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

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(TM) 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)/1, 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 (R(2)greater than or equal to0.90). 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 mycloid pDC and is suitable for routine enumeration of circulating myeloid pDC. (C) 2004 Elsevier B.V. All rights reserved.