一种用于测定外周血中髓样前体树突状细胞绝对计数的流式细胞术方法。

A flow cytometric method for determination of absolute counts of myeloid precursor dendritic cells in peripheral blood.

作者信息

Ma Ling, Scheers Werner, Vandenberghe Peter

机构信息

Laboratory for Experimental Hematology, University of Leuven, University Hospital Leuven, Leuven, Belgium.

出版信息

J Immunol Methods. 2004 Feb 15;285(2):215-21. doi: 10.1016/j.jim.2003.12.006.

DOI:10.1016/j.jim.2003.12.006

PMID:14980435

Abstract

Circulating precursor dendritic cells (pDCs) constitute a rare population in peripheral blood. They have a typical immunophenotypic profile, yet, they cannot be identified by pDC-specific immunophenotypic markers and therefore, their accurate and absolute enumeration poses a challenge. Here, we report a method for the evaluation of absolute counts of myeloid pDC in minimally manipulated blood samples on a flow cytometer as a single platform. Three-color flow cytometry was done to identify myeloid pDC as CD33+ HLA-DR+ CD14/CD16(dim/negative) cells in commercially available TruCount trade mark tubes that contain a defined number of brightly fluorescent polystyrene beads. The normal range in peripheral blood of 41 healthy adults, as determined by this single-platform method, was 17.0+/-5.7 x 10(6)/l, or 0.64+/-0.23% of mononuclear cells (MNCs). In parallel experiments, we have compared our procedure with two published 'dual-platform' methods that derive the absolute pDC count from a relative number obtained by flow cytometry, and from absolute counts obtained from a haematological analyser. Regression analysis showed an excellent correlation between results obtained with our single-platform protocol and these double-platform procedures (R2 > or = 0.90). However, the values obtained by the single-platform method were significantly higher than those obtained by the dual-platform methods. The higher myeloid pDC numbers in this single-platform procedure are likely due to reduced cell loss in this 'lyse-no-wash' protocol compared with the other methods which include density gradient separation and centrifugation steps. The intra- and interassay variability were 4.4% (range, 2.04-8.96%) and 5.8% (range, 2.59-9.65%), respectively. Thus, the single-platform method described here allows accurate, rapid and simple measurement of circulating blood myeloid pDC and is suitable for routine enumeration of circulating myeloid pDC.

摘要

循环前体树突状细胞（pDCs）在外周血中占比稀少。它们具有典型的免疫表型特征，然而，无法通过pDC特异性免疫表型标志物进行识别，因此，对其进行准确的绝对计数颇具挑战。在此，我们报告一种在流式细胞仪上作为单一平台评估微量处理血样中髓样pDC绝对计数的方法。采用三色流式细胞术，在含有一定数量明亮荧光聚苯乙烯微球的市售TruCount商标管中，将髓样pDC鉴定为CD33+HLA-DR+CD14/CD16（弱阳性/阴性）细胞。通过这种单一平台方法测定，41名健康成年人外周血中的正常范围为17.0±5.7×10⁶/l，或占单核细胞（MNCs）的0.64±0.23%。在平行实验中，我们将我们的方法与两种已发表的“双平台”方法进行了比较，这两种方法从流式细胞术获得的相对数量以及血液分析仪获得的绝对计数中得出pDC绝对计数。回归分析表明，我们的单一平台方案与这些双平台方法获得的结果之间具有极好的相关性（R²≥0.90）。然而，单一平台方法获得的值显著高于双平台方法获得的值。与包括密度梯度分离和离心步骤在内的其他方法相比，这种单一平台方法中髓样pDC数量较高可能是由于在这种“裂解不洗涤”方案中细胞损失减少。批内和批间变异分别为4.4%（范围，2.04 - 8.96%）和5.8%（范围，2.59 - 9.65%）。因此，本文所述的单一平台方法能够准确、快速且简单地测量循环血中的髓样pDC，适用于循环髓样pDC的常规计数。