Search

Human leukocyte antigen (HLA) incompatibilities are the most important immunological barriers to bone marrow transplant success when using unrelated donors. Until recently, standards for donor selection included serological methods for HLA class I antigens and DNA-based typing for HLA class II alleles. In our center cytotoxic T-lymphocyte precursor (CTLp) assays have been an integrated part of the search selection procedure as well. More recently, DNA-based typing for HLA class I became available. This allowed us to determine the correlation of CTLp frequencies directed against incompatibilities at the HLA-A, -B, and -C locus in 211 donor-recipient pairs. HLA class I incompatibilities are significantly (p < 0.001) associated with high CTLp frequencies. Exceptions did occur, high CTLp frequencies are seen in 14% of the HLA-A, -B, and -C allele matched pairs, whereas in 7% of the pairs mismatched for HLA-A or -B a low CTLp frequency occurred. The successful outcome of transplants performed in the latter cases suggest that the CTLp test can be used as a tool to detect permissible mismatches when no fully matched donor is available. The influence of HLA-C mismatches on the CTLp outcome was less clear. Although in the majority of mismatched pairs (64%) the CTLp frequency was high, in 36% of the pairs the CTLp frequency was low. Analysis of HLA amino acid sequences was performed on the HLA-C allele mismatched group. An amino acid difference was always found at five polymorphic positions 97, 99, 113, 114, and 116 situated at the peptide binding groove in the high CTLp frequency group, whereas in the low CTLp frequency group this was observed in only 9 of 17 combinations (p < 0.001). However, this is mainly due to Cw*0303-0304 mismatches. In conclusion, although there is a highly significant correlation between the outcome of the CTLp frequency test and HLA allele class I typing, exceptions occur. It is unclear whether they are all clinically relevant but they certainly provide additional insight in allograft recognition.

Organ exchange organizations such as Eurotransplant allocate organs on the basis of histocompatibility testing results. For this reason it is essential that all data reported by the affiliated laboratories are accurate and reliable. The Eurotransplant Reference Laboratory (ETRL) organizes proficiency testing schemes for the tissue-typing centers of the respective renal transplantation units participating in Eurotransplant. Each year, the ETRL sends out 8 peripheral blood samples of healthy blood donors for serological typing and crossmatching, 16 sera to screen for the presence and definition of HLA alloantibodies and 20 DNA samples for molecular typing to the 49 participating centers. The results are collected centrally and reported back to the participants in an open way. These exercises show that the quality of HLA typing, screening and crossmatching improved significantly over the years. In particular, the introduction of molecular typing for HLA-DR resulted in an increase of reliability. The clinical relevance of a reliable HLA typing was demonstrated in a selected group of transplants, the zero HLA-A,-B,-DR- mismatched group. After retyping the donors, 146 of the 3,458 matched transplants appeared to have a mismatch and those transplants had a significantly lower graft survival rate. A continuing problem, however, is the result of screening for panel reactive antibodies (PRA), where the percentage PRA reported for each serum varies significantly from center to center. The results indicate that the use of a PRA value for classification of patients and allocation of organs should be revisited.

High resolution typing for HLA-DR4 is required to identify the individual subtypes. In this study a panel of DR4-positive samples was typed by both sequencing-based typing (SBT) and oligohybridization (PCR sequence-specific oligonucleotide; PCR-SSO). SBT reveals the highest resolution; moreover, ambiguous DRB1*04 allelic combinations can be resolved by a selective amplification of the individual alleles and subsequent sequencing. An extended DR4-specific PCR-SSO makes high resolution typing possible; however, an additional protocol is required to resolve ambiguities.

A novel HLA-DR typing method was applied using PCR-amplified fragments and biotin-labeled oligonucleotides (PCR-biotin-SSO). The PCR-biotin-SSO method can be used efficiently to perform HLA-DR typing for a large number of individuals when time is not the limiting factor. The reliability of HLA typing of cadaveric organ donors is of vital importance for organ exchange organizations such as ET. Due to lack of time, these typings are usually performed by the complement-dependent cytotoxicity. The individual donor center typings are immediately reported to ET, where the recipient selection procedure is started. DNA isolated from donor spleen material, sent to the ETRL for retyping purposes, was subjected to PCR-biotin-SSO typing. The results were compared with the serological HLA-DR typings as reported to ET. The analysis of 1052 donor samples for the broad DR1-DR10 antigens revealed a concordance rate of over 90% between the donor center and the ETRL. The majority of the discrepancies involved specificities of the HLA-DR5, DR6, and DR8 cross-reacting group, with DR6 as the predominant discordant specificity. The results indicate (a) that PCR-biotin-SSO is a reliable technique for DNA-based HLA-DR typing and (b) that HLA-DR serology is still a useful technique when time is limited, such as for cadaveric donor typing.