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Phagocytosis assay

ISSN 2398-2977

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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)   Blood: biochemistry - gamma globulins   or complement (C3b).

Uses

Alone

  • Primary immunodeficiency or secondary suppression of phagocytic ability may be indicated by an impaired ability of test cells to phagocytose, in comparison to cells from control normal animals.

In combination

  • Assessment of phagocytic function is generally performed as part of an overall assessment of immunological competence. 
  • A screen of immune function might also include quantification of serum immunoglobulins and complement, and measurement of the numbers and functional capacity of blood lymphocytes.
  • Other tests can be used to measure the ability of phagocytes to respond to chemotactic stimuli, to adhere to various surfaces, to activate internal biochemical pathways when stimulated (eg the respiratory burst, inducible nitric oxide), or to kill phagocytosed micro-organisms.
  • Fluorescence activated cell sorting (FACS) may be employed to assess the expression of essential cell surface adhesion molecules, eg CD11b/CD18.
  • Technologies such as PCR   Polymerase chain reaction (PCR)  , northern and southern blotting can be used to determine gene mutations. Inherited forms of neutrophil dysfunction have not yet been described in the horse, but do occur in man and other species.

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 Staphylococcusspp   Staphylococcus spp  , Candidaspp   Candida albicans  ) 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/132.8°F for 30 min) 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 ratio 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   Staining techniques: Diffquick stain  .
  • 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 PubMed and VetMedResource.
  • Michelotto P V, Muehlmann L A, Zanatta A L, Bieberbach E W, Fernandes L C & Nishiyama A (2010) Platelet-activating factor and evidence of oxidative stress in the bronchoalveolar fluid of Thoroughbred colts during race training. J Vet Intern Med 24 (2), 414-419 PubMed.
  • Gardner R B, Nydam D V, Luna J A, Bicalho M L, Matychak M B & Flaminio M J (2007) Serum opsonization capacity, phagocytosis, and oxidative burst activity in neonatal foals in the intensive care unit. J Vet Intern Med 21 (4), 797-805 PubMed.
  • McTaggart C, Penhale J & Raidala S L (2005) Effect of plasma transfusion on neutrophil function in healthy and septic foals. Aust Vet J 83 (8), 499-505 PubMed.
  • Demmers S, Johannisson A, Gröndahl G & Jensen-Waern M (2001) Neutrophil functions and serum IgG in growing foals. Equine Vet J 33 (7), 676-680 PubMed.
  • McTaggart C, Yovich J V, Penhale J & Raidal S L (2001) A comparison of foal and adult horse neutrophil function using flow cytometric techniques. Res Vet Sci 71 (1), 73-79 PubMed.
  • Raidal S L, Rose R J & Love D N (2001) Effects of training on resting peripheral blood and BAL-derived leucocyte function in horses. Equine Vet J 33 (3), 238-243 PubMed.
  • Chanter N, Talbot N C, Newton J R, Hewson D & Verheyen K (2000) Streptococcus equiwith truncated M-proteins isolated from outwardly healthy horses. Microbiology 146, 1361-1369 PubMed.
  • Flaminio M J, Rush B R, Davis E G, Hennessy K, Shuman W & Wilkerson M J (2000) Characterization of peripheral blood and pulmonary leukocyte function in healthy foals. Vet Immunol Immunopathol 73 (3-4), 267-285 PubMed.
  • Anzai T, Timoney J F, Kuwamoto Y, Fujita Y, Wada R & Inoue T (1999) In vivo pathogenicity and resistance to phagocytosis of Streptococcus equistrains with different levels of capsule expression. Vet Microbiol 67 (4), 277-286 PubMed.