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Clinical Chemistry 54: 911-915, 2008; 10.1373/clinchem.2007.101337
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(Clinical Chemistry. 2008;54:911-915.)
© 2008 American Association for Clinical Chemistry, Inc.

Brief Communications

A Simple Assay to Measure Phagocytosis of Live Bacteria

Heike Bicker1, Conny Höflich1, Kerstin Wolk2, Katrin Vogt1, Hans-Dieter Volk1 and Robert Sabat2,a

1 Institute of Medical Immunology and 2 Interdisciplinary Group of Molecular Immunopathology, Dermatology/Medical Immunology, University Hospital Charité, Berlin, Germany;

aaddress correspondence to this author at: Interdisciplinary Group of Molecular Immunopathology, Dermatology/Medical Immunology, University Hospital Charité, Campus Charité Mitte, Charitéplatz 1, D-10117 Berlin, Germany. Fax +49 30 450 518964; e-mail robert.sabat{at}charite.de.


Abstract

Background: The phagocytosis of pathogens is essential for fighting infections. No assay is available, however, to measure both engulfment and degradation of bacteria under conditions similar to those in vivo. We sought to develop a flow cytometric assay to measure the engulfment and degradation of live bacteria by human blood monocytes and granulocytes.

Methods: We generated enhanced green fluorescent protein (EGFP)-expressing Eschericha coli by transforming E. coli with the plasmid vector pEGFP. We used these bacteria in a flow cytometric assay to measure both engulfment and degradation of living bacteria by monocytes and granulocytes in human whole blood from fresh, heparinized venous blood samples. To determine whether the test detected differences between healthy individuals and patients with secondary immunodeficiencies, we compared the phagocytosis of monocytes and granulocytes measured in blood samples from immunosuppressed kidney transplantation patients and from patients with postoperative sepsis in immunoparalysis with phagocytosis measured in samples from age-matched healthy individuals.

Results: In samples from healthy individuals, we found that in both monocytes and granulocytes bacterial degradation was negatively correlated with the age of the sample donor. Furthermore, we detected decreased bacterial engulfment in granulocytes from septic patients and decreased bacterial degradation in monocytes from immunosuppressed kidney transplantation patients.

Conclusions: This flow cytometric assay measures the engulfment and degradation of live bacteria by human blood monocytes and granulocytes. By means of this assay we detected significant differences between healthy controls and patients with secondary immunodeficiencies that may contribute to the increased incidence of infection complications seen in these patients.






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Copyright © 2008 by the American Association for Clinical Chemistry.