Evaluation of Flow Cytometric Crossmatch Results in Comparison with Donor-specific Antibodies Detected by Luminex-PRA Tests in Organ Transplantation Patients / 대한이식학회지

BACKGROUND: Two of the most sensitive methods for detecting donor-specific HLA antibodies (DSAs) are solid phase panel reactive antibody (PRA) assay using Luminex platform (Luminex-PRA), and a cell-based flow cytometric crossmatch (FCXM) test. We evaluated FCXM results in relation to DSAs detected by the Luminex-PRA method in solid organ transplantation candidates or post-transplant follow-up patients. METHODS: A total of 171 donor-recipient pairs were evaluated by Luminex-PRA (LIFECODES Class I and Class II ID kits; Gen-Probe, USA) and FCXM (T- and B-cells) tests. DSA levels were analyzed using a sum of median fluorescence intensity (MFI) values, and FCXM results were analyzed using MFI ratios. RESULTS: Class I and II DSAs were detected in 11.7% (20/171) and 11.1% (19/171) of tested sera, respectively. T-FCXM was negative in 97.4% (147/151) of Class I DSA negative sera, and B-FCXM was negative in 99.3% (137/138) of Class I and II DSA negative sera. T-FCXM was positive in 91.7% (11/12) of sera with moderate to strong Class I DSAs and B-FCXM was positive in 88.9% (16/18) of sera with moderate to strong Class II and/or Class I DSAs in the evaluation of sensitivities of FCXM in relation to DSA. There were significant correlations between FCXM ratios and DSA levels for both T-FCXM (P=0.008) and B-FCXM (P97%) and the sensitivities of T- and B-FCXM were satisfactory (>88%) in detecting moderate to strong DSAs.

Discrepancies between Human Leukocyte Antigen Registry Typing and Confirmatory Typing Results of Unrelated Hematopoietic Stem Cell Donors / 대한진단검사의학회지

BACKGROUND: In unrelated hematopoietic stem cell transplantation, the accuracy of HLA registry typing (RT) of donors is important for timely search and coordination of HLA-matched donors. We analyzed discrepancies between HLA RT and confirmatory typing (CT) results of stem cell donors in Korean and foreign registries. METHODS: We analyzed the HLA typing results of 834 donors for whom CT was performed at Seoul National University Hospital between April 1997 and March 2010. For CT, DNA typing was used in majority of the cases and HLA-A and HLA-B serological typing was used in some early cases. The discrepancies between the typing results were analyzed at the serological/generic level. RESULTS: The overall discrepancy rate (RT error rate) was 3.2%, and the rate was similar in the Korean and foreign registries. The discrepancy rates in the Korean and foreign registries were more than 10% in the 1997-2001 searches, but decreased to less than 3% in the 2002-2010 searches. Analysis of 19 cases of RT errors in the Korean registry revealed 3 cases of sample switchover errors and 16 cases of typing errors in one of the HLA-A, HLA-B, or HLA-DR loci. The RT error rate in Japan Marrow Donor Program was lower than those in other foreign registries. CONCLUSIONS: The error rate of HLA RT results of unrelated stem cell donors in the Korean registry was similar to those in the foreign registries, and has decreased in the recent searches following the change in the typing method from serological to DNA typing.

Allelic and Haplotypic Diversity of HLA-A, -B, -C, and -DRB1 Genes in Koreans Defined by High-resolution DNA Typing / 대한진단검사의학회지

BACKGROUND: In this study, we used high-resolution DNA typing to investigate the distribution of HLA alleles and haplotypes in Koreans. METHODS: HLA-A, -B, -C, and -DRB1 alleles were genotyped at the allelic (4-digit) level in 474 healthy Koreans. HLA genotyping was performed in two steps. Initially, serologic typing or generic-level DNA typing was performed using the PCR-sequence-specific oligonucleotide method, and then allelic DNA typing (exons 2 and 3 for class I, and exon 2 for DRB1) was carried out using the PCR-single-strand conformation polymorphism method or sequence-based typing. HLA allele and haplotype frequencies and linkage disequilibrium values were calculated by the maximum likelihood method using a computer program developed for the 11th International Histocompatibility Workshop. RESULTS: A total of 21 HLA-A, 40 HLA-B, 22 HLA-C, and 29 HLA-DRB1 alleles were found in Koreans. The most frequent alleles in each locus with frequencies of > or =10% were, in decreasing order of frequency, as follows: A*24:02, A*02:01, A*33:03; B*51:01; C*01:02, C*03:03; and DRB1*09:01. The numbers of two- and three-locus haplotypes with frequencies of >0.5% were as follows: 44 A-C, 42 B-C, 51 A-B, 52 B-DRB1, 42 A-C-B, and 34 A-B-DRB1. Thirteen A-B-DRB1 haplotypes with frequencies of > or =1.0% comprised 26.0% of the total haplotypes. The six most common haplotypes were as follows: A*33:03-B*44:03-DRB1*13:02 (3.7%), A*33:03-B*44:03-DRB1*07:01 (3.0%), A*33:03-B*58:01-DRB1*13:02 (3.0%), A*24:02-B*07:02-DRB1*01:01 (2.8%), A*30:01-B*13:02-DRB1*07:01 (2.3%), and A*11:01-B*15:01-DRB1*04:06 (2.2%). CONCLUSIONS: The information obtained in this study can be used as basic data for Koreans in the fields of organ transplantation, disease association, and anthropologic studies.

An Evaluation of Platelet Transfusion Response Using HLA Crossmatch-compatible Donors in Patients with Platelet Refractoriness / 대한진단검사의학회지

BACKGROUND: Majority of immune-mediated platelet refractoriness is caused by HLA alloimmunization and can be effectively managed by HLA-matched platelet transfusions. However, HLA class I-typed large-sized donor registry has not been well established in Korea. We evaluated the effectiveness of platelet transfusion using HLA crossmatch-compatible donors without HLA typing. METHODS: Sixteen patients showing platelet refractoriness to random donor platelets (1 hr corrected count increment [CCI] 60%) were crossmatched with 78 platelet apheresis-eligible donors using National Institute of Health (NIH) and anti-human globulin (AHG) lymphocytotoxicity methods. NIH negative/AHG negative and NIH negative/AHG positive donors were selected as best and second choice donors, respectively. RESULTS: Eleven patients (11/16, 69%) could find NIH-crossmatch negative donors and 27 donors (27/78, 35%) belonged to the best donors. To 8 patients, 32 apheresis platelet products from 19 donors were transfused. The mean 1 hr and 24 hr CCI values from the best donors were significantly higher than those from random donors (17,893 vs 2,358, P=0.003; 8,292 vs -614, P<0.001), whereas such differences were not observed for those from the second choice donors. Platelet storage time was inversely correlated with CCI values and platelets stored < or =10 hr after collection gave significantly higher CCI values. Neither ABO match nor donor status (related vs unrelated) affected the transfusion effectiveness. CONCLUSIONS: Effective post-transfusion platelet increment using HLA crossmatch-compatible donors was attained in patients with platelet refractoriness due to HLA antibodies, and this method can be used effectively where HLA-typed platelet donor registry is not available.

HLA-A, -B, -DR Allele Frequencies and Haplotypic Associations in Koreans Defined by Generic-Level DNA Typing / 대한진단검사의학회지

BACKGROUND: HLA allele and haplotype distribution varies widely among different ethnic groups. For organ transplantation, anthropology and disease association studies, reliable data on the HLA distribution in each ethnic group is needed. In recent years, more accurate DNA typing methods are increasingly used in place of the serologic typing method. METHODS: We examined HLA-A, -B, and -DR alleles at the generic (serologic) level in 1, 600 Koreans registered for the Korea Marrow Donor Program (KMDP) using the PCR-sequence specific oligonucleotide (SSO) method (Dynal RELI(TM) kit). Allele and haplotype frequencies were estimated by the maximum likelihood method using the computer program developed for the 11th International Histocompatibility Workshop. RESULTS: HLA alleles found in Koreans were 13 in A, 31 in B, and 13 in DR locus. Most frequent alleles with frequencies > or =10% were: A2, A24, A33, A11; B62, B44, B51; DR4, DR15, DR13, and DR8 in each locus in decreasing order of frequency. Subtype frequencies of B61 and B75 serologic specificities were identified: B*4002 (51.1%), *4003 (7.6%) and *4006 (41.3%) for B61, and B*1502 (9.5%) and *1511 (90.5%) for B75. Two-locus haplotypes with frequencies> or =0.1% were presented (99 A-B, 115 B-DR), among which those with frequencies> or =1.0% showing significant positive linkage disequilibrium (P or =0.1% were identified in Koreans, among which 38 haplotypes showed frequencies> or =0.5%. We compared the results of this study with those of our previous study of serologically typed HLA-A, -B and DNA typed HLA-DR in 2, 000 Koreans. Results from the two studies were similar, but blank frequencies were decreased to 0% for HLA-A, -B, and -DR locus compared with the frequencies of 0.3-0.8% in the previous study (A, 0.3%; B, 0.8%; DR, 0.3%) and all of the serologic splits could be assigned in this study. CONCLUSIONS: In this study, we provided the allele and haplotype frequencies of HLA-A, -B, and -DR in Koreans defined by a DNA typing method, which can be used as basic data on Koreans for organ transplantation and disease association studies.

Analysis of Ambiguities of HLA-B Typing using the Dynal RELI(TM) SSO HLA-B Test and Development of an`Interpretation Program for Koreans' / 대한진단검사의학회지

BACKGROUND: In recent years, DNA typing has been increasingly used in HLA-A and B typing, and commercial kits based on the PCR-SSO method are most commonly used in Korea. However, SSO typing kits show ambiguities to some extent in the generic level typing of HLA-B alleles. We analyzed the ambiguities in the Dynal RELI(TM) SSO HLA-B test (Dynal B test) with confirmatory typing results, and developed and evaluated the accuracy and efficacy of an `Interpretation Program for Koreans'. METHODS: A total of 2, 169 Korean marrow donor registry samples were typed for HLA-B alleles using the Dynal B test (56 probes) and all of the 222 cases showing ambiguities were subjected to confirmatory typing. We have developed an `Interpretation Program for Koreans' for the Dynal B test on the basis of the allele frequencies of Korean, Japanese and Asian populations. The samples showing ambiguities in the Dynal B test were interpreted using the `Interpretation Program for Koreans' and the results were compared with confirmatory typing results. RESULTS: The Dynal B test showed 10.2% (222/2, 169) of ambiguities and these ambiguities were classified into 47 different band patterns. These ambiguity patterns were interpreted using the `Interpretation Program for Koreans', which showed ambiguities in 14 band patterns and 3.4% (73/2, 169) of the total samples. Among these ambiguities, 4 band patterns (55 samples) arose from those alleles which are not found in Koreans and rarely found in Japanese or Asians (B*1522, *3521, *7802). Thus, excluding these rarities, only less than 1% (18/2, 169) of samples resulted in ambiguities, and most (16/18) of these were B55 vs. B56 ambiguities. The results from the `Interpretation Program for Koreans' were fully concordant with the confirmatory typing results. CONCLUSIONS: The Dynal B test showed around 10% ambiguities and the `Interpretation Program for Koreans' showed 3.4% of ambiguities. Excluding the ambiguities with extremely low probabilities arising from rare alleles in Japanese or Asians, actually >S99% of the samples could be typed accurately using the program without additional confirmatory tests.