检验医学
檢驗醫學
검험의학
Laboratory Medicine
2015年
9期
916-920
,共5页
单核细胞-血小板聚集体%流式细胞术%处理方法
單覈細胞-血小闆聚集體%流式細胞術%處理方法
단핵세포-혈소판취집체%류식세포술%처리방법
Monocyte-platelet aggregation%Flow cytometry%Processing
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。
目的：探討實際工作中不同處理方法對流式細胞術檢測單覈細胞-血小闆聚集體（ MPA）的影響，為準確檢測MPA提供依據。方法穫取枸櫞痠鈉抗凝外週靜脈全血，分彆以CD14-藻紅蛋白（ PE）、CD61-異硫氰痠熒光素（ FITC）標記單覈細胞、血小闆，以CD62P-PE標記活化血小闆，使用流式細胞術檢測CD14-PE／CD61-FITC雙暘性MPA佔單覈細胞百分比及血小闆CD62P暘性率。採用不同處理方法[全血標本處理前後放置不同時間、全血固定後立即標記抗體或放置不同時間後標記抗體、抗體標記前全血離心處理、不同抗體組閤（抗CD14-PE和抗CD61-FITC ，抗CD14-PE和抗CD41-ECD，抗CD45-ECD和抗CD61-FITC）]檢測MPA，比較各種處理方法檢測的MPA是否有差異。結果與穫取標本後立即處理比較，放置于室溫（18～25℃）和低溫（2～8℃）， MPA百分比、血小闆CD62P暘性率均隨放置時間的延長而增加（P＜0．05），兩者呈正相關（室溫r＝0．82，P＜0．05；低溫r＝0．83，P＜0．05）。標本處理後立即檢測或低溫保存放置至24 h再檢測， MPA百分率差異無統計學意義（P＞0．05）。與固定前標記抗體的處理方法比較，1％多聚甲醛低溫固定全血標本後標記抗體，或者低溫保存放置至24 h再標記抗體，MPA百分率差異無統計學意義（P＞0．05）。全血標本離心處理後，MPA百分率和血小闆CD62P暘性率均明顯增加（P＜0．05）。不同的抗體組閤檢測MPA百分比的差異均無統計學意義（P均＞0．05）。結論穫取全血標本後應儘快處理，不能立即處理的應使用固定劑固定後可低溫保存24 h。在標記新鮮全血標本前，應避免離心，處理完畢的標本使用固定劑固定後可低溫保存24 h再用流式細胞術檢測。不同抗體組閤應驗證後使用，CD14／CD61、CD14／CD41、CD45／CD61組閤均適用于檢測MPA。
목적：탐토실제공작중불동처리방법대류식세포술검측단핵세포-혈소판취집체（ MPA）적영향，위준학검측MPA제공의거。방법획취구연산납항응외주정맥전혈，분별이CD14-조홍단백（ PE）、CD61-이류청산형광소（ FITC）표기단핵세포、혈소판，이CD62P-PE표기활화혈소판，사용류식세포술검측CD14-PE／CD61-FITC쌍양성MPA점단핵세포백분비급혈소판CD62P양성솔。채용불동처리방법[전혈표본처리전후방치불동시간、전혈고정후립즉표기항체혹방치불동시간후표기항체、항체표기전전혈리심처리、불동항체조합（항CD14-PE화항CD61-FITC ，항CD14-PE화항CD41-ECD，항CD45-ECD화항CD61-FITC）]검측MPA，비교각충처리방법검측적MPA시부유차이。결과여획취표본후립즉처리비교，방치우실온（18～25℃）화저온（2～8℃）， MPA백분비、혈소판CD62P양성솔균수방치시간적연장이증가（P＜0．05），량자정정상관（실온r＝0．82，P＜0．05；저온r＝0．83，P＜0．05）。표본처리후립즉검측혹저온보존방치지24 h재검측， MPA백분솔차이무통계학의의（P＞0．05）。여고정전표기항체적처리방법비교，1％다취갑철저온고정전혈표본후표기항체，혹자저온보존방치지24 h재표기항체，MPA백분솔차이무통계학의의（P＞0．05）。전혈표본리심처리후，MPA백분솔화혈소판CD62P양성솔균명현증가（P＜0．05）。불동적항체조합검측MPA백분비적차이균무통계학의의（P균＞0．05）。결론획취전혈표본후응진쾌처리，불능립즉처리적응사용고정제고정후가저온보존24 h。재표기신선전혈표본전，응피면리심，처리완필적표본사용고정제고정후가저온보존24 h재용류식세포술검측。불동항체조합응험증후사용，CD14／CD61、CD14／CD41、CD45／CD61조합균괄용우검측MPA。
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.
