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 <or=0.1 WBC per microL 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).


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 (32)P-labeled probe and autoradiography. All specimens were amplified for conserved HLA DQ(alpha) sequences.


RESULTS
Standards prepared by both methods gave reproducible and equivalent results. Quantitation of standards representing a dynamic range of 8 x 10(o) 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 (32)P-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.


CONCLUSIONS
kPCR is a high-throughput, sensitive assay that could prove useful in routine quality assurance of the WBC reduction process.
Lee
Tzong-Hae
TH
Molecular Biology Blood Centers of the Pacific, Irwin Center, Research Division, San Francisco, California 94118, USA. thlee@qpcr.com
Wen
Li
L
Chrebtow
Vera
V
Higuchi
Russell
R
Watson
Robert M
RM
Sninsky
John J
JJ
Busch
Michael P
MP
D003199:Computer Systems; D004247:DNA / Q000097:blood; D005374:Filtration; D006801:Humans; D007700:Kinetics; D007958:Leukocyte Count / Q000592:standards; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004247:DNA