Complement Activation during Blood Sampling Procedures Alters the Expression of CD11b/CD18 on Human Neutrophils

Abstract

Background and objectives: Differences in blood sampling and separation 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, the CD11b/CD18 antigen and the EG2 epitope on intracellular eosinophilic cationic protein (ECP), in neutrophils and eosinophils, respectively. Materials and methods: Sample I was collected directly after completion of blood donation by an open technique and constant flow from the transfer tube directly into EDTA tubes. After sampling, the transfer tube was manually closed with a clamp. Sample II was collected 45 s later by the same technique by opening the clamp. Results: We found a significantly (p < 0.01) higher expression of CD11b/CD18 on neutrophils collected by sampling procedure II than on those collected by sampling procedure I. In contrast, we did not find any difference in the intracellular ECP expression between sampling procedures I and II. To further explore the mechanisms for the observed upregulation of CD11b/CD18, fragments of a transfer tube were incubated with normal human serum (NHS) and heat-inactivated NHS (NHS₅₆), respectively, for 60 min at +37 °C. Leukocytes from healthy blood donors were then incubated for 15 min at +37 °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, when leukocytes were incubated with transfer tube activated NHS₅₆, no difference was observed compared with incubation with NHS alone. In addition, by using confocal laser scanning microscopy, we could identify complement (C3c) deposits on the inner surface of the transfer tube fragments incubated in NHS, but not in NHS₅₆. Conclusions: The quantitative level of the activation marker CD11b/CD18 on neutrophils, but not the EG2 epitope on intracellular ECP in eosinophils is significantly increased by a slight modification of the blood sampling procedure. It is suggested that the observed upregulation of CD11b/CD18 is caused by complement activation within the transfer tube. The results emphasize the importance of in-house data on the effect of variations in sampling procedures, particularly when data from healthy blood donors are included in clinical studies.