This work was partly supported by the NSC 98-2320-B-182A-006 and NSC 99-2320-B-182A-006MY3 from the National Science Council, Taiwan.
Real-time amplification of glyceraldehyde-3-phosphate dehydrogenase gene for quality control of leukopoor platelets
Article first published online: 19 JUN 2013
© 2013 American Association of Blood Banks
Volume 54, Issue 2, pages 445–450, February 2014
How to Cite
Chen, D.-P., Sun, C.-F., Ning, H.-C., Peng, C.-T., Wang, W.-T. and Tseng, C.-P. (2014), Real-time amplification of glyceraldehyde-3-phosphate dehydrogenase gene for quality control of leukopoor platelets. Transfusion, 54: 445–450. doi: 10.1111/trf.12294
- Issue published online: 11 FEB 2014
- Article first published online: 19 JUN 2013
- Manuscript Accepted: 1 MAY 2013
- Manuscript Revised: 29 APR 2013
- Manuscript Received: 6 FEB 2013
- National Science Council, Taiwan. Grant Numbers: NSC 98-2320-B-182A-006, NSC 99-2320-B-182A-006MY3
Leukoreduction of blood products is crucial to prevent white blood cell (WBC)-associated complications during transfusion. Of the widely accepted methods for quantifying WBCs in blood components, Nageotte hemocytometry is time-consuming and laborious whereas a specialized instrument is required for flow cytometry. A reliable and affordable method to assess WBC count in blood products is of particular interest.
Study Design and Methods
Real-time polymerase chain reaction (PCR) of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was developed for quantifying WBCs in leukopoor platelets (LPPs). After normalization by the cell-free prefiltrated and postfiltrated plasma DNA, the relative copy number of GAPDH gene in the platelet (PLT) concentrate and its corresponding LPPs was calculated according to the equation of 2−ΔΔCt of which Ct is defined as the threshold cycle. The percentage and the number of WBCs that remained in LPPs were consequently determined. This method was compared to Nageotte hemocytometry and was validated by using serially diluted PLT concentrate and 10 pairs of PLT concentrate-LPP samples.
Consistent with the removal of WBCs after filtration, the Ct values for the LPP samples were increased when compared to their corresponding PLT concentrate. As revealed by real-time PCR of GAPDH gene, there is a correlation between the calculated and theoretical WBC count in the serially diluted PLT concentrate (correlation coefficient, 0.9532). The WBC counts for the 10 LPP samples were comparable between Nageotte and real-time PCR method and were all below 3.3 × 106 WBCs/L.
The real-time PCR method we report in this study is applicable for routine quality assurance during leukoreduction process.