Flow cytometric "rare event analysis": a standardized approach to the analysis of donor cell chimerism

Lack of standardization of methods and detection limits contributes to the controversial results regarding microchimerism after organ transplantation and has prompted the development of a standardized, reproducible, "easy-to-use" flow cytometric method for this type of "rare event analysis".EDTA-blood of healthy, HLA-typed donors was stained simultaneously with FITC- and biotin-labeled HLA-class I antibodies (One Lambda) as well as Cy5-PE-labeled CD45 (Medac, Hamburg) according to a standard protocol and analysed on a Coulter EPICS XL Flow cytometer (FCM). An absolute range of positivity (mean MESF+/- 1 STD) was determined for 22 HLA-specific antibodies. The range of positivity ranged between 5000 and 20,000 MESF (anti-A23, 24(9) FITC) and 40,000-140,000 (anti-Bw6 FITC). The frequency of nonspecific positive signals using nonstained cells, isotype-controls and irrelevant HLA antibodies was between 0.01% and about 0.5%, in some samples up to 1.4%, with an MESF between 8000 and 150,000, thus interfering clearly with the defined positive range of most antibodies tested. Using an "HLA antibody cocktail", combining FITC- and PE-labeled antibodies for different HLA specificities and thereby creating an internal control, the identification of donor cells was improved but was only rarely applicable. Due to the lack of highly reactive antibodies, FCM analysis was not suitable for the reliable identification of very low numbers of donor hematopoetic cells despite the theoretical advantages of flow cytometric detection of rare events. The single parameter approach was hampered by a significant frequency of nonspecific positive signals, which were easily mistaken as specific (true) positive signals, whereas the multiparameter approach could only be used in selected cases.