Pinto Ligia A, Trivett Matthew T, Wallace Dora, Higgins Jeanette, Baseler Michael, Terabe Masaki, Belyakov Igor M, Berzofsky Jay A, Hildesheim Allan
NCI-Frederick/SAIC-Frederick, Frederick, Maryland, USA.
Cytometry B Clin Cytom. 2005 Jan;63(1):47-55. doi: 10.1002/cyto.b.20038.
Immunophenotyping of whole blood (WB) and isolated peripheral blood mononuclear cells (PBMCs) is a common tool used to evaluate immune system changes in clinical studies. The development of methods that would allow preservation of samples for flow cytometric analysis is important for the extension of this technology to field testing in settings where the equipment might be not readily accessible.
Three-color flow cytometric analysis was used to determine percentages of T cells and their subsets (CD3(+), CD4(+), CD8(+)), B cells (CD19(+)), and natural killer cells (CD16(+)/56(+)) in WB and PBMCs using variations of a standard stain/fix WB staining procedure (Optilyse) that included staining following fixation and freezing of fixed samples before or after staining.
Comparable lymphocyte subset percentages in WB or PBMCs were observed regardless of Optliyse method used (all Ps >/= 0.8). However, differences in fluorescence intensity for several markers were observed across procedures. Compared with the standard stain/fix procedures, fix/stain decreased the mean fluorescence intensities for CD4, CD8, CD19 and CD16/56 in WB and PBMCs (P </= 0.03 for these markers P = 0.105 for CD8 in PBMCs). Further decreases in mean fluorescence intensity were seen with the fix/stain/freeze procedure. The stain/fix/freeze yielded intensities largely comparable to those seen with standard stain/fix procedure (P >/= 0.13), suggesting that, when the markers of interest are known at the time of field collection, implementation of this procedure might be desirable. Fix/freeze/stain resulted in diminution of intensity in general, but they tended to be more modest than those seen for fix/stain/freeze and therefore might be applicable to field studies in instances when the specific markers of interest cannot be defined upfront.
Freezing of fixed WB and PBMCs before or after cell surface staining is a reliable method for preserving specimens in field sites for later determination of lymphocyte subset percentages, which are commonly assessed in immunodeficient and cancer patients.
全血（WB）和分离的外周血单核细胞（PBMC）的免疫表型分析是临床研究中用于评估免疫系统变化的常用工具。开发能够保存样本用于流式细胞术分析的方法，对于将该技术扩展到设备可能不易获取的现场检测非常重要。
采用三色流式细胞术分析，使用标准染色/固定全血染色程序（Optilyse）的变体，包括固定后染色以及固定样本在染色前后冷冻，来测定全血和外周血单核细胞中T细胞及其亚群（CD3(+)、CD4(+)、CD8(+)）、B细胞（CD19(+)）和自然杀伤细胞（CD16(+)/56(+)）的百分比。
无论使用何种Optliyse方法，在全血或外周血单核细胞中观察到的淋巴细胞亚群百分比具有可比性（所有P值≥0.8）。然而，在不同程序中观察到几种标志物的荧光强度存在差异。与标准染色/固定程序相比，固定/染色降低了全血和外周血单核细胞中CD4、CD8、CD19和CD16/56的平均荧光强度（这些标志物的P值≤0.03，外周血单核细胞中CD8的P值 = 0.105）。固定/染色/冷冻程序使平均荧光强度进一步降低。染色/固定/冷冻产生的强度与标准染色/固定程序产生的强度基本相当（P值≥0.13），这表明，当在现场采集时已知感兴趣的标志物时，采用该程序可能是可取的。固定/冷冻/染色总体上导致强度降低，但往往比固定/染色/冷冻的程度更轻，因此在无法预先确定感兴趣的特定标志物的情况下，可能适用于现场研究。
在细胞表面染色之前或之后冷冻固定的全血和外周血单核细胞，是在现场保存标本以便日后测定淋巴细胞亚群百分比的可靠方法，淋巴细胞亚群百分比常用于免疫缺陷和癌症患者的评估。
