DNA ENZYME-IMMUNOASSAY OF THE PCR-AMPLIFIED HLA-DQ ALPHA-GENE FOR ESTIMATING RESIDUAL LEUKOCYTES IN FILTERED BLOOD

Blood is filtered for selective removal of leukocytes (WBC) to reduce the immunological and virological risks of transfusion, Exceedingly lo w numbers of residual WBC in leukodepleted blood cannot be enumerated by conventional hematologic methods, Therefore, we investigated the ap plication of a DNA enzyme immunoassay (DEIA) for detecting a region of the HLA-DQ alpha gene following amplification by PCR. After hybridiza tion with a specific probe coated onto the wells of a microtiter plate , the PCR-amplified DNA was detected by adding monoclonal antibodies t o double-stranded DNA, enzyme tracer, and chromogen substrate for colo rimetric measurement, The sensitivities of DEIA and radioisotopic liqu id hybridization were similar in five sets of experiments performed wi th a known number of human WBC. The optical density and the number of spiked human WBC in the range of 1.0 to 0.05 cells per mu l showed goo d correlation in five calibration experiments performed with human WBC suspended in heterologous blood, Using a calibration curve for DEIA, we estimated the concentration of residual WBC in five individual unit s of erythrocytes passed through blood filters. The postfiltration WBC count was 1.6 WBC per mu l in one unit, while in four other units it was below the lower detection limit (<0.05 WBC per mu l) of the DEIA. DEIA obviates the use of radioisotopes in PCR for detection of exceedi ngly low numbers of residual WBC in filtered blood.