Normal human blood density gradient lymphocyte subset analysis: I. An interlaboratory flow cytometric comparison of 85 normal adults

An interlaboratory flow cytometric comparison of several commercially available human lymphocyte subset reagents was undertaken in three different laboratories. Fresh Hypaque-Ficoll purified blood mononuclear cells were stained at 4°C or 22°C. Direct or indirect surface immunofluorescence was carried out at all sites using an EPICS V flow cytometer. Fullbright, 10-μm fluorescent polystyrene microspheres were used for optical alignment and standardization. A log integral fluorescent histogram gated on forward and right angle scatter was collected on 1–2 × 104 cells for each reagent and the proportion, of positive cell determined for each reagent. With the exception of one reagent, anti-B1, which showed an approximately twofold variation, all three laboratories showed remarkable agreement. Thus there was no significant difference noted for the following reagents: OKT4, CCT4, Leu 3a, Leu 2a, OKT8, or CCT8. We attribute these findings to the availability of quality reagents, precision instrumentation, and a standard lymphocyte preparation.