Investigation of specimen mislabelling in paraffin-embedded tissue using a rapid, allele-specific, PCR-based HLA class II typing method.

Mislabelling of surgical specimens can seriously affect the accuracy of histopathology reports. We describe two cases in which clinically suspected mislabelling was investigated by polymerase chain reaction (PCR)-based HLA DRB and DQB tissue typing of paraffin biopsy-derived DNA, using sequence specific primers (PCR-SSP HLA typing). In the first case, two patients underwent surgery for breast carcinoma. A subcutaneous lymph node containing metastatic carcinoma was received with the breast specimen from one patient, but was clinically considered more likely to originate from the other patient who underwent breast surgery on the same day. In the second case, histological examination of retained products of conception from a young woman revealed adenocarcinoma, but a repeat curettage specimen consisted of secretory phase endometrium. In case 1, PCR-SSP HLA typing confirmed the clinical suspicion that the subcutaneous lymph node received with tissue from one patient originated from the other patient. This result converted the first patient from lymph node positive breast carcinoma to lymph node negative disease. In case 2, there was no evidence from PCR-SSP HLA typing that the endometrial samples had originated from different patients. PCR-SSP HLA typing requires fewer steps than methods based on PCR amplification followed by oligonucleotide probing (PCR-SSOP HLA typing), and relies on the amplification of shorter sequences of DNA. Therefore, this technique can produce more rapid results than PCR-SSOP HLA typing, and is ideally suited to typing partially degraded DNA derived from formalin-fixed and paraffin-embedded tissue.

HLA class I A and B typing in the clinical laboratory using DNA-based techniques.

The potential for clinical HLA class I A and B typing utilizing the polymerase chain reaction combined with sequence-specific oligonucleotide probes (PCR-SSOP) was investigated. Two hundred and ten clinical samples for the HLA-B locus and 100 clinical samples for the HLA-A locus were typed by DNA-based methods and serology. For the HLA-B locus an improved SSOP typing system was developed which involved using HLA-B specific 5' primers and two 3' primers, in separate reactions. Using a panel of 30 digoxigenin-labelled SSOPs, HLA-B types were assigned for all 210 individuals with an improvement in resolution over previously described DNA-based systems and confirming serologically assigned types in all cases except one. In addition, using a single primer pair and a panel of 16 SSOPs, 100 samples were successfully HLA-A typed by PCR-SSOP resolving ambiguous serological types, including HLA-A19 subtypes and A2 homozygosity. In 25 samples, the assigned types were also confirmed by the amplification refractory mutation system (ARMS-PCR). These results indicate that non-urgent clinical HLA-A and -B typing may be performed by PCR-SSOP with a resolution at least equal to that of serology.

UHG crossmatching. A comparison with PCR-SSO typing in the selection of HLA-DPB1-compatible bone marrow donors.

The technique of universal heteroduplex generator (UHG) crossmatching has been developed to permit comparison of HLA-DPB1 alleles between two or more individuals. It offers a rapid and simple method of screening prospective bone marrow donors for HLA-DPB1 compatibility with the recipient. We present the nucleotide sequence and describe the method of construction of the DPB1 UHG. To test its effectiveness, 56 patient-bone marrow donor pairs previously HLA-DPB1-typed by PCR-SSO probing, were tested by UHG crossmatching. In 52/56 (93%) pairs there was concordance between PCR-SSO typing and UHG crossmatching. All 32 pairs that were defined as mismatched by PCR-SSO typing were also mismatched by UHG crossmatching. We conclude that UHG crossmatching is a simple, sensitive, and cost effective method of HLA-DPB1 matching for the rapid selection of compatible bone marrow donors.

Absence of strong HLA-DR/DQ-DP linkage disequilibrium in the British and French Canadian Caucasoid populations.

HLA-DR/DQ-DP linkage disequilibrium was investigated in healthy, unrelated British (n = 150) and French Canadian (n = 67) Caucasoid subjects. HLA-DR and DQ typing was performed by Taq I DNA-RFLP analysis, while DPB1 typing was performed by PCR-SSOP. chi 2 and Fisher's exact tests were performed for all 2-locus biallelic comparisons and coefficients of linkage disequilibrium determined. In the British population, only one example of linkage disequilibrium, significant at P = 0.05 (after correction for the number of comparisons made) was seen (DPB1*0101-DRB1*0301[17(1)]). Additional associations, significant at P = 0.05 before correction for the number of comparisons were also seen, including DPB1*0401-DRB1*15, DPB1*1101-DRB1*0701(7(1)), DPB1*1701-DRB1*0701/ 2(7(2)), DPB1*0101-DQA1*0501, DPB1*0401-DQA1*0102, DPB1*0501-DQA1*0102, DPB1*0101-DQB1*0201, DPB1*0401-DQB1*0602/0603 and DPB1*1101-DQB1*0201. With one exception (DPB1*1101-DQB1*0201), none of these associations was seen in the French Canadian group. These results indicate that although more frequent than thought hitherto, HLA class II linkage disequilibrium involving DPB1 alleles is generally weak, and can differ even between different caucasoid populations. This may have implications for HLA and disease studies.

HLA DPB1 alleles and susceptibility to rheumatoid arthritis.

HLA-DPB1 genotypic and phenotypic frequencies were investigated in a series of 35 adult rheumatoid arthritis (RA) patients and 42 controls. No significant associations between DPB1 alleles and susceptibility to RA were demonstrated, although some non-significant differences in DPB1*0301 and 0401 allele frequencies between patients and controls were observed.

No association between susceptibility to multiple sclerosis and HLA-DPB1 alleles in the French Canadian population.

HLA-DPB1 typing was performed using polymerase chain reaction DNA amplification and sequence-specific oligonucleotide probing (PCR-SSOP) which permitted identification of 17 distinct DPB alleles using 15 oligonucleotide probes. The accuracy of this approach was confirmed in an initial study of 26 human B-lymphoblastoid cell lines which demonstrated close agreement between PCR-SSOP and PLT assigned types. A cohort of 47 adult French Canadians was then studied to provide an estimate of DPB1 allelic frequencies in an ethnically homogeneous population. DPB1*0401 was the most frequent phenotype (61.5%) and only DPB1*0101, 0301 and 0402 were also present at frequencies greater than 10%. HLA-DPw4 has been reported to be associated with multiple sclerosis (MS) but our PCR-SSOP analysis of 52 French Canadian MS patients showed no association with either the DPB1*0401 or DPB1*0402 splits of DPw4 or with any other DPB1 allele.