ICSH mandated that populations to be identified by FCM were neutrophils, basophils, eosinophils, monocytes, lymphocytes, immature granulocytes, blasts, and nucleated red blood cells (nRBCs). Furthermore, the total number of colors was limited to 5 so the method might be widely performed in clinical laboratories. Published candidates protocols were not all in agreement with these goals, hence each of the three tested protocols were modified to meet these aims. The three candidate protocols were referred to by the country of origin of the authors (7–10). Dr. Björnsson (Malmö, Sweden) proposed to use CD123 instead of CD203c in the Swedish (SWE) protocol to allow more accurate basophil detection (9). Therefore, the SWE combination derived from the Bjornsson's publication included CD36, CD123, CD138, DRAQ5, CD16, CD56, CD45 in a single 5 color tube; Table 2). The U.S. combination derived from Dr. Cherian's publication included 2 tubes in 5 colors, respectively, US1: CD19, CD16, CD123, HLA-DR, CD33, CD64, CD45 and US2: Syto16, CD34, CD117, CD45, CD33, CD64, CD38 (8; Table 2). Finally, a second tube was designed to complete the French (FR) protocol as the published one could not identify some requested cell types such as nRBCs or myeloid blasts (7, 10). Therefore, the FR combination derived from Faucher's publication included 2 tubes in 5 colors with respectively FR1: CD36, CD2, CD294, CD19, CD16, CD45 and FR2: Syto16, CD13, CD20, HLA-DR, CD34, CD117, CD45 (10; Table 2). Fluorochromes where then adapted to the clinical models of cytometers provided by two major instrument manufacturers using fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE-cyanine 5 (PC5), and PE-cyanine 7 (PC7) for all panels and PE-Texas Red (ECD) or APC-H7 for, respectively, FC500 or CANTO/LSRII models (Table 2). Antibody cocktails were titered, centrally prepared (BLW, Seattle) and distributed. The choice was made to include in this comparison antibodies and reagents from various manufacturers (Table 2). Each leukocyte-flagged sample was processed using a “lyse-no wash” protocol. Briefly, 100 microliters of blood was labeled with 100 microliters of antibody cocktail. After a 15 min incubation, red blood cells were lysed with Versalyse, Optilyse or NH4Cl buffer (Table 2) and after a further 10 min the samples were ready for analysis by flow cytometry. The lysing reagents used were selected by the authors of the published methods for use in these modified protocols. Counting beads were also added to the prepared sample as indicated (Table 2).
Before analysis, flow cytometer settings were standardized (i) FC500 from Rennes and Seattle and (ii) CANTO from Rennes and LSRII from Seattle) using Rainbow Beads (Spherotech, Liberty, IL) or Flow Set Fluorosphere (Beckman Coulter) for respectively for CANTO/LSRII and FC500. Beads were used principally to standardize the settings between similar instruments at the two sites. No attempts were made to standardize intensities between instrument platforms for a given assay other than to perform global instrument optimization prior to defining settings. For each panel, targets defined in the Table 3 were used. For each sample, 100,000 nucleated cells were analyzed after cell debris exclusion.