Nationwide quality control trial on lymphocyte immunophenotyping and flow cytometer performance in Italy

The Italian Society for Cytometry (Gruppo Italiano di Citometria, GIC) promoted this nationwide large-scale quality control trial on cellular immunophenotyping and flow cytometer performance in 1991. The aim of this independent study was to evaluate instrument performance with calibrated fluorescence microbeads (minimum threshold for FITC, linearity, coefficients of variation and resolution indexes for fluorescence), and to correlate it to the measurement of lyophilized lymphocyte surface immunofluorescence staining with a wide spectrum of antibodies (percentage of positive cells and fluorescence mean intensity). A single send-out was made to 306 laboratories with 350 instruments throughout Italy. Each kit included anonymous vials containing premixed calibrated microbeads, lyophilized human lymphocytes and small aliquots of conjugated monoclonal antibodies, for both single FITC and double FITC/PE staining. Participants were also asked to use their own anti-CD4 monoclonal. A valid answer was returned by 209 laboratories with 221 instruments. Gating was not an assay variable. The minimum sensitivity threshold for FITC ranged from 26.6 to 11,293 MESF, with marked instrument heterogeneity as far as the relationship between FITC threshold and coefficients of variation for fluorescence was concerned. A low FITC threshold also correlated with a good resolution index for low intensity stainings. No performance comparisons among instrument brands and models were made. The lyophilized lymphocyte analysis showed an overall frequency of outliers ranging from 3.2% to 19.7%, with a maximum for CD2 FITC (19.7%) and CD3 + HLA-DR + (12.1%). A strongly negative relationship was evident between the FITC threshold and the number of CD2+ cells, whereas the sensitivity threshold had virtually no effects on the measured level of higher antigen density markers like CD4 and many others. Absolute fluorescence intensity measurements of CD4+ and CD19+ were also made. This study design proved valid and suitable for a large-scale evaluation of both instrument performance and cytometer operators' skill. A number of practical problems were identified among participants, thus stressing the need for more effective educational programmes and technical guidelines.