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 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.