| Background and Objective Residual leukocytes in platelet concentrates could induce alloimmunizations and cause several transfusion associated complications in recipients, such as nonhemolytic febrile transfusion reactions(NHFTRs), transfusion related immunomodulation(TRIM), transfusion-related acute lung injury(TRALI), transfusion associated graft versus host disease (TAGVHD), HLA alloimmunisation and platelet refractoriness. Some investigations implicated that leukocyte fragments (LFs),which derived from intact leucocytes, might be competent to play the same role in the etiology of these complications as the intact leucocytes did. In order to determine the concentrations of LFs in widely used apheresis platelet concentrates (AP-PCs) , and discuss the influences of storage time, filtration and PLT concentration on it, a new method will be established in this study, by adopting real-time quantitative polymerase chain reaction (RQ-PCR) and flow cytometry(FCM).Methods 67 qualified donors were selected .Each of them donated 1 therapeutic dose of AP-PCs.Then the AP-PCs were sampled as soon as possible and each of the samples was divided into 6 parts.The one was analyzed by hematology analyzer. Each of others underwent 4 DNA extractions in different conditions ( filtered or unfiltered , before and after centrifugation) at the time of wthin 4 hours,24hours,48 hours , 72 houres and 96 hours after donation respectively. Then the amounts of total DNA of the AP-PCs and the cell-free DNA in supernatant were mensurated by using RQ-PCR for the albumin gene,and the results were calculated into leukocytes equivalent. Intact leucocytes were counted by using FCM. The number of LFs was calculated by subtracting cell-free DNA and intact leucocytes from the total DNA amount. Then the differences of LFs concentrations among groups of different storage time ,and between unfiltered and filtered groups were analyzed. All the AP-PCs were grouped according to their PLT concentrations, the LFs contents of all the groups were compared. Meanwhile, a bivariate correlation analysis was carried out between PLT concentration and LFs content. In order to investigate the accuracy and precision of the RQ-PCR and FCM technique for quantifying the number of WBCs presented in AP-PCs, WBC concentrates diluted with different volume of double filtered AP-PCs or PBS were measured respectively. The results were compared with the real concentrtions separately.Results The PCR results showed that the relative errors of PBS diluted series samples were fairly low (≤7.0%) when the real concentrtions were above the level of 5WBCs/μl; In AP-PCs diluted series samples ,due to the residual WBCs came from the solvent ,the acceptable results (≤15.0%) appeared only while the expected concentrtions were above 50WBCs/μl. The FCM results showed ,when the background was deducted , the relative errors of AP-PCs diluted series samples were <25.0% at all the levels. The LFs contents of all the AP-PCs samples were quantitated successfully.The amounts of LFs in unfiltered AP-PCs at <4hours,24 hours,48 hours ,72 hours and 96 houres after collection were 31.4±17.6, 47.5±25.3, 100.7±53.5, 89.5±47.2 and 16.1±7.8 WBCs/μl ; The counterparts of filtered 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(F=472.756,P < 0.01).The concentrations of LFs kept on increasing within 48 hours after donations,then decreased sharply.The peaks appeared between 48 hours and 72 hours after collections. The differences of LFs content between unfiltered and filtered AP-PCs at <4hours,24 hours,48 hours ,72 hours and 96 houres after collection were 14.5,23.2,17.6,11.3 and 2.5 WBCs/μl respectively.There were statistically significant differences between unfiltered and filtered samples(F=9.216,P<0.05). The differences were considerable within 48 hours,and then declined gradually. The amounts of LFs among AP-PCs with different concentrations of platelet had no statistically significant differences(F=0.020,P>0.05). The results of bivariate correlation analyses showed there were no statistically significant correlation between PLT concentrations and LFs contents (at <4hours,24 hours,48 hours ,72 hours and 96 houres after collections the correlation coefficients rs were -0.002,0.015,0.027,0.042 and 0.037 respectively,under all the conditions P2-tailed>0.05).Conclusions The method established here is competent for quantitating the LFs in AP-PCs. There is considerable amount of LFs in AP-PCs, which can reach 20% of the total residual WBCs.The number of LFs increases rapidly between 48 to 72 hours after collection,and then falls sharply during further storage.The LFs content will decline while the AP-PCs is filtered, but the difference will be slighter when the filtration is conducted after 48 hours of storage. The concentration of platelets in AP-PCs have no influce on the concentration of LFs .
|
PDF Full Text Request