Fiebig E W, Johnson D K, Hirschkorn D F, Knape C C, Webster H K, Lowder J, Busch M P
Irwin Memorial Blood Centers, San Francisco, California 94118, USA.
Cytometry. 1997 Dec 1;29(4):340-50. doi: 10.1002/(sici)1097-0320(19971201)29:4<340::aid-cyto11>3.0.co;2-u.
An approach to perform lymphocyte subset analysis on frozen-thawed whole blood (F/T WB) is described. WB from 24 human immunodeficiency virus type 1 (HIV-1) seropositive individuals and 21 controls was analyzed fresh and after frozen storage (with or without dimethyl sulfoxide) at -80 degrees C, in liquid nitrogen (LN2), and at -20 degrees C. Analysis of F/T WB utilized 3-color flow cytometry with CD45 and right angle light scatter gating. Absolute cell counts were obtained for 30 samples by using staining tubes containing internal bead standards [TruCount, Becton Dickinson Immunocytometry Systems (BDIS), San Jose, CA]. The mean difference between CD3+4+ percentages for F/T (-80 degrees C storage for up to 1 year) and fresh WB was less than -0.2% (95% limits +/-3%, P = 0.5) with 39 of 45 (87%) results falling within 2% of the fresh values (P = 0.74). Absolute CD3+4+ cell counts for F/T WB were generally lower than corresponding results for fresh aliquots (median difference was 33 cells/microl, P < 0.0001), but the results were highly correlated (r2 = 0.975, P < 0.0001). Results were more variable, although still highly correlated, for CD3+8+ cells, and with other freezing and storage conditions. It is concluded that lymphocyte subset analysis using F/T WB yields comparable results to fresh samples, which should prove useful for a number of practical applications.
本文描述了一种对冻融全血（F/T WB）进行淋巴细胞亚群分析的方法。对来自24名1型人类免疫缺陷病毒（HIV-1）血清阳性个体和21名对照的全血进行新鲜样本分析，并在-80℃、液氮（LN2）和-20℃下冷冻保存（添加或不添加二甲基亚砜）后进行分析。F/T WB分析采用三色流式细胞术，通过CD45和直角光散射门控进行。使用含有内部微珠标准品的染色管[TruCount，Becton Dickinson免疫细胞分析系统（BDIS），加利福尼亚州圣何塞]对30个样本进行绝对细胞计数。F/T WB（-80℃保存长达1年）和新鲜全血的CD3 + 4 +百分比的平均差异小于-0.2%（95%置信区间为±3%，P = 0.5），45个结果中有39个（87%）落在新鲜样本值的2%范围内（P = 0.74）。F/T WB的绝对CD3 + 4 +细胞计数通常低于新鲜样本的相应结果（中位数差异为33个细胞/微升，P < 0.0001），但结果高度相关（r2 = 0.975，P < 0.0001）。对于CD3 + 8 +细胞以及其他冷冻和保存条件，结果的变异性更大，尽管仍高度相关。结论是，使用F/T WB进行淋巴细胞亚群分析产生的结果与新鲜样本相当，这对于许多实际应用应该是有用的。
