Canis ISSN: 2398-2942

Phagocytosis assay

Contributor(s): Michael Day, Helen Milner

Overview

  • Phagocytic cells (neutrophils and macrophages) take up particulate antigen for processing (breakdown) within specialized cytoplasmic compartments.
  • The ability of a phagocyte to take up antigen is enhanced in the presence of serum opsonins (IgG and complement C3) that coat the antigen and interact with specific receptors (Fc and C3b receptors, respectively) expressed on the cell membrane of the phagocyte.
  • The phagocytic assay tests the ability of neutrophils or macrophages to phagocytose particulate antigen in vitro. The test may also be used to measure the ability of serum to opsonize particulate antigen for phagocytosis.
  • Serum which is inefficient at opsonization may have low levels of immunoglobulin G (IgG) or complement (C3b).

Sampling

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Tests

Methodologies

  • In simplest terms, these tests involve incubating neutrophils or macrophages from the patient with particulate antigen, and after a predetermined period of incubation, assessing the numbers of internalized particles.
  • Testing neutrophil uptake is more readily performed than tests of macrophage function, for reasons as follows.
  • Neutrophils may be isolated from whole blood using a range of methods and set numbers of purified viable neutrophils can then be incubated with a set number of antigenic particles.
  • Alternatively, and simpler, the antigenic particles can be incubated with a whole blood sample.
  • In contrast, for tests of macrophage phagocytosis, blood mononuclear cells must first be isolated from whole blood, and then cultured on a glass surface.
  • The tests are then performed with the adherent fraction of cells which will include macrophages that have developed from the monocytes.
  • A range of particulate antigens have been utilised, including micro-organisms (eg Staphylococcus spp, Candida spp) or latex particles.
  • Biological antigens may be opsonized before incubation with the phagocytes.
  • Opsonization may be performed with a pool of normal serum or with autologous serum obtained from the patient.
  • The serum should be fresh, or fresh-frozen to retain complement activity.
  • Alternatively, the serum may be heat-inactivated (at 56°C for 30 minutes) to neutralize complement activity. The latter would thus test only the opsonic activity of immunoglobulin.
  • Antigen particles (opsonized or not) are incubated with phagocytic cells under defined conditions (duration, temperature) and at a predetermined ratios of particles to cells.
  • Efficiency of phagocytosis may be determined using a variety of different methods. The most simple is by microscopic examination of the phagocytes after culture.
  • Cells can be cytocentrifuged and stained, eg Diff Quik.
  • If 100 phagocytes are counted, the percentage of phagocytes that have engulfed antigen can be determined.
  • Additionally, the average number of particles per phagocyte might be determined.
  • Other, more automated systems, have been developed, eg the antigen may be labeled with a fluorochrome and the proportion of cells that have taken up antigen be determined by examination under a fluorescence microscope or by flow cytometry.

Control

  • The test should include appropriate controls, particularly the concomitant testing of samples from clinically normal control animals.

Availability

  • These tests will rarely be available outside research institutions.

Result Data

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Further Reading

Publications

Refereed papers

  • Recent references from VetMed Resource and PubMed.
  • Chamas P P C & Hagiwara M K (1998) Evaluation of neutrophilic function (chemotaxis, phagocytosis and microbicidal activity) in healthy dogs and in dogs suffering from recurrent deep pyoderma. Vet Immunol Immunopathol 64, 123-131.
  • Shearer D H & Day M J (1997) An investigation of phagocytosis and intracellular killing of Staphylococcus intermedius by canine neutrophils in vitroVet Immunol Immunopathol 58, 219-230.
  • Wisselink M A, van Kessel K P M and Willemse T (1997) Leukocyte mobilization to skin lesions, determination of cell surface receptors (CD11b/CD18) and phagocytic capacities of neutrophils in dogs with chronic deep pyoderma. Vet Immunol Immunopathol 57, 179-186.
  • DeBowes L J & Anderson N V (1991) Phagocytosis and erythrocyte antibody-rosette formation by three populations of mononuclear phagocytes obtained from dogs treated with glucocorticoids. Am J Vet Res 52, 869-872.

Other sources of information

  • Day M J (1999) Clinical Immunology of the Dog and Cat. Manson Publishing, London.


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