Nested polymerase chain reaction with sequence-specific primers typing forHLA-A, -B, and -C alleles: Detection of microchimerism in DR-matched individuals

It is widely accepted that donor leukocytes survive within the recipient pe riphery after blood transfusion or solid organ transplantation. The signifi cance of this microchimerism remains unclear, partially because of the inse curity of assays used to detect the donor-derived material. The techniques used to detect donor-derived DNA within recipient peripheral blood rely lar gely on major histocompatibility complex class II polymorphism, We and othe rs have shown that the sensitivity of polymerase chain reaction with sequen ce-specific primers (PCR-SSP) typing for HLA class II alleles can be increa sed 100-fold by the addition of a primary amplification step (nested PCR-SS P). We have now extended this technique to encompass typing for HLA class I alleles, thereby adding flexibility to microchimerism testing by enabling testing of recipients HLA-DR matched with their donors. However, the high l evel of sensitivity achieved with the technique (1:100,000) leads to a conc omitant decrease in the specificity that results in the amplification of un expected products, a phenomenon we encountered in the development of our ne sted PCR-SSP typing system for HLA class II alleles. We describe here how i t is possible to compensate for these anomalies by including multiple testi ng of a pretransfusion sample that acts as a specificity control, establish ing a rigorous baseline for subsequent analysis, (C) 1999 by The American S ociety of Hematology.