Polymerase chain reaction based assessment of leukoreduction efficacy using a cytomegalovirus DNA transfected human T-cell line.

Abstract

Detection of CMV DNA by PCR in seropositive blood donors is affected by nucleic acid extraction, amplification conditions and PCR product detection sensitivity. Clinical studies have shown that leukoreduced blood products are as effective as CMV-seronegative blood products in minimizing transfusion-associated CMV infection. We developed a PCR-based test model to assess the efficacy of leukoreduction on the removal of CMV DNA from blood donations. Whole blood units were spiked with a human Jurkat T-lymphocyte cell line which had been stably transfected with a CMV DNA immediate early sequence. All blood units were either CMV seronegative or shown to be CMV PCR negative. The amount of CMV DNA by PCR before, during and after filtration of blood units with third-generation leukocyte reduction filters were determined using a semi-quantitative adaptation of the Digene SHARP Signal System Assay (DSSSA). Whole blood units spiked with 1-2 x 10(6) CMV transfected Jurkat cells contained no detectable CMV DNA after leukoreduction. With a higher inoculum of 2.9 x 10(8) transfected Jurkat cells per unit, CMV DNA was detected after leukoreduction; however, there was an approximate 3 log decrease in the amount of CMV DNA detected. This CMV DNA transfected human Jurkat cell line in conjunction with semi-quantitative CMV PCR may be used to model leukoreduction efficacy and provides evidence that leukoreduction of blood products can decrease the amount of cellular CMV DNA by approximately 3 logs.