机采血小板中白细胞碎片定量检测方法的建立及其影响因素

Establishment of a quantitative method for leukocyte fragments in apheresis platelet concentrates and its influential factors

目的采用实时定量PCR（RQ-PCR）和流式细胞术（FCM）建立机采血小板（AP—PCs）中白细胞碎片（LFs）的定量检测方法，并探讨保存时间、是否过滤和PLT浓度等因素对LFs含量的影响。方法选取合格献血者67名，各采集AP—PCs 1个治疗剂量，并尽快留取标本。然后将每份标本分成6份，其中1份用于血液分析仪检测，另外5份分别于采集后4h内、24、48、72、96h时，分别在过滤前后和离心前后等不同状态下进行4次DNA抽提。然后运用RQ-PCR对所抽提DNA中人类白蛋白基因进行定量测定，并换算成白细胞当量（WBCs／μl）。完整白细胞用FCM进行计数。LFs含量通过从总DNA含量中减去游离DNA含量和完整白细胞含量而算出，比较不同保存时间组和过滤前后组间LFs含量的差别。并按照PLT浓度的不同进行分组，比较不同PLT浓度组间过滤前LFs含量的差别。同时对PLT浓度和LFs含量进行双变量相关分析。结果所有AP—PCs标本中LFs含量均被成功检测。过滤前LFs含量在采集后4h内、24、48、72、96h分别为（31．4±17．6）、（47．5±25．3）、（100．7±53．5）、（89．5±47．2）和（16．1±7．8）WBCs／μl，过滤后分别为（16．9±8．7）、（24．3±12．2）、（83．1±42．6）、（78．2±40．2）和（13．6±6．6）WBCs／μl，不同保存时间组间LFs含量差异有统计学意义（F组内=472．756，P〈0．01）。LFs含量在采集后48h内呈上升趋势，此后逐渐下降，其峰值出现在血小板采集后48～72h之间。过滤前后LFs含量差值在4h内、24、48、72、96h分别为14．5、23．2、17．6、11．3和2．5WBCs／μl，过滤前后差异有统计学意义（F组间=9．216，P〈0．05），其差值在48h内较大，随后逐渐减小。双变量相关分析结果显示，PLT浓度与LFs含量之间不呈相关关系，采集后4h内、24、48、72、96h相关系数rs分别为～0．002、0．015、0．027、0．042和0．037，P双侧均〉0．05。结论RQ—PCR和FCM可用于AP—PCs中LFs的定量检测，AP—PCs中LFs含量受保存时间和过滤因素的影响，但不受PLT浓度的影响。

Objective To establish a new method for quantitating leukocyte fragments （LFs） in apheresis platelet concentrates （ AP-PCs ） by using real-tlme 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 six fractions, One was analyzed by hematology analyzer. For the Others, DNA was extracted under different conditions （ filtrated or unfihrated, 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 supernatant 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-free 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 unfiltrated 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. Results LFs 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 r were - 0. 002, 0. 015, 0. 027, 0. 042 and 0. 037, respectively, P2-tailed 〉 0. 05 ）. Conclusions RQ-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.