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International archives of allergy and immunology

Direct measurement of neutrophil F-actin content in microvolume whole blood samples.


PMID 8768799

Abstract

The traditional methods of measuring F-actin content in neutrophils required a large blood sample and a series of isolation procedures. To reduce the disturbing effect on neutrophils, a new method was designed to measure the neutrophil F-actin content directly in microvolume whole blood samples. The neutrophil F-actin content was measured in 100 microliters of whole blood directly after formyl-Met-Leu-Phe (FMLP) stimulation and lysis of red blood cells (with FACS lysing solution, Becton, Dickinson Immunocytometry Systems, San Jose, Calif.). This method resulted in a typical dose-dependent increase in the neutrophil F-actin content in response to FMLP similar to that using isolated neutrophils. However, the relative F-actin content of whole blood samples was significantly higher than those of isolated neutrophils at 60 and 300 s after stimulation (2.44 +/- 0.21 vs. 2.00 +/- 0.22 at 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 stimulation showed a subpopulation (17.5 +/- 2.7%) of lower F-actin content, which cannot be definitely identified in the isolated neutrophil samples. Furthermore, the neutrophil actin response to FMLP in a pair of premature twins was analyzed using this method. The response of the healthy twin is similar to that of the adult volunteers while that of the septic one is characterized by an increasing number of nonresponsive cells as the clinical condition deteriorated. This new method is effective in evaluating the neutrophil F-actin content even in premature infants. It is characterized by avoiding most of the isolation procedures which might have an activating effect on neutrophils. Besides, the study of the twins also indicated the usefulness of this method in monitoring the clinical course of neonatal sepsis.

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