Preliminary evaluation of a flow cytometric assay with microsphere controls for the detection of platelet-bound antibodies in canine immune thrombocytopenia.
autoimmune diseases
blood platelets
hematologic diseases
Journal
Veterinary clinical pathology
ISSN: 1939-165X
Titre abrégé: Vet Clin Pathol
Pays: United States
ID NLM: 9880575
Informations de publication
Date de publication:
Sep 2022
Sep 2022
Historique:
revised:
26
10
2021
received:
03
08
2021
accepted:
11
11
2021
pubmed:
17
3
2022
medline:
15
9
2022
entrez:
16
3
2022
Statut:
ppublish
Résumé
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. We aimed to develop control reagents to facilitate the characterization of canine platelet surface-associated immunoglobulin (PSAIg) in flow cytometric assays. 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. 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). 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.
Sections du résumé
BACKGROUND
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
OBJECTIVE
We aimed to develop control reagents to facilitate the characterization of canine platelet surface-associated immunoglobulin (PSAIg) in flow cytometric assays.
METHODS
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
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
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.
Substances chimiques
Immunoglobulin G
0
Immunoglobulin M
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
330-338Subventions
Organisme : American Kennel Club Canine Health Foundation
ID : 02536-MOU
Organisme : American Kennel Club Canine Health Foundation
ID : CHF 2052
Informations de copyright
© 2022 American Society for Veterinary Clinical Pathology.
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