HLA class I DNA typing of 215 "HLA-A, -B, -DR zero mismatched" kidney transplants

DNA typing for HLA class II improves the typing quality and this was shown previously to be relevant for kidney graft survival. In this project we addressed the question whether molecular typing for HLA class I also increases the efficacy of HLA matching in kidney transplantation. 215 HLA-A,-B,-DR zero-mismatched donor/recipient pairs as defined by serological typing were selected. Retrospective HLA-A and HLA-B typing was performed both by the PCR-SSP and the PCR-SSOP method. DNA typing for HLA-A revealed discrepant results to serology in 5.7% of the donors and 2.8% of the recipients. HLA-B typing discrepancies were found in 6.6% of the donors and 5.6% of the recipients. 10.4% of the donors and 6.5% of the recipients showed either an HLA-A or an HLA-B discrepancy. Nearly one-third of the HLA-A discrepancies affected A19 splits. The most common reason for HLA-A discrepancies was the erroneous assignment of serological blanks, whereas HLA-B errors were caused mainly by the assignment of incorrect specificities. DNA typing allowed the definition of HLA-A and -B split specificities in all 118 "splitable" cases for which only broad specificities were reported based on serological typing. A total of 183 DNA class I compatible transplants had a 15% higher one-year graft survival rate than 32 transplants for which DNA typing revealed a class I incompatibility.     

Long-term follow-up in London Transplant Group recipients of cadaver renal allografts. The influence of HLA matching on transplant outcome

The London Transplant Group followed 1341 patients with cadaver renal transplants, none of whom received cyclosporine, for six months to 14 years to determine the effect on graft survival of matching donor and recipient for HLA Class I antigens (HLA-A, -B, and -C) and Class II antigens (HLA-DR, -MT, and -DQ). Long-term graft survival was greatly improved by matching for HLA Class I antigens, especially HLA-B. Transplants that could not be matched for both B-locus antigens but were completely matched for Bw4/Bw6 also did very well. In addition, since 1978, excellent results have been obtained with HLA-DR and -DRw52/53 (HLA-MT) matching, but not with HLA-DQ matching. Multivariate analysis using the Cox regression model confirmed that combination Class I and Class II matching produced significant improvements in graft survival. Thus, transplants matched for HLA-DR plus HLA-B and those matched for HLA-MT plus HLA-B had excellent results--even better than those reported with cyclosporine treatment. Double HLA-MT incompatibilities yielded the poorest results. We conclude that this approach of combining the broad and narrow specificities of Class I and II is extremely practical and that appropriate matching of tissue types is clinically important.     

Validation of DNA-based HLA-A and HLA-B testing of volunteers for a bone marrow registry through parallel testing with serology

A total of 42,160 individuals were typed for HLA-A and HLA-B by both serology and PCR-based typing. The HLA assignments included all of the known serological equivalents. The majority of the individuals (99.9%) were from U.S. minority population groups. The serologic typing was performed between 1993 and 1997 at the time of recruitment for the National Bone Marrow Program (NMDP) registry. The polymerase chain reaction (PCR)-based typing was carried out in two phases. In phase I, DNA typing was performed by PCR using sequence-specific oligonucleotide probes (PCR-SSOP) or PCR using sequence-specific primers (PCR-SSP) without knowledge of the serologic assignments. Discrepancies were identified between the serologic and DNA assignments in 24% of the volunteers (8% of volunteers differed for only HLA-A assignments, 13% for HLA-B, and 3% for both HLA-A and -B) and a potential explanation was assigned each discrepant serology/DNA pair. In phase II, a random sampling scheme was used to select a statistically significant number of individuals for repeat DNA typing from each of these categories. The categories included antigens missed by serology, nonexpressed (null) alleles, PCR amplification failures, misassignment of antigens and nomenclature issues. Only a single individual was found to carry a null allele. DNA-based testing correctly typed nearly 99% of the donors at HLA-A, more than 98% at HLA-B, and more than 97% at both HLA-A and -B validating this methodology for registry typing.     

Comparison of HLA-A antigen typing by serology with two polymerase chain reaction based DNA typing methods: implications for proficiency testing

Serology has been routinely used for class I HLA typing for the selection of donors for allotransplantation. However, serology is not adequate for the assignment of all class I specificities especially when testing non-Caucasians subjects and it is necessary to adopt new strategies for routine testing. At the present time the extent of incorrect serologic HLA-A assignments in clinical testing is not known. The polymerase chain reaction (PCR) based techniques have become useful standard clinical typing methods of HLA class II alleles but most laboratories still use serology for class I typing. In this report we have compared two PCR based techniques, PCR amplification with sequencespecific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP), for the assignment of HLA-A specificities in 56 blood samples from patients and families serologically typed for HLA-A. This side-by-side comparison of PCR methods showed 100% correlation between them. However, serology showed 7.1% misassignments and, in an additional panel of 19 cells where serology produced equivocal results, the PCR-SSP and SSOP methods identified the correct HLA-A specificity. Our results emphasize the need to complement routine serologic testing of HLA specificities with a small number of primers designed to test HLA-A34, A36, A43, A66, A74 and A80, that are not detected with high precision by serology. We concluded that the PCR-SSP and -SSOP methods can be used in routine HLA-A typing of patients and donors for transplantation with a greater precision than serology.     

Heterogeneity of systemic lupus erythematosus elucidated by cluster analysis. The influence of HLA

For 75 patients with systemic lupus erythematosus (SLE), 39 laboratory and clinical characteristics, including HLA-A, B, C and DR typing, were analysed using a cluster analysis technique. Three groups were identified. Group I (46 patients) was characterized by infrequently severe disease, good response to therapy and infrequent multisystem involvement. Group II (24 patients) was characterized by a severe course of disease (although the tendency to remit after therapy was not unusual), and frequently, renal involvement and pericarditis. Group III (5 patients) was characterized by more severe renal disease. Of the 75 patients studied, 38.7% possessed HLA-DR3, compared to 17.4% of controls. Group I patients did not differ from controls but 80% of Group II patients and 4/5 Group III patients had DR3. Cluster analysis identifies subsets of SLE patients who show marked differences in disease course and severity, correlated with possession of the HLA B8, DR3 phenotype.     

HLA-A antigen incompatibility and HLA-B antigen compatibility associated with enhanced kidney allograft survival in blood-transfused patients

The combined effect on 2-year primary kidney allograft survival of pretransplant blood transfusion, HLA-DR matching, HLA-A matching, HLA-B matching and other factors was analyzed in 116 azathioprine- and prednisolone-treated patients. The results show an hitherto unreported, statistically significant association between incompatibility for HLA-A antigens and enhanced graft survival in transfused recipients of transplants mismatched for HLA-DR (p = 0.005) and for HLA-B (p = 0.008). As expected, compatibility for HLA-B antigens was significantly associated with increased graft survival in transfused recipients of both HLA-DR compatible (p = 0.004) and HLA-DR incompatible (p = 0.01) grafts. If confirmed, the findings foreshadow new aspects of HLA matching in kidney transplantation and might explain the beneficial effect of blood transfusion on renal allograft survival.     

Comparison of serological and molecular typing for HLA-A and -B on cord blood lymphocytes

Abstract: HLA class I typing by standard microcytotoxicity testing has been unsatisfactory for 14.5% of 1644 cord blood samples. In this study, we evaluated the capacity of PCR-SSP in solving problems in HLA-A,B typing with serological methods. With this aim we have compared serology with PCR-SSP in 100 cord blood samples with doubtful or unreliable HLA-A,B typing. PCR-SSP was successful in amplifying HLA-A,B alleles in all 100 cord blood samples. Forty-six typings gave discrepant results with the 2 methods (serology and PCR-SSP). Typings were considered discrepant also in the case of inability to define a split. For 19 specimens, no serological conclusion was drawn due to high mortality of the cell suspension, while PCR-SSP allowed the definition of a clear typing. In 6 cases it was necessary to infer information from serology to define the current typing. Finally, in 3 other cases it was impossible to exclude or attribute the antigen/allele B67 or B4802. PCR-SSP for HLA-A,B can improve the overall reliability of HLA-A,B typing requiring a small amount of blood although, with the set of sequence specific primers adopted, a number of alleles are still poorly defined.     

Error rate for HLA-B antigen assignment by serology: implications for proficiency testing and utilization of DNA-based typing methods

Until recently, the majority of HLA class I typing has been performed by serology. Expensive commercial typing trays are frequently used for testing non-Caucasian subjects and new strategies using DNA-based methods have been adopted for improving clinical histocompatibility testing results and adapted as supplements in proficiency testing. A double-blind comparison of the typing of HLA-B specificities in 40 samples was carried out between serology and two polymerase chain reaction (PCR) methods, PCR amplification with sequence-specific primers (PCR-SSP) and PCR amplification and subsequent hybridization with sequence-specific oligonucleotide probes (PCR-SSOP). The results demonstrated 22.5% misassignments of HLA-B antigens by serology. There was complete concordance between the results obtained with the two PCR based typing methods. A second panel of 20 donor samples with incomplete or ambiguous serologic results was analyzed by PCR-SSP and SSOP. Both PCR methods identified correctly the HLA-B antigens. Our results suggest that more accurate typing results can be achieved by complementing serologic testing with DNA-based typing techniques. The level of resolution for HLA-B antigen assignment can be obtained by this combination of serology and limited DNA-based typing is equivalent to the HLA-B specificities defined by the WHO-HLA Committee. This level of resolution cannot routinely be achieved in clinical histocompatibility testing or in proficiency testing using serologic reagents only.     

Adverse effect of blood transfusion on the long-term outcome of kidney transplantation

In 116 patients treated with azathioprine and prednisolone, 2-year patient survival and primary renal allograft survival was analyzed with regard to the effects of pretransplant blood transfusion and of matching for HLA-A, HLA-B, and HLA-DR. Two years after transplantation, patient survival was significantly lower in transfused patients than in non-transfused patients (81 vs. 97%, p less than 0.05). A detrimental effect of blood transfusion on graft survival was found in conjunction with HLA-A compatibility (p = 0.01 overall, and p = 0.003 for HLA-B mismatched transplants). Among transfused patients, enhanced graft survival was associated with incompatibility for HLA-A (p = 0.02 overall, and p = 0.008 for HLA-B mismatched transplants), and with compatibility for HLA-B (p = 0.0006 overall, and p = 0.005 for HLA-A mismatched transplants). The present results indicate that blood transfusion may have detrimental effect on patient survival.     

HETEROGENEITY OF SYSTEMIC LUPUS ERYTHEMATOSUS ELUCIDATED BY CLUSTER ANALYSIS

For 75 patients with systemic lupus erythematosus (SLE), 39 laboratory and clinical characteristics, including HLA-A, B, C and DR typing, were analysed using a cluster analysis technique. Three groups were identified. Group I (46 patients) was characterized by infrequently severe disease, good response to therapy and infrequent multisystem involvement. Group I1 (24 patients) was characterized by a severe course of disease (although the tendency to remit after therapy was not unusual), and, frequently, renal involvement and pericarditis. Group 111 (5 patients) was characterized by more severe renal disease. Of the 75 patients studied, 38.7% possessed HLA-DR3, compared to 17.4% of controls. Group I patients did not differ from controls but 80% of Group II patients and 4/5 Group III patients had DR3. Cluster analysis identifies subsets of SLE patients who show marked differences in disease course and severity, correlated with possession of the HLA B8, DR3 phenotype.     

Comprehensive, serologically equivalent DNA typing for HLA-B by PCR using sequence-specific primers (PCR-SSP)

Abstract: Polymorphic products of HLA class I genes from the human major histocompatibility complex (MHC) are traditionally assigned by serology with additional heterogeneity detectable using one-dimensional isoelectric focusing (1D-IEF). With the increased availability of HLA class I DNA sequence information it has become feasible to genotype for class I by polymerase chain reaction utilising sequence-specific primers (PCR-SSP). We describe here a comprehensive HLA-B PCR-SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one-step PCR reactions. In addition, four new unsequenced variants have been identified. DNA samples from 57 International Histocompatibility Workshop reference cell lines and 160 control individuals have been typed by the HLA-B PCR-SSP technique. 3/57 cell line types and 12/160 normal control individuals types were discrepant with the reported serological types. The SSP system has been designed to be higher resolution than serology but is not a complete allele-specific PCR although many single alleles can be identified. The system is entirely complementary to previous published PCR-SSP systems for HLA-Class II and HLA-Class I in that the same PCR conditions and controls are used which allows us to do one step PCR-SSP for all relevant HLA loci in under 3 hours in a system suitable for the typing of cadaver donors.     

Effect of HLA class I or class II incompatibility in pediatric marrow transplantation from unrelated and related donors

The degree of histoincompatibility that can be tolerated, and the relative importance of matching at individual HLA class I and class II locus in bone marrow transplantation (BMT) has not been established. We hypothesized that matching for HLA-DR may not be more important than matching for HLA-A or HLA-B in selection of a donor for successful BMT. We retrospectively analyzed the outcomes of 248 consecutive pediatric patients who received allogeneic BMT from related donors (RD, n = 119) or unrelated donors (URD, n = 129). HLA-A and HLA-B were serologically matched, and HLA-DRB1 were identical by DNA typing in 69% of donor-recipient pairs. Most patients (89%) had hematologic malignancies; the rest had aplastic anemia or a congenital disorder. One HLA-A antigen mismatch was associated with a decrease in survival (p = 0.003) and a delay in granulocyte engraftment (p = 0.02) in recipients of RD marrow; as well as a decrease in survival (p = 0.02) and the development of severe acute graft-versus-host disease (GVHD) (p = 0.03) in recipients of URD marrow. One HLA-B antigen mismatch was associated with a decrease in the survival (p = 0.05) and the development of severe GVHD (p = 0.0007) in recipients of RD marrow. One HLA-DRB1 allele mismatch was associated only with a decrease in the survival (p = 0.0003) of recipients of RD marrow. Results of this study suggest that disparity in HLA-A and HLA-B antigens may not be better tolerated than disparity in HLA-DR allele in allogeneic BMT. Further studies are warranted to confirm our results.     

Comparison of speedy PCR-ssp method and serological typing of HLA-A24 for Japanese cancer patients

Human leukocyte antigen (HLA) typing is essential to carry out HLA-class I restricted antigenic peptide-based cancer immunotherapy. To establish a one-step polymerase chain reaction-sequence-specific primer (PCR-SSP) method, we designed two novel HLA-A24-specific primer sets and determined the optimal conditions for specific amplification. Then, we performed HLA-A24 typing of two healthy donors' and 17 cancer patients' peripheral blood with serological typing and PCR-SSP typing. Eleven of the 19 cases were determined HLA-A24-positive by the PCR-SSP method precisely; however, five cases showed false positive with serological analysis. Thus, for HLA-A24 typing in the Japanese population, the PCR-SSP method is faster and more accurate than serological typing.     

HLA compatibility and organ transplant survival. Collaborative Transplant Study

The influence of HLA compatibility on organ transplant survival was analyzed in more than 150,000 recipients transplanted from 1987 to 1997 at transplant centers participating in the Collaborative Transplant Study. A statistically highly significant effect of HLA matching on graft and patient survival rates was found in the analysis of kidney transplants (P < 0.0001). Ten years after transplantation, the graft survival rate of first cadaver kidney transplants with a complete mismatch (6 HLA-A+B+DR mismatches) was 17% lower than that of grafts with no mismatch. During the first post-transplant year, the class II HLA-DR locus had a stronger impact than the class I HLA-A and HLA-B loci. During subsequent years, however, the influence on graft survival of the three loci was found to be equivalent and additive. For optimal graft outcome, compatibility at all three HLA loci is, therefore, desirable. The excellent correlation of HLA matching observed in recipients of cadaver kidneys with very short ischemic preservation (0-6 hours) or recipients of kidneys from living unrelated donors contradicts reports that short ischemia can eliminate the influence of matching. Although HLA has a significant effect on graft outcome regardless of the state of presensitization, the matching effect is potentiated in patients with highly reactive preformed lymphocytotoxic antibodies. Among first cadaver transplant recipients with an antibody reactivity against > 50% of the test panel, the difference in graft survival at 5 years between patients with 0 or 6 mismatches reached 30%. A collaborative project, in which molecular DNA typing methods were employed, showed that the correction of serological HLA typing errors by more accurate DNA typing results in a significantly improved HLA matching effect. Moreover, matching for the class II locus HLA-DP, a locus that can be typed reliably only by DNA methods, showed a significant effect in cadaver kidney retransplants, especially in the presence of preformed lymphocytotoxic antibodies. The analysis of heart transplants showed a highly significant impact of HLA compatibility on graft outcome (P < 0.0001). This result is of particular interest because donor hearts are not allocated according to the HLA match. A biasing influence of donor organ allocation (i.e. a preferential allocation of good matches to good risk recipients) can, therefore, be excluded. In liver transplantation, neither matching for HLA class I nor HLA class II could be shown to influence transplant outcome.     

HIV高度暴露血清学指标持续阴性者的流行病学调查和HLA分型研究

目的 了解中国有偿献血人群中的HIV高度暴露血清学指标持续阴性者（highlyexposed but persistently seronegative,HEPS）流行病学和HLA分型情况.方法 采用队列研究方法对有偿献血人群进行流行病学追踪、血清流行病学调查研究.利用序列特异性引物PCR（PCR with sequencespecific primer,PCR-SSP）进行HLA分型.结果 流行病学研究发现8例HIV高度暴露血清学指标持续阴性者,HLA分型HEPS人群与HIV抗体阳性配偶比较,HLA-A24、HLA-40基因型占优势.结论 在中国有偿献血人群中存在HIV高度暴露血清学指标持续阴性者,初步分析中国HEPS人群HLA分型,结 HLA-A24、HLA-B40基因型占优势.     

HLA-AB typing by polymerase-chain reaction with sequence-specific primers: more accurate, less errors, and increased resolution compared to serological typing

Until recently, serological typing has been the primary technique used for HLA class I analysis. But because of limitations, molecular-typing techniques have replaced or supplemented the microlymphocytotoxicity test. It has been assumed that HLA class I serological typing was more accurate than serological HLA-DR typing; the latter has been shown to have 10-25% errors. But several studies have shown that HLA-AB typing was poorer than expected, and error frequencies between 5-25% were reported. This study systematically investigated the accuracy of HLA class I serological AB typing in healthy, bone-marrow registry donors, necrokidney donors, kidney-transplantation patients (on waiting lists), and haematological disorder patients. Genomic HLA class I typing, which uses polymerase-chain reaction with sequence-specific primers (PCR-SSP), gave discrepant results in 3-24% of the patients, compared to serological typings. The highest error rate (24%) was found among haematological disorder patients. Among the kidney waiting-list patients and necrokidney donors, 11% discrepancies were found. In the consecutively typed bone-marrow donors group, 3% errors were found. But among those with only one detected HLA-A specificity, 12% discrepancies were found, and among donors with only one detected HLA-B specificity, 19% errors were found. Based on these results, we recommend that patients with haematological disorders should be typed using genomic techniques. In investigations of bone-marrow registry donors and kidney patients, in which only one serological specificity is found, additional typing by genomic methods should be done.     

Association of human leucocyte antigen Class I (HLA-A and HLA-B) with chronic hepatitis C virus infection in Egyptian patients

Egypt has the highest prevalence of hepatitis C virus (HCV) in the world, ranging from 6% to 28% with an average of approximately 13.8% in the general population. It has been reported that human leucocyte antigen (HLA) alleles are associated with the outcome of HCV infection, but this associations showed ethnic and geographical differences. The objective of this study is to investigate the association between the frequencies of HLA Class I and chronic HCV infection in Egyptian patients and to find out whether there is a relation between certain HLA Class I antigens and HCV viral load, degree of fibrosis, activity and alanine aminotransferase (ALT) level. A case control study was conducted on 100 patients with chronic HCV infection and 150 healthy controls. HLA-A and HLA-B typing by complement-dependent micro-lympho-cytotoxicity assay was performed for both groups. HLA-A11 antigen was significantly increased in patients with chronic HCV infection versus controls (OR 3.98; 95% CI = 1.85-8.89; P = 0.001; and Pc = 0.021). HLA-B12, HLA-B13, HLA-B17 and HLA-B40 were higher in patients, and HLA-A32 and HLA-B14 were higher in controls, although the significance was lost after correction for multiple testing. HLA-A9 was significantly associated with low viral load (P = 0.008, Pc = 0.048). The results of this work implicate that HLA-A11 antigen may influence chronic HCV infection and may play a role in viral persistence. Different HLA Class I antigens are not associated with degree of liver fibrosis, grades of activity or level of ALT. However, HLA-A9 is associated with low HCV viral load in chronic HCV Egyptian patients.     

Allele assignment for HLA-A, -B, and -C genes to the Tenth International Histocompatibility Workshop cell lines

Development of DNA typing for Class I HLA alleles has lagged behind that of class II for a variety of technical reasons. Following the recognition of locus specific sequences in the first and the third intron, and acquiring the ability to amplify genomic DNA by intron-based PCR primer, we have devised DNA typing of class I alleles by SSOP and direct sequencing. In this study using these techniques we provide the allelic typing of HLA-A, -B, and -C genes for the B-lymphoblastoid reference cell lines from the Tenth International Histocompatibility Workshop. We also describe some common associations of the C alleles with HLA-A and HLA-B alleles.     

HLA-B*15 subtypes in the population of north-eastern Thailand.

The HLA-B*15 group is the most polymorphic HLA-B allele and so has several subtypes. These subtypes have not been defined in the population of north-eastern Thailand (NET). In a previous study, using polymerase chain reaction-sequence-specific primers (PCR-SSP), subtypes were categorized into four groups, namely: group I: HLA-B*15 (01, 04-07, 12, 14, 19, 20, 24, 25, 26N, 27, 32, 33, 34 and 35); group II: HLA-B*15 (02, 08, 11, 15, 28 and 30); group III: HLA-B*1503/4802; group IV: HLA-B*1521. Groups I and II occurred frequently (allele frequency = 8.0 and 2.5%), and thus we optimized the polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) method to identify HLA-B*15 subtypes of groups I and II. Eighty samples of DNA carrying HLA-B*15 from 300 healthy unrelated individuals were tested. B*1502 (52.5%) and B*1525 (13.8%) were the most common subtypes found in NET. They also showed strong linkage disequilibrium with HLA-Cw and heterogeneity of HLA-A, DR, DQ haplotypes. Although limited conclusions can be drawn from this study because of the small number of DNA references used, the baseline data will be useful in the selection of common HLA-B*15 alleles when subtyping for unrelated donor transplantations.     

The utility of class I HLA antibodies screening in a Tunisian obstetric department in order to select good HLA typing reagents

In order to reduce the cost of the serologic class I HLA typing, we have established a screening program for HLA antibodies among obstetric patients. Class I HLA antibodies were identified by standard microlymphocytotoxicity test using a panel of 100 lymphocytes. In the study period, carried out between January 2000 and June 2002, 817 women were tested, most of them (62%) during the last trimester of pregnancy and in the other cases after delivery. These patients, aged between 18 and 45 years, were in the majority (88%) multiparous. From the total number of 817 maternal serum samples screened, 194 specimens (23,74%) tested positive for HLA antibodies. Thirty eight HLA specificities were characterised in 110 maternal sera of which 62 contained monospecific HLA antibodies. HLA-A2 and HLA-B51(5) were the most common specificities characterised in this study. HLA alloimmunization was present since the first pregnancy. There was no statistically significant linear correlation between HLA alloimmunization and the number of pregnancies. Fifty maternal serum samples (6,11%) could be used as HLA typing reagents. Of these 50 sera, 36 had monospecific HLA antibodies. These useful antisera covered a total of 30 specificities: 8 HLA-A and 22 HLA-B. The cost of self - screening for useful antisera as estimated at dollars 62/mL, the personnel was not considered. However, the lowest cost of commercial HLA typing sera is approximately dollars 400/mL. Our study showed the utility and the net economic advantage to use maternal sera as reagents in our new HLA laboratory with limited budget.