Quantitation of residual white cells in filtered blood components by polymerase chain reaction amplification of HLA DQ-A DNA.

BACKGROUND: Over the past several years, blood filtration technology has improved dramatically, such that currently available experimental filters are capable of reducing white cells (WBCs) in blood components to less than 0.1 WBC per microL. These residual WBC concentrations are below the sensitivity of automated cell counters, as well as of large-volume (Nageotte) hemocytometers. STUDY DESIGN AND METHODS: A quantitative polymerase chain reaction (PCR) amplification assay directed at HLA DQ-A DNA sequences has been developed for the enumeration of WBCs in filtered blood. To ensure quantitative recovery of WBCs at very low residual cell concentrations, a direct red cell lysis and WBC concentration protocol using 0.5 mL of filtered blood was perfected. Amplified product is detected by oligomer hybridization using 32P-labeled probes, with quantitation by image analysis of autoradiographic signals relative to a standardized dilution series processed in parallel. RESULTS: Recovery of residual WBCs in filtrates was shown to be enhanced by the addition of xenogeneic WBCs or polystyrene beads, which served as "carrier" particles during red cell lysis and wash steps. A contribution of nuclear fragments in filtered blood to PCR signal in the range of 0.01 to 0.5 WBCs per microL was observed; a modified protocol was developed to minimize this effect. Parallel analysis of spiked dilution series and evaluations of 39 red cell components filtered through commercial filters indicated good correlation between PCR and standard Nageotte counts in the range of 0.1 to 10 WBCs per microL (r2 = 0.94); only PCR was able to detect residual WBCs in filtrates from prototype 6 log10 WBC-reduction filters. CONCLUSION: This assay should prove useful for the development and quality assurance of increasingly efficient WBC-reduction filters.

Duke Scholars

Author Joseph Heitman Molecular Genetics and Microbiology