DETECTION OF MICROCHIMERISM AFTER ALLOGENEIC BLOOD-TRANSFUSION USING NESTED POLYMERASE-CHAIN-REACTION AMPLIFICATION WITH SEQUENCE-SPECIFIC PRIMERS (PCR-SSP) - A CAUTIONARY TALE

In bone marrow transplantation, the detection of chimerism is an impor tant adjunct to the repertoire of tests available for determining acce ptance of the graft, In solid organ transplantation, there is currentl y intense interest in the role that chimerism plays in both short- and long-term host reactivity to the graft. Allogeneic blood transfusion has been associated with a subtle immunosuppressive effect in renal tr ansplantation and chimerism is implicated as a possible mechanism for this effect. To assess the survival of allogeneic cells after blood tr ansfusion or transplantation, we have developed a technique based on m olecular typing for HLA class II alleles, which enables the detection of donor-derived cells in patients receiving blood transfusions. While developing this technology, we investigated why we and others observe false amplification. Sequencing of false products has shown that they arise from amplification of both pseudogenes and non-pseudogenes pres ent in the DNA under test, Elucidation of this phenomenon allows the a mplification of these false products to be predicted in any given comb ination and hence avoided by the judicious selection of primers. Valid ation has been achieved by following donor alleles after transfusion o f blood containing defined numbers of leukocytes expressing selected m ismatched antigens. (C) 1998 by The American Society of Hematology.