COMPLEMENT ACTIVATION DURING BLOOD-SAMPLING PROCEDURES ALTERS THE EXPRESSION OF CD11B CD18 ON HUMAN NEUTROPHILS/

Background and objectives: Differences in blood sampling and separatio n techniques can affect the quantitative levels of activation markers on different leukocyte subsets. We examined the effect of two sampling procedures of EDTA blood on the quantitative levels of two markers, t he CD11b/CD18 antigen and the EG2 epitope on intracellular eosinophili c cationic protein (ECP), in neutrophils and eosinophils, respectively . Materials and methods: Sample I was collected directly after complet ion of blood donation by an open technique and constant flow from the transfer tube directly into EDTA tubes. After sampling, the transfer t ube was manually closed with a clamp. Sample II was collected 45 s lat er by the same technique by opening the clamp. Results: We found a sig nificantly (p<0.01) higher expression of CD11b/CD18 on neutrophils col lected by sampling procedure II than on those collected by sampling pr ocedure I. In contrast, we did not find any difference in the intracel lular ECP expression between sampling procedures I and II. To further explore the mechanisms for the observed upregulation of CD11b/CD18, fr agments of a transfer tube were incubated with normal human serum (NHS ) and heat-inactivated NHS (NHS56), respectively, for 60 min at +37 de grees C. Leukocytes from healthy blood donors were then incubated for 15 min at +37 degrees C with these serum preparations. The CD11b/CD18 expression was significantly higher (p<0.01) on neutrophils incubated with transfer-tube-activated NHS compared with NHS alone. However, whe n leukocytes were incubated with transfer tube activated NHS56, no dif ference was observed compared with incubation with NHS alone. In addit ion, by using confocal laser scanning microscopy, we could identify co mplement (C3c) deposits on the inner surface of the transfer tube frag ments incubated in NHS, but not in NHS56. Conclusions: The quantitativ e level of the activation marker CD11b/CD18 on neutrophils, but not th e EG2 epitope on intracellular ECP in eosinophils is significantly inc reased by a slight modification of the blood sampling procedure. It is suggested that the observed upregulation of CD11b/CD18 is caused by c omplement activation within the transfer tube. The results emphasize t he importance of in-house data on the effect of variations in sampling procedures, particularly when data from healthy blood donors are incl uded in clinical studies.