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Analytic characterization of flow cytometric assays for detection of immunoglobulin G on canine erythroid cells, including detection of dog erythrocyte antigen 1 on erythroid precursors

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  • 1 Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824.
  • | 2 Department of Medicine, College of Human Medicine, Michigan State University, East Lansing, MI 48824.
  • | 3 Department of Medicine, College of Human Medicine, Michigan State University, East Lansing, MI 48824.
  • | 4 Department of Small Animal Clinical Sciences, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824.
  • | 5 Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824.

Abstract

OBJECTIVE To develop and characterize flow cytometric assays for detecting IgG bound to canine erythrocytes and bone marrow erythroid precursors.

SAMPLE Blood samples from 20 healthy and 61 sick dogs with (n = 33) or without (28) immune-mediated hemolytic anemia (IMHA) and bone marrow samples from 14 healthy dogs.

PROCEDURES A flow cytometric assay for measurement of IgG on RBCs was developed, and appropriate positive control cells were generated. Analytic and diagnostic performance were characterized. The RBC IgG assay was then combined with density-gradient fractionation of aspirated bone marrow cells and a 2-color process to yield an assay for detecting IgG on nucleated RBCs (nRBCs). Cell sorting and cytologic examination confirmed target cell populations, and anti–dog erythrocyte antigen 1 (DEA1) blood-typing serum was used to generate IgG-positive nRBCs.

RESULTS Within- and between-run coefficients of variation for the RBC IgG assay were 0.1% to 13.9%, and > 90% of spiked IgG-positive RBCs were detected. Diagnostic sensitivity and specificity of the assay for detection of IMHA were 88% and 93%, respectively. Cytologic findings for sorted bone marrow fractions rich in early-, mid-, and late-stage nRBCs from 3 healthy dogs indicated 89% to 98% nRBC purity. After IgG coating with anti-DEA1 blood-typing serum, IgG was detected on nRBCs from DEA1-positive, but not DEA1-negative, healthy dogs.

CONCLUSIONS AND CLINICAL RELEVANCE The developed RBC IgG assay had favorable analytic and diagnostic performance for detection of IMHA in dogs and was successfully adapted to detect IgG on canine nRBCs of various maturation stages. The findings supported the presence of DEA1 on canine nRBCs.

Abstract

OBJECTIVE To develop and characterize flow cytometric assays for detecting IgG bound to canine erythrocytes and bone marrow erythroid precursors.

SAMPLE Blood samples from 20 healthy and 61 sick dogs with (n = 33) or without (28) immune-mediated hemolytic anemia (IMHA) and bone marrow samples from 14 healthy dogs.

PROCEDURES A flow cytometric assay for measurement of IgG on RBCs was developed, and appropriate positive control cells were generated. Analytic and diagnostic performance were characterized. The RBC IgG assay was then combined with density-gradient fractionation of aspirated bone marrow cells and a 2-color process to yield an assay for detecting IgG on nucleated RBCs (nRBCs). Cell sorting and cytologic examination confirmed target cell populations, and anti–dog erythrocyte antigen 1 (DEA1) blood-typing serum was used to generate IgG-positive nRBCs.

RESULTS Within- and between-run coefficients of variation for the RBC IgG assay were 0.1% to 13.9%, and > 90% of spiked IgG-positive RBCs were detected. Diagnostic sensitivity and specificity of the assay for detection of IMHA were 88% and 93%, respectively. Cytologic findings for sorted bone marrow fractions rich in early-, mid-, and late-stage nRBCs from 3 healthy dogs indicated 89% to 98% nRBC purity. After IgG coating with anti-DEA1 blood-typing serum, IgG was detected on nRBCs from DEA1-positive, but not DEA1-negative, healthy dogs.

CONCLUSIONS AND CLINICAL RELEVANCE The developed RBC IgG assay had favorable analytic and diagnostic performance for detection of IMHA in dogs and was successfully adapted to detect IgG on canine nRBCs of various maturation stages. The findings supported the presence of DEA1 on canine nRBCs.

Supplementary Materials

    • Supplementary Appendix S1 (PDF 217 kb)
    • Supplementary Appendix S2 (PDF 239 kb)
    • Supplementary Table S1 (PDF 204 kb)
    • Supplementary Table S2 (PDF 177 kb)
    • Supplementary Table S3 (PDF 169 kb)

Contributor Notes

Dr. Lucidi's present address is Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706.

Address correspondence to Dr. Lucidi (lucidi@wisc.edu).