Hernández-Campo Pilar M, Martín-Ayuso Marta, Almeida Julia, López Antonio, Orfao Alberto
Servicio General de Citometría, Departmento de Medicina y Centro de Investigaciones del Cáncer, Universidad de Salamanca, Paseo de San Vicente, 58-182 37007 Salamanca, Spain.
Cytometry. 2002 Jun 15;50(3):191-201. doi: 10.1002/cyto.10072.
Flow cytometry-based immunophenotypic techniques for the analysis of CD55 and CD59 expression on the major cell populations present in blood are the preferred method for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH).
In the present study, we comparatively analyze the effects of stain-lyse-and-then-wash techniques and lyse-wash-and-then-stain procedures on the detection of both CD55 and CD59 expression on the major peripheral blood (PB) leucocyte subsets, as analyzed by flow cytometry. Our major goal was to establish the minimum amounts of anti-CD55 and anti-CD59 reagents required to be added to a minimum volume of blood, which would allow an optimal staining for both antigens on red cells, platelets, and leucocytes present in a single tube.
Our results show that upon comparing stain-lyse-and-then-wash techniques with lyse-wash-and-then-stain protocols, the presence of important amounts of red cells at the time peripheral blood leucocytes are stained for CD55 and CD59 is associated with a significantly (P < 0.01) lower and more heterogeneous pattern of antigen expression on almost all major PB leucocyte subsets, supporting the need to use red cell lysing procedures prior to the staining of leucocytes. Identical, optimal patterns of antigen staining for CD55 and CD59 were obtained upon incubating 3 microL of blood with 10 microL of each of these monoclonal antibody (mAb) reagents (protein concentration of 0.05 microg/microL and 0.2 microg/microL respectively) for 30 min (room temperature [RT]) using a non-lyse-non-wash sample preparation procedure. This latter procedure allowed for the simultaneous analysis of CD55 and CD59 expression on red cells, platelets, neutrophils, monocytes, and lymphocytes present in the sample through the combined staining of CD55 and CD59 with CD64-fluorescein isothiocyante (FITC) plus CD61-peridinin chlorophyll protein (PerCP) and CD45-PerCP.
In summary, our results show that the sample preparation protocol has a significant impact on the quality of the staining obtained for the CD55 and CD59 antigens on the major PB leucocyte subsets; additionally, we propose a simple and reliable stain-non-lyse-non-wash method for the simultaneous analysis of CD55 and CD59 expression on PB red cells, platelets, neutrophils, monocytes, and lymphocytes, which could be reached through the use of two triple stainings.
基于流式细胞术的免疫表型技术用于分析血液中主要细胞群体上CD55和CD59的表达，是阵发性夜间血红蛋白尿（PNH）诊断筛查的首选方法。
在本研究中，我们通过流式细胞术比较分析了先染色-再裂解-然后洗涤技术和先裂解-洗涤-然后染色程序对主要外周血（PB）白细胞亚群上CD55和CD59表达检测的影响。我们的主要目标是确定添加到最小体积血液中的抗CD55和抗CD59试剂的最小量，这将允许对单个试管中存在的红细胞、血小板和白细胞上的两种抗原进行最佳染色。
我们的结果表明，将先染色-再裂解-然后洗涤技术与先裂解-洗涤-然后染色方案进行比较时，在外周血白细胞进行CD55和CD59染色时存在大量红细胞，与几乎所有主要PB白细胞亚群上抗原表达的显著降低（P < 0.01）和更不均匀模式相关，这支持在白细胞染色前使用红细胞裂解程序的必要性。使用非裂解-非洗涤样品制备程序，将3微升血液与10微升每种这些单克隆抗体（mAb）试剂（蛋白质浓度分别为0.05微克/微升和0.2微克/微升）在室温（RT）下孵育30分钟后，获得了相同的CD55和CD59抗原最佳染色模式。后一种程序允许通过将CD55和CD59与CD64-异硫氰酸荧光素（FITC）加CD61-叶绿素蛋白（PerCP）和CD45-PerCP联合染色，同时分析样品中红细胞、血小板、中性粒细胞、单核细胞和淋巴细胞上CD55和CD59的表达。
总之，我们的结果表明样品制备方案对主要PB白细胞亚群上CD55和CD59抗原的染色质量有显著影响；此外，我们提出了一种简单可靠的非裂解-非洗涤染色方法，用于同时分析PB红细胞、血小板、中性粒细胞、单核细胞和淋巴细胞上CD55和CD59的表达，这可以通过使用两种三重染色来实现。
