Leucocount

At CDL determination of rWBC levels was performed using a flow cytometer (FACSCalibur, BD Biosciences) with QC performed with Calibrite beads, (BD Biosciences). QM‐Manchester used Navios analyzers with QC performed using Flow‐Check Pro and Flow‐Set Pro beads (Beckman Coulter). Both sites used WBC enumeration kits (Leucocount, BD Biosciences) for staining rWBCs before flow cytometric analysis. At QM‐Manchester, flow cytometry results were recorded in a QM database, with results of 1 WBC/μL or less being recorded as 1 WBC/μL.

Residual cells (platelets, leucocytes, erythrocytes, and erythrocytes ghosts) were quantified in frozen samples by flow cytometric (FACS) analysis using a no‐lyse/no‐wash preparation procedure [37 (link)]. The manufacturer's instructions were followed for leucocyte enumeration with LeucoCount (BD Biosciences, Sunnyvale, California). To quantify platelets and erythrocytes, 100 μl of plasma were added to TrueCount tubes (BD Biosciences) and incubated with 10 μl of a monoclonal antibody cocktail against CD42a (FITC, BD Biosciences) and glycophorin‐A (PE, Dako, Glostrup, Denmark). After 30 min of incubation at 4°C, 300 μl PBS were added before measurement on a FACSCalibur (BD Biosciences). Platelets and erythrocytes were subsequently measured separately from the same tube. Cell debris signals were removed. Logarithmic amplification was used for all variables. Data were evaluated by multi‐parameter analysis using the Paint‐A‐Gate software (BD Biosciences).