Establishment of a quantitative method for leukocyte fragments in apheresis platelet concentrates and its influential factors / 中华检验医学杂志

ABSTRACT

Objective To establish a new method for quantitating leukocyte fragments (LFs) in apheresis platelet concentrates (AP-PCs) by using real-time quantitative polymerase chain reaction (RQ-PCR) and flow cytometry(FCM) and discuss the factors influencing LFs concentrations such as storage time, filtration and PLT concentration. Methods 67 qualified donors were selected. Each of them donated one therapeutic dose of AP-PCs. AP-PCs samples were collected as soon as possible and divided into si xfractions. One was analyzed by hematology analyzer. For the Others, DNA was extracted under differen tconditions (filtrated or unfiltrated, before or after centrifugation) at 4 hours, 24 hours, 48 hours, 72 hours, 96 hours after blood draw, respectively. Then the amounts of albumin gene of the AP-PCs and the cell-free DNA in supematant were quantitatively determined using RQ-PCR and the results were calculated into leukocytes equivalent(WBCs/μl). Intact leucocytes were counted by FCM. The concentrations of LFs were calculated by subtracting cell-frce DNA and intact leucocytes from the total DNA amount. Then the differences of LFs concentrations among groups with different storage time were compared and the differences of LFs concentrations between unfihrated and filtrated groups were also compared. After grouping all the AP-PCs according to their PLT concentrations, LFs contents of AP-PCs before filtration among groups were compared. Meanwhile, bivariate correlation analysis between PLT concentrations and LFs contents was carried out. ResultsLFs contents of all the AP-PCs samples were quantitated successfully.The concentrations of LFs in AP-PCs before filtration in 4 hours,24 hours,48 hours,72 hours , 96 houres after blood draw were(31.4±17. 6), (47.5±25.3), (100.7±53.5), (89.5 ±47.2) and (16.1±7.8) WBCs/μl ; After filtration the results were (16. 9±8. 7), (24. 3 ± 12. 2), (83. 1±42. 6), (78.2 ±40. 2) and (13.6 ± 6. 6) WBCs/μl respectively. There were statistically significant differences among groups of different storage time (Fwithin subjects = 472. 756,P < 0.01). The concentrations of LFs kept on increasing within 48 hours after collections, and then decreased gradually. The peaks appeared between 48 hours and 72 hours after collections. The differences of LFs contents between unfiltered and filtered AP-PCs in 4 hours, 24 hours, 48 hours, 72 hours, 96 hours after collections were 14. 5, 23. 2, 17. 6, 11.3 and 2. 5 WBCs/μl, respectively.There was statistically significant difference between unfiltered and filtered samples (Fbetween subjects=9. 216,P < 0. 05). The differences were considerable within 48 hours, and then declined gradually. The results of bivariate correlation analysis showed that there were no statistically significant correlation between PLT concentrations and LFs contents (at 4, 24, 48, 72, 96 hours after collections the correlation coefficients rs were -0.002, 0.015, 0.027, 0.042 and 0. 037,respectively,P2-tailed>0.05). ConclusionsRQ-PCR and FCM can be used to quantitate LFs in AP-PCs. The concentration of LFs in AP-PCs is influenced by storage time and filtration, but it is not affected by PLT concentration.