Quantitation of residual WBCs in filtered blood components by high-throughput, real-time kinetic PCR

BACKGROUND: The effort to eliminate transfusion complications associated with WBCs has led to the widespread use of filters able to reduce WBC concentrations to less than or equal to 0.1 WBC per muL blood. This has necessitated sensitive QC methods to quantitate residual WBCs in filtered units. One fast, effective method is DNA amplification using real-time kinetic PCR (kPCR). STUDY DESIGN AND METHODS: Two methods of preparation of standards were compared and used for the optimization of quantitative kPCR. The first involved spiking genomic DNA cell lysate into a diluent, followed by a series of 1 in 10 dilutions. The second involved spiking serial 1 in 10 dilutions of WBCs into twice-filtered fresh whole blood. Two hundred fifty filtered frozen whole-blood samples were amplified in duplicate to show the kPCR assay's reproducibility. Another 359 filtered frozen whole blood samples were used to compare data from kPCR with data from a standard PCR protocol using P-32-labeled probe and autoradiography. All specimens were amplified for conserved HLA DQalpha sequences. RESULTS: Standards prepared by both methods gave reproducible and equivalent results. Quantitation of standards representing a dynamic range of 8 x 10(0) to 8 x 10(5) WBCs per mL, yielded standard deviations ranging from 0.59 cycle to 1.04 cycles (a one-cycle increase is equivalent to a twofold increase in WBC concentration). The scatter graph of the 250 samples tested in duplicate by kPCR generated a slope of 1.0122 and an R-2 value of 0.9265. The comparison of kPCR and P-32-probe hybridization results on 359 clinical samples gave a scatter-graph slope of 0.9428 and an R 2 value of 0.8718, indicating excellent agreement of the methods over a 4-log dynamic range. CONCLUSION: kPCR is a high-throughput, sensitive assay that could prove useful in routine quality assurance of the WBC reduction process.