A flow cytometric method performing a five‐part leukocyte differential based on three‐color staining with anti‐CD45‐fluorescein isothiocyanate (FITC), anti‐CD‐14‐phycoerythrin (PE)/Cy5, and a cocktail of PE‐labeled anti‐CD2, anti‐CD16, and anti‐HLA‐DR antibodies was evaluated. Results 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 excellent correlations with the manual differential for all leukocyte subclasses and even higher correlations between the different flow cytometric methods. Flow cytometric basophil results were identical to the manual counts, regardless of which sample preparation technique or flow cytometer 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 explained by cell loss during washing steps. Moreover, a small influence of the flow cytometer was also observed. Assessing the influence of sample storage, we found only minimal changes within 24 h. In establishing reference values, high precision of flow cytometric results facilitated detection of a significantly higher monocyte count for males (relative count: 7.08 ± 1.73% vs. 6.44 ± 1.33%, P < 0.05; absolute count: 0.536 ± 0.181 × 109/liter vs. 0.456 ± 139 × 109/liter, P < 0.01). Our data indicate that monoclonal antibody‐based flow cytometry is a highly suitable reference method for the five‐part differential: It also shows, however, that studies will have to put more emphasis on methodological issues to define a method that shows a high interlaboratory reproducibility. Cytometry 30:72–84, 1997. © 1997 Wiley‐Liss, Inc.

Typing for HLA-B27 is routinely performed in patients with seronegative spondarthritides. Besides the microlymphocytotoxic test (MLCT), other serological techniques have been developed such as enzyme immunoassays (EIA) using serum or plasma as a source for the determination of soluble HLA-B27 (sHLA-B27) and flow cytometric (FC) methods. The aim of the present study was to check the accuracy and reliability of the EIA for sHLA-B27 in comparison to the MLCT using antibodies against HLA-B27 and cross-reacting specificities (CRS), as well as an FC method and a molecular biological method. Any discrepant results should be typed with the MLCT using a complete panel of anti-HLA-class I antibodies, with FC and with a molecular biological technique. The EIA should also be repeated in those patients, using serum and plasma from a new venipuncture. In 81 patients with rheumatic disorders, the EIA and the MLCT using antibodies against HLA-B27 and CRS were performed. Based on the MLCT with a complete panel of anti-HLA-class I antibodies as a standard, discrepant test results were obtained for 9 out of 81 patients with the MLCT using antibodies against HLA-B27 and CRS and with the EIA. The following wrong results occurred: in the MLCT with anti-HLA-B27 and CRS, there were two false-negative results; in the EIA there were four false-negative and one false-positive results; one sample was undeterminable. In comparison with the MLCT, including the complete panel of HLA-class I antibodies, as well as with a molecular biological technique, typing with FC showed a complete concordance. Our investigations demonstrated that for routine typing for HLA-B27 the MLCT cannot be replaced by EIA because of a significant number of mistypings. The MLCT performed only with antibodies against HLA-B27 and CRS may also lead to typing errors. No errors were detected using flow cytometry. If only serological methods can be performed in a laboratory a combination of flow cytometry and MLCT could therefore enhance the safety of HLA-B27 typing.

Additional key phrase: inflammatory rheumatic diseasesSerological typing of HLA-B27 is routinely done by the microlymphocytotoxicity test (MLCT).l This method is easy and allows a daily typing of a relatively large number of samples. Nevertheless, the interpretation requires some experience in the exclusion of false-positive typing results caused by cross-reactions. A significant disadvantage of the MLCT is the subjective microscopic evaluation of the percentage of killed and viable lymphocytes. In order to overcome the difficulties with the MLCT, other serological methods such as flow cytometry (FC)2 or enzyme immunoassay (EIA)3 have been developed. Recently molecular biological methods have also been introduced.v? The aim of our study was to compare the accuracy of FC and EIA with MLCT for HLA-B27 typing. MATERIALS AND METHODS PatientsEighty-one patients, 35 women and 46 men (aged between 19 and 89 years) with a suspected diagnosis of inflammatory disorders (ankylosing spondylitis, seronegative rheumatoid arthritis, reactive arthritis, psoriatic arthritis, psoriasis vulgaris and other unclassified rheumatic disorders) were included in the study. Determination of HLA-B27 was by MLCT, ElA and occasionally (if discrepant typing results were obtained) by Fe. In addition 20 healthy probands were typed for HLA-B27 by FC only in order to obtain appropriate values for

Autoimmune diseases are a clinically heterogeneous group of disorders that represent a challenge for the general practitioner in daily routine. Except for rheumatoid arthritis, which is one of the most frequent autoimmune diseases with a prevalence of approximately 1 % of the population, systemic autoimmune disorders are rare. Thus outside specialized wards it might be a challenge to diagnose the underlying autoimmune disease considering the often kaleidoscopic clinical manifestations. Together with careful anamnesis and suspicious clinical symptoms determination of specific autoantibodies can support the suspected diagnosis. The Austrian group of the European autoimmune standardization initiative (EASI) firstly published this guide 2009 with the aim to provide a map through the jungle of the biomarkers for autoimmune diseases for the general practitioner.