PHAGOTEST® is a complete test kit for the investigation of the phagocytic function of granulocytes and monocytes [1, 2] in whole blood. This test kit allows the quantitative determination of leukocyte phagocytosis in heparinized whole blood. It contains fluorescein (FITC) labelled opsonized bacteria (E. coli-FITC) and necessary reagents. It measures the overall percentage of monocytes and granulocytes showing phagocytosis in general (ingestion of one or more bacteria per cell) and the individual cellular phagocytic activity (number of bacteria per cell). The evaluation of the results can be done either by flow cytometry or by fluorescence microscopy.

APPLICATIONS

PHAGOTEST® is intended to investigate the phagocytic activity found in various disorders and to evaluate the effects of drugs.

Abnormal phagocytosis can occur with a variety of clincal disorders (3, 4). The defects can be associated with the neutrophil itself or with an immunoglobulin or complement defect. Known inborne defects are actin dysfunction, tuftsin deficiency and complement receptor C3bi deficiency. These deficiencies can result in increased susceptibility to infection due to defective neutrophil phagocytosis.
Acquired defects associated with altered phagocytic activity can be observed in trauma, diabetes, renal failure, and infection. Reduced phagocytosis was found in patients with recurrent bacterial skin and sinopulmonary infections (5), in patients with wound infections from burns (6), in patients with AIDS (7), in neonates or in elderly people.

Various immunomodulators (cytokines such as interleukin-2 or interferon-, lactic acid bacteria and plant extracts such as from Echinaceae Purpureae herba) can increase the phagocytic activity of neutrophils and monocytes. These effects can be investigated in vitro or ex vivo (8, 9).

PHAGOTEST® also allows the study of the phagocytic activity of HL-60 promyelocytic leukemia cells or of isolated monocytes or macrophages.

The test kit is compatible with blood of mice, rats, rabbits, dogs, cattle and other species.

TEST PRINCIPLES

Phagocytosis by polymorphonuclear neutrophils and monocytes constitutes an essential arm of host defense against bacterial or fungal infections. The phagocytic process can be separated into several major stages: chemotaxis (migration of phagocytes to inflammatory sites), attachment of particles to the cell surface of phagocytes, ingestion (phagocytosis) and intracellular killing by oxygen-dependent and oxygen-independent mechanisms (1, 2).

PHAGOTEST® allows the quantitative determination of leukocyte phagocytosis (ingestion of bacteria). It measures the percentage of phagocytes which have ingested bacteria and their activity (number of bacteria per cell). The phagocytosis test kit contains fluorescein-labelled opsonized Escherichia coli bacteria and other necessary reagents. Heparinzed whole blood is incubated with the fluorescein-labelled E.coli bacteria at 37°C, a negative control sample remains on ice. The phagocytosis is stopped by placing the samples on ice and adding QUENCHING SOLUTION. This solution allows the discrimination between attachment and internalization of bacteria by quenching the FITC fluorescence of surface bound bacteria leaving the fluorescence of internalized particles unaltered. After two washing steps with WASHING SOLUTION erythrocytes are then removed by addition of LYSING SOLUTION. The DNA STAINING SOLUTION, which is added just prior flow cytometric analysis, excludes aggregation artifacts of bacteria or cells. The percentage of cells having performed phagocytosis are then analyzed as well as their mean fluorescence intensity (number of ingested bacteria).

The E.coli bacteria are opsonized with immunoglobulin and complement of pooled sera. Cells of the phagocytic system (monocytes, polymorphonuclear neutrophils) have receptors for a complement component (C3b) and for the constant part of the immunoglobulin molecule (Fc) mediating the adhesion of the bacteria to the cell surface. By utilizing both opsonized and nonopsonized bacteria, which are also available, both opsonic capacity and phagocytosis can be measured at the same time. Thus, it can be determined whether abnormal phagocytosis is due to a failure in the opsonization process or to a defect in the ingestion capability of the phagocyte.

REAGENTS PROVIDED

PHAGOTEST® contains reagents for 100 tests:

• Fluorescein-labelled opsonized Escherichia coli bacteria
• QUENCHING SOLUTION
• DNA STAINING SOLUTION
• LYSING SOLUTION
• WASHING SOLUTION

REFERENCES

(1)	Roitt, I.M., Brostoff, J. & D.K. Male. 1996. Immunology, 4th ed. Gower Medical Publishing Ltd., London.
(2)	Sawyer, D.W., Donowitz, G.R. & G.L. Mandell. 1989. Polymorphonuclear neutrophils: An effective antimicrobial force. Rev. Infect. Dis. 11: S1532-S1544.
(3)	Donadebian, H. D. 1989. Congenital and aquired neutrophil abnormalities. In: Klempner, M.S. et al. (eds) Phagocytes and Disease. Kluwer, Dordrecht Boston New York, pp 103-118.
(4)	Robinson, J.P. & W.O. Carter. 1993. Flow cytometric analysis of granulocytes. In: Bauer, K.D. et al. (eds) Clinical Flow Cytometry, Principles and Applications. Williams & Wilkins, Baltimore, pp 405-433.
(5)	Bassoe, C.-F. 1984. Flow cytometric studies on phagocyte function in bacterial infections. Acta path. microbiol. immunol. scand. Sect. C, 92: 167 - 171.
(6)	Crogan, J.B. 1976. Altered neutrophil phagocytic function in burn patients. J. Trauma 16: 734-738.
(7)	Dobmeyer, T.S., Raffel, B. Dobmeyer, J.M., Findhammer, S., Klein, S.A., Kabelitz, D. Hoelzer, D., Helm, E.B. & Rossol.1995. Decreased function of monocytes and granulocytes during HIV-1 infection correlates with CD4 cell counts. Eur. J. Med. Res. 1: 9-15.
(8)	Schiffrin, E.J., Rochat, F., Link-Amster, H., Aeschlimann, J.M. & A. Donnet-Hughes. 1995. Immunomodulation of human blood cells following ingestion of lactic acid bacteria. J. Dairy Sci (USA) 78: 491-497.
(9)	Brosche, T. & D. Platt. 1995. Nutritional factors and age-associated changes in cellular immunity and phagocytosis: A mini review. Aging Immunol Infect Dis 6: 31-42.

For further information please contact:

Dr. Werner Hirt
Head of Immunology, Cell Biology and Flow Cytometry
+49 6221 9105-50
e-Mail: w.hirt@orpegen.com