairs (50,8%) differed in DP alleles, 1 pair (1,6%) has a DQ mismatch only and 22 (36,1%) pairs differed in more alleles. More than one potential donor was found for 26 patients and 5 of them have several donors with DP mismatches only. Among the 31 donor-recipient pairs differing only in DP alleles, 0, 1 and 2 mismatches were observed in GvH direction in 7, 10 and 14 cases, respectively. In the MLC assay no proliferative response was observed when no DP mismatch has been found. Among the 1 and 2 DP mismatched cases 11 (35,5%) gave negative and 13 (41,9%) gave positive MLR results. We have found a large scatter in RR values. On the basis of the DNA typing and MLR results we have found that HLA-DPB1*0101-0201 stimulator-responder combination always gave negative MLR in both direction. HLA-DPB1*0201-0301 and DPB1*0301-0401 allele combinations were reactive in all cases. In conclusion MLC assay might indicate the low immunogeneicity in certain DP allele combinations as well as the avoidable positive combinations, which may help to select the best fitting donor for BMT.
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HLA-DPB1 mismatch and acute graft-versus host disease in HLA-identical sibling donors
Article
  • Feb 2005
Allogeneic hematopoietic stem cell transplantation (HSCT) is an established curative therapy for a variety of hematologic malignancies. Genotypically HLA-identical sibling donors (ISDs)--who are available for about 30% of white patients are still considered as the best donors for HSCT. HLA-DPB1 is characterized by a high polymorphism, weak linkage disequilibrium with HLA-DR and -DQ loci, and its role as a transplantation antigen is controversial. We investigated the impact of HLA-DPB1 mismatch in HLA-identical sibling donor transplantation on a Graft-versus-host disease (GVHD). We Typed HLA-DPB1 by Innolipa in 33 patient-donor pairs with different hematologic diseases. Four (12.2 %) pairs with HLA-DPB1 mismatched were identified without identity. The incidence of grades II-IV aGVHD was higher in the HLA-DPB1 mismatched group (p=0.014, OR=26). Univariate analysis of risk factor for aGVHD II-IV showed that HLA-DPB1 is the only significant association (p=0.014), while non significant association was found between the age and sex of the patient, age of the donor, disease of the patient, and ABO compatibility, and positive CMV serology in patients and donors. It is concluded that HLA-DPB1 can mediate alloreactive responses and that HLA-DPB1 mismatch increases the risk of aGVHD in sibling donor stem cell transplantation.
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