DIRECT MEASUREMENT OF NEUTROPHIL F-ACTIN CONTENT IN MICROVOLUME WHOLE-BLOOD SAMPLES

Background: The traditional methods of measuring F-actin content in ne utrophils required a large blood sample and a series of isolation proc edures. To reduce the disturbing effect on neutrophils, a new method w as designed to measure the neutrophil F-actin content directly in micr ovolume whole blood samples. Method: The neutrophil F-actin content wa s measured in 100 mu l of whole blood directly after formyl-Met-Leu-Ph e (FMLP) stimulation and lysis of red blood cells (with FAGS lysing so lution, Becton, Dickinson Immunocytometry Systems, San Jose, Calif.). Results: This method resulted in a typical dose-dependent increase in the neutrophil F-actin content in response to FMLP similar to that usi ng isolated neutrophils. However, the relative F-actin content of whol e blood samples was significantly higher than those of isolated neutro phils at 60 and 300 s after stimulation (2.44+/-0.21 vs. 2.00+/-0.22 a t 60 s, p<0.05, n=16; 1.77+/-0.19 vs. 1.48+/-0.19 at 300 s, p<0.05, n= 16). The histograms of whole blood samples at 30 and 60 s after stimul ation showed a subpopulation (17.5+/-2.7%) of lower F-actin content, w hich cannot be definitely identified in the isolated neutrophil sample s. Furthermore, the neutrophil actin response to FMLP in a pair of pre mature twins was analyzed using this method. The response of the healt hy twin is similar to that of the adult volunteers while that of the s eptic one is characterized by an increasing number of nonresponsive ce lls as the clinical condition deteriorated. Conclusion: This new metho d is effective in evaluating the neutrophil F-actin content even in pr emature infants. It is characterized by avoiding most of the isolation procedures which might have an activating effect on neutrophils. Besi des, the study of the twins also indicated the usefulness of this meth od in monitoring the clinical course of neonatal sepsis.