TOWARD A NEW REFERENCE METHOD, FOR THE LEUKOCYTE 5-PART DIFFERENTIAL

A flow cytometric method performing a five-part leukocyte differential based on three-color staining with anti-CD45-fluorescein isothiocyana te (FITC), anti-CD-14-phycoerythrin (PE)/Cy5, and a cocktail of PE-lab eled anti-CD2, anti-CD16, and anti-HLA-DR antibodies was evaluated. Re sults obtained by using three different sample preparation procedures and two different flow cytometers were compared with those of a 1,000- cell manual differential for evaluation of accuracy. We observed excel lent correlations with the manual differential for all leukocyte subcl asses and even higher correlations between the different flow cytometr ic methods. Flow cytometric basophil results were identical to the man ual counts, regardless of which sample preparation technique or flow c ytometer was used. Therefore, we propose our flow cytometric method as the first acceptable automated reference method for basophil counting . The flow cytometric results for the other leukocyte subclasses were apparently influenced by the sample preparation, which could not be ex plained by cell loss during washing steps. Moreover, a small influence of the flow cytometer was also observed. Assessing the influence of s ample storage, we found only minimal changes within 24 h. In establish ing reference values, high precision of flow cytometric results facili tated detection of a significantly higher monocyte count for males (re lative count: 7.08 +/- 1.73% vs. 6.44 +/- 1.33%, P < 0.05; absolute co unt: 0.536 +/- 0.181 x 10(9)/liter vs. 0.456 +/- 139 x 10(9)/liter, P < 0.01). Our data indicate that monoclonal antibody-based flow cytomet ry is a highly suitable reference method for the five-part differentia l: It also shows, however, that studies will have to put more emphasis on methodological issues to define a method that shows a high interla boratory reproducibility. (C) 1997 Wiley-Liss, Inc.