DNA enzyme immunoassay of the PCR-amplified HLA-DQ alpha gene for estimating residual leukocytes in filtered blood

Abstract

Blood is filtered for selective removal of leukocytes (WBC) to reduce the immunological and virological risks of transfusion. Exceedingly low numbers of residual WBC in leukodepleted blood cannot be enumerated by conventional hematologic methods. Therefore, we investigated the application of a DNA enzyme immunoassay (DEIA) for detecting a region of the HLA-DQ alpha gene following amplification by PCR. After hybridization with a specific probe coated onto the wells of a microtiter plate, the PCR-amplified DNA was detected by adding monoclonal antibodies to double-stranded DNA, enzyme tracer, and chromogen substrate for colorimetric measurement. The sensitivities of DEIA and radioisotopic liquid hybridization were similar in five sets of experiments performed with 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 microliter showed good 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 units of erythrocytes passed through blood filters. The postfiltration WBC count was 1.6 WBC per microliter in one unit, while in four other units it was below the lower detection limit (< 0.05 WBC per microliter) of the DEIA. DEIA obviates the use of radioisotopes in PCR for detection of exceedingly low numbers of residual WBC in filtered blood.