PROPOSED REFERENCE METHOD FOR PERIPHERAL-BLOOD MONOCYTE COUNTING USING FLUORESCENCE-LABELED MONOCLONAL-ANTIBODIES

Flow cytometry using fluorescence-labelled monoclonal antibodies has b een proposed as a possible new reference method to evaluate the monocy te counting performance of automated hematology analyzers. Since in pr evious studies only one such technique was applied, we investigated ho w different flow cytometric techniques compared to the manual differen tial and a hematology analyzer. Relative monocyte counts of 60 samples of the daily routine were determined on a Coulter Profile II flow cyt ometer after incubation with two different CD45-FITC/CD14-PE antibody combinations and subsequent preparation with two whole-blood lysis tec hniques, including one no-wash technique, Results were compared to tho se of a 600-cell manual differential and to those of the Coulter STKS hematology analyzer, All flow cytometric methods correlated very well with the manual differential (r greater than or equal to 0.925) and no ne showed a significant bias. The Coulter STKS relative monocyte count s were slightly higher than those of the manual differential (8.76% vs , 8.18%). The correlations between the methods employing monoclonal an tibodies were excellent (r greater than or equal to 0.995) and the mea n monocyte counts identical although a small, non-systematic influence of sample preparation techniques was noted, An influence of the antib ody clones was not observed. The precision of the Profile II results w as far superior to that of the manual differential and the STKS. Our d ata show that flow cytometry employing fluorescence-labelled monoclona l antibodies is a potentially ideal new reference method for monocyte counting, However, they also show that establishing a new reference me thod will require extensive investigation and exact definition of the sample preparation procedure to be used. (C) 1996 Wiley-Liss, Inc.