不同处理方法对流式细胞术检测外周血单核细胞-血小板聚集体的影响

摘要

Objective To investigate the influence of different processing techniques on monocyte-platelet aggregation(MPA) by flow cytometry in order to provide the reference for the determination of MPA.Methods Sodium citrate anticoagulation whole blood samples were collected randomly.CD14-phycoerythrin( PE) and CD61-fluorescein isothiocyanate (FITC) were used to label monocytes and platelets.CD62P-PE was used to label activated platelets.The percentages of CD14-PE/CD61-FITC-double-positive MPA in monocytes and CD62P-positive platelets were determined by flow cytometry.Different processing techniques were performed as follows.Whole blood samples were prepared immediately after collection or delayed for different times.After preparation, samples were analyzed immediately or delayed for different times.Antibody immunolabeling was performed before fixation of whole blood samples, immediately after fixation or at different delayed times after fixation.Before immunolabeling, whole blood samples were centrifugated or not.Different antibodies, anti-CD14-PE with anti-CD61-FITC, anti-CD14-PE with anti-CD41-ECD and anti-CD45-ECD with anti-CD61-FITC, were used to label MPA.The results of MPA from different processing techniques were compared.Results Compared with immediate preparation after blood collection, the percentages of MPA and CD62P-positive platelets increased with the delayed time at room temperature ( 18-25℃) and low temperature ( 2-8℃) ( P<0.05), and there was a positive correlation(room temperature: r=0.82, P<0.05; low temperature: r=0.83, P<0.05).Compared with immediate determination after preparation, the percentages of MPA of samples stored at low temperature for 24h after preparation did not alter significantly(P >0.05).Compared with immunolabeling samples before fixation with 1% paraformaldehyde, no significant change was observed in MPA percentages from immediate immunolabeling after fixation or immunolabeling samples stored at low temperature for 24 h after fixation(P>0.05). Centrifugation of whole blood samples before immunolabeling resulted in significant increase of the percentages of MPA and CD62P-positive platelets(P<0.05).MPA percentages resulted from different antibodies had no significant variance (P >0.05).Conclusions Whole blood samples should be handled as soon as possible.Fixation with 1%paraformaldehyde at low temperature could store samples for 24h before handling.Centrifugation should be avoided before immunolabeling.After preparation and fixation, samples could be stored at low temperature for 24h before analysis.Different antibody combinations, including anti-CD14-PE/anti-CD61-FITC, anti-CD14-PE/anti-CD41-ECD and anti-CD45-ECD/anti-CD61-FITC, are all suitable for immunolabeling MPA.%目的：探讨实际工作中不同处理方法对流式细胞术检测单核细胞-血小板聚集体（ MPA）的影响，为准确检测MPA提供依据。方法获取枸橼酸钠抗凝外周静脉全血，分别以CD14-藻红蛋白（ PE）、CD61-异硫氰酸荧光素（ FITC）标记单核细胞、血小板，以CD62P-PE标记活化血小板，使用流式细胞术检测CD14-PE／CD61-FITC双阳性MPA占单核细胞百分比及血小板CD62P阳性率。采用不同处理方法[全血标本处理前后放置不同时间、全血固定后立即标记抗体或放置不同时间后标记抗体、抗体标记前全血离心处理、不同抗体组合（抗CD14-PE和抗CD61-FITC ，抗CD14-PE和抗CD41-ECD，抗CD45-ECD和抗CD61-FITC）]检测MPA，比较各种处理方法检测的MPA是否有差异。结果与获取标本后立即处理比较，放置于室温（18～25℃）和低温（2～8℃）， MPA百分比、血小板CD62P阳性率均随放置时间的延长而增加（P＜0．05），两者呈正相关（室温r＝0．82，P＜0．05；低温r＝0．83，P＜0．05）。标本处理后立即检测或低温保存放置至24 h再检测， MPA百分率差异无统计学意义（P＞0．05）。与固定前标记抗体的处理方法比较，1％多聚甲醛低温固定全血标本后标记抗体，或者低温保存放置至24 h再标记抗体，MPA百分率差异无统计学意义（P＞0．05）。全血标本离心处理后，MPA百分率和血小板CD62P阳性率均明显增加（P＜0．05）。不同的抗体组合检测MPA百分比的差异均无统计学意义（P均＞0．05）。结论获取全血标本后应尽快处理，不能立即处理的应使用固定剂固定后可低温保存24 h。在标记新鲜全血标本前，应避免离心，处理完毕的标本使用固定剂固定后可低温保存24 h再用流式细胞术检测。不同抗体组合应验证后使用，CD14／CD61、CD14／CD41、CD45／CD61组合均适用于检测MPA。