Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Sep;51(3):330-338.
doi: 10.1111/vcp.13093. Epub 2022 Mar 15.

Preliminary evaluation of a flow cytometric assay with microsphere controls for the detection of platelet-bound antibodies in canine immune thrombocytopenia

Marjory B Brooks  1 , Haruhiko Maruyama  2 , Signe E Cremer  3 , Robert Goggs  4 , Marnin A Forman  5 , Michael Koch  6 , Julia Merriam  7 , Kelly Makielski  8 , Austin Viall  9 , Dana N LeVine  8
Affiliations

Preliminary evaluation of a flow cytometric assay with microsphere controls for the detection of platelet-bound antibodies in canine immune thrombocytopenia

Marjory B Brooks et al. Vet Clin Pathol. 2022 Sep.

Abstract

Background: Canine immune thrombocytopenia (ITP) ranges from a mild to severe bleeding disorder, and platelet counts do not reliably predict clinical disease course. The detection of platelet autoantibodies may further define the disease phenotype, but variability in assay configurations and a lack of well-characterized controls limit the diagnostic utility of anti-platelet antibody assays.

Objectives: We aimed to develop control reagents to facilitate the characterization of canine platelet surface-associated immunoglobulin (PSAIg) in flow cytometric assays.

Methods: Silica microspheres were coated with canine IgG and IgM to assess the reactivity of goat and rabbit origin anti-canine immunoglobulin reagents. They were also used as positive controls in the PSAIg assay. Preliminary assay evaluation and determination of sample stability used PRP isolated from seven healthy dogs and 26 dogs newly diagnosed with thrombocytopenia.

Results: Blood sample stability was established for up to a 48-hour storage time. The conjugated positive control microspheres demonstrated stable fluorescent labeling over a 2-year observation period. Rabbit and goat origin anti-dog IgM fluorescent antibody labels reacted nonspecifically with canine IgG. Rabbit origin anti-dog IgG antibody demonstrated greater class specificity for canine IgG than a goat origin antibody. Thrombocytopenic dogs had a broad range of membrane-bound immunoglobulin. Median PSAIgG for dogs with primary or secondary ITP (18.4%, 34.1%, respectively) were significantly higher than controls (3.8%, P < .05).

Conclusions: The described assay reagents and procedures provide positive controls and allow consistent thresholding to define a positive test result, suitable for any flow cytometer. A rabbit anti-dog IgG fluorescent label demonstrated specificity for canine IgG and was useful for the detection of PSAIgG in thrombocytopenic dogs.

Keywords: autoimmune diseases; blood platelets; hematologic diseases.

PubMed Disclaimer

References

REFERENCES

    1. Botsch V, Küchenhoff H, Hartmann K, Hirschberger J. Retrospective study of 871 dogs with thrombocytopenia. Vet Rec. 2009;164:647-651.
    1. Dircks BH, Schuberth H-J, Mischke R. Underlying diseases and clinicopathologic variables of thrombocytopenic dogs with and without platelet-bound antibodies detected by use of a flow cytometric assay: 83 cases (2004-2006). J Am Vet Med Assoc. 2009;235:960-966.
    1. Lewis DC, Meyers KM. Canine idiopathic thrombocytopenic purpura. J Vet Intern Med. 1996;10:207-218.
    1. Bachman DE, Forman MA, Hostutler RA, Corn S, Lin J, Kociba GJ. Prospective diagnostic accuracy evaluation and clinical utilization of a modified assay for platelet-associated immunoglobulin in thrombocytopenic and nonthrombocytopenic dogs. Vet Clin Pathol. 2015;44:355-368.
    1. Grindem CB, Breitschwerdt EB, Perkins PC, Cullins LD, Thomas TJ, Hegarty BC. Platelet-associated immunoglobulin (antiplatelet antibody) in canine Rocky Mountain spotted fever and ehrlichiosis. J Am Anim Hosp Assoc. 1999;35:56-61.

LinkOut - more resources