Detection of specimen contamination in routine histopathology by HLA class II typing using the polymerase chain reaction and sequence specific oligonucleotide probing

Abstract

This study evaluates the use of human leukocyte antigen (HLA) class II PCR-SSO typing for the investigation of suspected specimen contamination in four routine surgical histopathology cases. Two cases were of patients undergoing transurethral resection of the prostate gland. The third case was a patient undergoing excision of a subcutaneous small cell carcinoma. The fourth case was a patient undergoing reversal of a Hartman's procedure for adenocarcinoma of the colon. Tissue was extracted from routinely processed formalin-fixed, paraffin-embedded material and HLA class II typed, using a non-radioactive polymerase chain reaction-sequence specific oligonucleotide probe technique (PCR-SSO), using probes specific for the DRB, DQA, and DQB loci. The accuracy of PCR-SSO typing of DNA extracted from paraffin biopsy material was confirmed by concordant results for DRB, DQA, and DQB typing in five separate control individuals from whom frozen and paraffin lymph node biopsies were available. PCR-SSO typing was also successful in the four study cases and revealed that contamination had occurred in two cases, eliminating this possibility in the remaining cases. This study demonstrates that DNA can be reliably amplified and accurately typed from routinely processed material, and reveals a useful new application for PCR-SSO tissue typing.

Polymerase chain reaction based human leucocyte antigen genotyping for the investigation of suspected gastrointestinal biopsy contamination

“…We have previously developed PCR based HLA class II tissue genotyping methods that were derived from routine DNA based tissue typing techniques for solid organ and bone marrow transplantation matching and disease association studies3 4 and are applicable to the relatively degraded DNA extractable from formalin fixed and paraffin wax embedded tissue 12 We have also developed related PCR based techniques, again applicable to paraffin wax biopsy specimens, for tissue identification and HLA-disease association studies 1213 These techniques are particularly useful for the investigation of individual tissue identity as HLA molecules, involved in modulation of the immune response, are encoded by the most polymorphic loci in the human genome, within the major histocompatibility complex on chromosome six.…”

Symposium on ’Nutrition in the post-genomic era’ Plenary session 4: Genetic variation and diet-related disease

“…Due to this exuberant HLA polymorphism and its central role in the immune response, characterisation of this polymorphism within the HLA genes and encoded molecules is of critical importance in the clinical laboratory in both bone marrow and solid organ transplant patient-donor matching and in HLA-disease association studies (Bidwell & Navarrete, 2000). In our laboratory, characterisation of HLA polymorphism has also provided a tool for determination of individual identity during the investigation of potentiallycontaminated or mislabelled surgical biopsy specimens (Bateman et al 1994(Bateman et al , 1996(Bateman et al , 1997.…”

Nested polymerase chain reaction-based HLA class II typing for the unique identification of formalin-fixed and paraffin-embedded tissue

“…8,9 These PCR-based techniques have also recently permitted HLA genotyping using DNA extracted from formalin-fixed and paraffinembedded tissue, with proven applications in HLAdisease association studies 10 and surgical biopsy identification. 11,12 However, the quantity and quality of paraffin biopsy-derived DNA are often low, especially when the biopsies under investigation are small, and this may limit the utility of current techniques. 11 We have developed a significantly more sensitive HLA class II genotyping method which utilizes nested PCR and reliably detects HLA DRB alleles within DNA extracted from even very small paraffin biopsies.…”

“…11,12 However, the quantity and quality of paraffin biopsy-derived DNA are often low, especially when the biopsies under investigation are small, and this may limit the utility of current techniques. 11 We have developed a significantly more sensitive HLA class II genotyping method which utilizes nested PCR and reliably detects HLA DRB alleles within DNA extracted from even very small paraffin biopsies. This method comprises amplification of the second variable exon within the HLA DRB1 gene, followed by identification of DRB1 alleles using PCR-SSP analysis of the amplified product derived from the initial PCR.…”