Routine Tissue Typing Research Articles

Identification of new HLA-C alleles by polymerase chain reaction-sequence specific oligonucleotide typing: Cw*0314 and Cw*1511*.

In this article, we report two new human leukocyte antigen-C (HLA-C) alleles, HLA-Cw*0314 and Cw*1511, which were identified during routine tissue typing of donors for the Australian Bone Marrow Donor Registry and Australian Cord Blood Bank. HLA-Cw*0314 shows six codon changes in exon 3 compared to Cw*030401 and shares some sequence homology with Cw*07 alleles. Cw*1511 has two nucleotide changes compared with Cw*150201 in exon 2, both resulting in amino acid changes in the protein sequence.

Provision of Epstein-Barr virus-transformed B-cell lines in a routine tissue typing laboratory: practicalities and applications.

A bank of Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (BCLs) has been established in this laboratory over the last 7 years. Novel, rare and unusual HLA phenotypes were highlighted during routine clinical testing and during typing of volunteer haematopoietic stem cell donors for the Welsh Bone Marrow Donor Registry. The BCLs are routinely used as laboratory reagents, as a source of DNA for reference purposes and for research work. Consenting donors are tested for hepatitis B surface antigens (HBsAg), human immunodeficiency virus (HIV) and hepatitis C virus (HCV) before their B-lymphocytes are transformed. Strict bio-identity checks are performed before and after transformation and after each BCL expansion. Each BCL is tested regularly for mycoplasma contamination. A total of 230 blood samples were transformed. One hundred and fifty-nine sterile samples produced 157 BCLs (98.7% success), while 71 non-sterile samples produced 50 BCLs (70.4% success), giving an overall success rate of 90.0%. Fifteen of the transformation failures have since been repeated successfully. Factors contributing to the high success rate and reasons for the 23 failed transformations are discussed. The successful development and use of pools of BCLs for HLA antibody screening by flow cytometry are described. Whilst certain training and health and safety issues require close attention, it is clear that EBV transformation of B lymphocytes and/or the use of BCLs is feasible in a routine tissue typing laboratory and that BCLs are a valuable resource. Careful adherence to the methods and procedures detailed here should virtually guarantee successful transformation (98.7%) from good quality, sterile whole blood samples.

Bone marrow transplantation with unrelated donors: what is the probability of identifying an HLA-A/B/Cw/DRB1/B3/B5/DQB1-matched donor?

Patients transplanted with marrow from an HLA-ABDR serologically matched unrelated donor suffer from more post-transplant complications than those who are transplanted with marrow from an HLA-identical sibling. This is most likely due to either HLA-ABDR incompatibilities not resolved by standard techniques and/or HLA polymorphisms not tested for by routine tissue typing (HLA-Cw,-DQ). By resolving these incompatibilities by molecular techniques combined with the in vitro cytotoxic T lymphocyte precursor frequency (CTLpf) test, we have shown that a high degree of HLA compatibility is associated with increased patient survival. However, higher requirements for HLA matching decrease the number of available donors. We have estimated the probability of finding an HLA-A/B/Cw/DRB1/DRB3/DRB5/DQB1 compatible donor based on 104 consecutive unrelated bone marrow donor searches initiated between January 1995 and December 1997, with December 1998 as the endpoint. For 96 patients (92.3%), one or more ABDR-identical donors were listed in the Bone Marrow Donor Worldwide Registry (BMDW). After contacting the registries, we obtained at least one (mean, 5.36; range, 1-20; total, 461) blood sample for 86 patients. A highly compatible donor was identified for 33/86 patients (38.4%), after testing an average number of 4.5 donors/patients (range, 1-13). However, by accepting an HLA-DRB3 or -DQB1 or -Cw incompatibility, this number would be as high as 68.6%. Approximately half of the patients (n = 40) for whom a search had been initiated have been transplanted: 22 patients with a perfectly matched donor, 15 patients with an HLA-DRB3 or -DQB1 or -Cw mismatch and three with other mismatches. The average time needed to identify the most compatible donor was 4 months. Extremely long searches seemed to be less useful, because after testing the first seven, a more compatible donor was seldom found. These results show that even when requirements for compatibility are high, the chances of finding a donor remain considerably low.

High Resolution HLA Matching Associated With Decreased Mortality After Unrelated Bone Marrow Transplantation

As compared with related HLA-identical sibling donors, bone marrow transplantation (BMT) with phenotypically HLA ABDR-compatible unrelated donors is associated with increased mortality. This may be due to hidden HLA incompatibilities not detected by conventional typing. We have analyzed 44 unrelated patient-donor pairs who were matched for HLA-A, -B, and -DR by routine tissue typing. Our comprehensive HLA typing approach consisted of serology, cytotoxic T-cell precursor (CTLp) tests, T-cell cloning, oligotyping, and DNA sequencing. Using these techniques, we identified numerous HLA allele mismatches not detected by the previously applied routine typing. Twenty-four patient-donor pairs were highly matched and had a low CTLp frequency, whereas the remaining 20 pairs were allele-mismatched for HLA-A, -B, -C, -DR, -DQ antigens and/or had a positive result of the CTLp test. Patient and donor age, diagnosis, and treatment did not differ significantly between the matched and mismatched transplants. The probability for severe acute graft-versus-host disease grades III-IV was 21% in the matched and 47% in the mismatched patients (P = .0464). Transplant-related mortality was 21% and 57% (P = .0072) and actuarial patient survival rates at 3 years were 61% and 13% (P = .0005). We conclude that both HLA class I and class II allele mismatches between unrelated phenotypically ABDR-compatible patient-donor pairs are frequent and associated with increased incidence of posttransplant complications.

P441 - High resolution HLA matching associated with decreased mortality after unrelated bone marrow transplantation

As compared with related HLA-identical sibling donors, bone marrow transplantation (BMT) with phenotypically HLA ABDR-compatible unrelated donors is associated with increased mortalii. This may be due to hidden HLA incompatibiliiies not detected by conventional typing. We have analyzed 44 unrelated patient-donor pairs who were matched for HLA-A, -B, and -DR by routine tissue typing. Our comprehensive HLA typing approach consisted of serology, cytotoxic T-cell precursor (CTLp) tests, T-cell cloning, oligotyping, and DNA sequencing. Using these techniques, we identified numerous HLA allele mismatches not detected by the previously applied routine typing. Twenty-four patientdonor pairs were highly matched and had a low CTLp frequency, whereas the remaining 20 pairs were allele-mis-

Applicability of HLA‐DQB oligonucleotide typing for the TA10 and 2B3 specificities in routine HLA typing

Our group recently developed oligonucleotide probes associated to the TA10 and 2B3 specificities (1). Unambiguous typings were observed in a panel of homozygous typing cells and a family, using 32P-end labeled probes and PCR-amplified DNA (DQB exon 2). To investigate whether these TA10 and 2B3 associated oligonucleotides could be used in routine HLA-typing we extended the study to a panel of healthy, unrelated individuals. When TA10 and 2B3 typings by oligonucleotides were compared with those obtained in routine serology a complete correlation was observed for TA10. A discrepancy was seen for 2B3 typing in material obtained from DQw5- (and possibly DQw4)-positive individuals which could be explained by the CYNAP (cytotoxicity-negative, absorption-positive) phenomenon, using the IIB3 monoclonal antibody in routine tissue typing. Our results suggest that HLA-DQB oligonucleotide typing for TA10 and 2B3 is an accurate and reliable method and can be used effectively in routine HLA typing.

A simple and rapid method for the detection of lymphocytotoxic antibodies using cell panels frozen on Terasaki plates.

A 40 cell panel of lymphocytes selected for HLA-A,B and DR antigens, frozen in Terasaki microtest trays can be used routinely to identify the presence of specific HLA antibodies within two hours. Blind testing using well defined HLA typing sera showed that specificities could be identified to a high degree of significance using this method. The method has proved successful for screening for T cell, including HLA-A,B and B cell, including HLA-DR antibodies. The method is particularly useful for the routine tissue typing laboratory as the frozen panel can be used without the need for complicated and time-consuming cell washing procedures which have been the downfall of previously published methods.

Two monoclonal antibodies detecting allotypic determinants of HLA-A.

Three mouse hybridomas producing cytotoxic antibodies against HLA were established. By standard microcytotoxicity test against panels of normal controls, the antigen defined by MA-9 antibody (IgM) showed a good correlation with HLA-A9 alloantigen detected by conventional typing alloantisera (r = 1.0). Family studies also showed that MA-9 determinant segregated with HLA-A9. MA-10 antibody (IgM) reacted with all HLA-A10 positive lymphocyte donors and cross-reacted with two thirds of HLA-AW33 positive donors. Ml-1 antibody (IgG2a) reacted with all the panel cells tested and immunoprecipitated a molecule of 43,000 daltons from Nonidet P-40 lysates of 3H-glucosamine-labelled cells. The results showed that MA-9 and MA-10 antibodies can be used as routine tissue typing reagents.