Precursor-targeted immune-mediated anemia has been described in dogs in various studies1–8 and is the clinical diagnosis for approximately 25% of dogs undergoing bone marrow biopsies at the Michigan State University Veterinary Medical Center. However, the pathogenesis of PIMA is unclear. The condition is characterized by nonregenerative anemia, ineffective erythropoiesis, and, in most affected dogs evaluated at the authors’ institution, rubriphagocytosis, which is the phagocytosis of intact erythroid precursors (ie, nRBCs). Rubriphagocytosis in dogs with PIMA appears to be quite stage selective, with the maturation stage of the phagocytized nRBCs (ie, early, mid, or late) aligning with a spectrum of cytologic and histologic bone marrow patterns that commonly include mild to severe myelofibrosis.8
An immune-mediated mechanism for the pathogenesis of PIMA has been proposed, but not established, on the basis of rubriphagocytosis,1,3,4 apparent responses to immunosuppressive treatment,1–6 apparent relapses after withdrawal of immunosuppressive treatment,1,2,5,6 and evidence of concurrent IMHA in some affected dogs.3–7 However, responses to immunosuppressive drugs are slow, relapses are common, and many affected dogs die or are euthanized because of clinical pessimism or financial constraints.
Flow cytometry has been used to diagnose and characterize IMHA in dogs9–12 and humans.13–15 This method appears more sensitive than standard agglutination tests for IgG detection.9–11,13–15 A potential PIMA counterpart that may be mediated by IgG directed against nRBCs has been identified in humans,16–18 and flow cytometry was used to identify IgG on bone marrow–derived nRBCs in 1 instance.18 These findings suggest that flow cytometry could also be used to assess for increases in the amounts of IgG bound to nRBCs in dogs with PIMA.
The purpose of the study reported here was to develop a method that would allow isolation and flow cytometric assessment of IgG on the surface of various maturation stages of nRBCs from canine bone marrow samples. The initial goal was to develop an assay for measurement of IgG bound to RBCs and then apply the assay to marrow-derived nRBCs. Our intention was that these assays would facilitate various blood and stage-selective marrow assessments for dogs with PIMA as well as other disorders and help determine whether IgG has a role in the pathogenesis of PIMA in dogs. A better understanding of the pathophysiologic mechanisms underlying PIMA may lead to improvements in management strategies and treatment responses for affected dogs.
This manuscript represents a portion of a dissertation submitted by Dr. Lucidi to the Department of Pathobiology and Diagnostic Investigation, Michigan State University, as partial fulfillment of the requirements for a Doctor of Philosophy degree.
Supported in part by the Immunology and Serology Laboratory, Michigan State University.
Presented in part in abstract form at the 65th Annual Meeting of the American College of Veterinary Pathologists and the 49th Annual Meeting of the American Society for Veterinary Clinical Pathology, Atlanta, November 2014.
The authors thank Dr. Adam Aulbach, Rose Wahl, Kristin Koehl, and Dr. Andras Komaromy for technical assistance with dogs and samples, and Dr. William Jackson for his help with statistical analyses.
Bovine serum albumin
Complement component 3
Cluster of differentiation
Coefficient of variation
Dog erythrocyte antigen 1
Immune-mediated hemolytic anemia
Median fluorescence intensity
Precursor-targeted immune-mediated anemia
BD K2-EDTA tubes, Becton-Dickinson, San Jose, Calif.
Animal Blood Resources International, Stockbridge, Mich.
Becton-Dickinson, San Jose, Calif.
BSA (heat shock fraction, protease free, essentially globulin free, pH 7, ≥ 98%), Sigma-Aldrich, St Louis, Mo.
Kirkegaard & Perry Laboratories Inc, Gaithersburg, Md.
FACSCalibur, Becton-Dickinson, San Jose, Calif.
CellQuest Pro software, Becton-Dickinson, San Jose, Calif.
Sigma-Aldrich Corp, St Louis, Mo.
Percoll (pH, 8.5 to 9.5; 25°C; cell culture tested), Sigma-Aldrich Corp, St Louis, Mo.
Life Technologies, Grand Island, NY.
Mouse anti–dog CD18 monoclonal antibody, clone CA1.4E9, isotype IgG1, AbD Serotec, Raleigh, NC.
Goat anti–mouse IgG:RPE (rat adsorbed) polyclonal IgG antibody, AbD Serotec Ltd, Raleigh, NC.
BD Influx, Becton-Dickinson, San Jose, Calif.
BD FACS Sortware sorter software, Becton-Dickinson, San Jose, Calif.
BSA solution (22% in saline solution), Sigma-Aldrich Corp, St Louis, Mo.
GraphPad Prism, GraphPad Software Inc, San Diego, Calif.
Analyse-it for Microsoft Excel, version 4.65.2, build 6012.38758, Analyse-it Software Ltd, Leeds, England.
Erythrolyse RBC lysing buffer (10X), diluted in ddH2O, AbD Serotec Ltd, Raleigh, NC.
1. Stockham SL, Ford RB, Weiss DJ. Canine autoimmune hemolytic disease with a delayed erythroid regeneration. J Am Anim Hosp Assoc 1980;16:927–931.
2. Weiss DJ, Stockham SL, Willard MD, et al. Transient erythroid hypoplasia in the dog: report of five cases. J Am Anim Hosp Assoc 1982;18:353–359.
3. Jonas LD, Thrall MA, Weiser MG. Nonregenerative form of immune-mediated hemolytic anemia in dogs. J Am Anim Hosp Assoc 1987;23:201–204.
4. Holloway SA, Meyer DJ, Mannella C. Prednisolone and danazol for treatment of immune-mediated anemia, thrombocytopenia, and ineffective erythropoiesis in a dog. J Am Vet Med Assoc 1990;197:1045–1048.
5. Scott-Moncrieff JC, Reagan WJ, Glickman LT, et al. Treatment of nonregenerative anemia with human γ-globulin in dogs. J Am Vet Med Assoc 1995;206:1895–1900.
6. Stokol T, Blue JT, French TW. Idiopathic pure red cell aplasia and nonregenerative immune-mediated anemia in dogs: 43 cases (1988–1999). J Am Vet Med Assoc 2000;216:1429–1436.
7. Weiss DJ. Bone marrow pathology in dogs and cats with non-regenerative immune-mediated haemolytic anaemia and pure red cell aplasia. J Comp Pathol 2008;138:46–53.
8. Lucidi CA, de Rezende CLE, Jutkowitz LA, et al. Histologic and cytologic bone marrow findings in dogs with suspected precursor-targeted immune-mediated anemia and associated phagocytosis of erythroid precursors. Vet Clin Pathol 2017;46:401–415.
9. Wilkerson MJ, Davis E, Shuman W, et al. Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia. J Vet Intern Med 2000;14:190–196.
10. Quigley KA, Chelack BJ, Haines DM, et al. Application of a flow cytometric erythrocyte immunofluorescence assay in dogs with immune-mediated hemolytic anemia and comparison to the direct antiglobulin test. J Vet Diagn Invest 2001;13:297–300.
11. Kucinskiene G, Schuberth H, Leibold W, et al. Flow cytometric evaluation of bound IgG on erythrocytes of anaemic dogs. Vet J 2005;169:303–307.
12. Morley P, Mathes M, Guth A, et al. Anti-erythrocyte antibodies and disease associations in anemic and nonanemic dogs. J Vet Intern Med 2008;22:886–892.
13. Chaudhary R, Das SS, Gupta R, et al. Application of flow cytometry in detection of red-cell-bound IgG in Coombs-negative AIHA. Hematology 2006;11:295–300.
14. Lin JS, Hao TC, Lyou JY, et al. Clinical application of a flow cytometric direct antiglobulin test. Transfusion 2009;49:1335–1346.
15. Wang Z, Shi J, Zhou Y, et al. Detection of red blood cell-bound immunoglobulin G by flow cytometry and its application in the diagnosis of autoimmune hemolytic anemia. Int J Hematol 2001;73:188–193.
16. Mangan KF, Besa EC, Shadduck RK, et al. Demonstration of two distinct antibodies in autoimmune hemolytic anemia with reticulocytopenia and red cell aplasia. Exp Hematol 1984;12:788–793.
17. Meyer RJ, Hoffman R, Zanjani ED. Autoimmune hemolytic anemia and periodic pure red cell aplasia in systemic lupus erythematosus. Am J Med 1978;65:342–345.
18. Van De Loosdrecht AA, Hendriks DW, Blom NR, et al. Excessive apoptosis of bone marrow erythroblasts in a patient with autoimmune haemolytic anaemia with reticulocytopenia. Br J Haematol 2000;108:313–315.
19. Lucidi CA, Takahira RK, Gerlach JA, et al. Flow cytometric assessment of canine erythrocytes and platelets for dog erythrocyte antigen 1.1. Vet Clin Pathol 2011;40:435–443.
20. Arndt PA, Garratty G. A critical review of published methods for analysis of red cell antigen-antibody reactions by flow cytometry, and approaches for resolving problems with red cell agglutination. Transfus Med Rev 2010;24:172–194.
21. Weiss DJ, Blauvelt M, Sykes J, et al. Flow cytometric evaluation of canine bone marrow differential counts. Vet Clin Pathol 2000;29:97–104.
24. Piek CJ, Teske E, van Leeuwen MW, et al. Good agreement of conventional and gel-based direct agglutination test in immune-mediated haemolytic anaemia. Acta Vet Scand 2012;54:10.
25. Slappendel RJ. The diagnostic significance of the direct antiglobulin test (DAT) in anemic dogs. Vet Immunol Immunopathol 1979;1:49–59.
26. Day MJ. Serial monitoring of clinical, haematological and immunological parameters in canine autoimmune haemolytic anaemia. J Small Anim Pract 1996;37:523–534.
27. Caviezel LL, Raj K, Giger U. Comparison of 4 direct Coombs' test methods with polyclonal antiglobulins in anemic and nonanemic dogs for in-clinic or laboratory use. J Vet Intern Med 2014;28:583–591.
28. Loken MR, Shah VO, Dattilio KL, et al. Flow cytometric analysis of human bone marrow: I. Normal erythroid development. Blood 1987; 69:255–263.
29. Civin CI, Loken MR. Cell surface antigens on human marrow cells: dissection of hematopoietic development using monoclonal antibodies and multiparameter flow cytometry. Int J Cell Cloning 1987;5:267–288.
30. Chen K, Liu J, Heck S, et al. Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis. Proc Natl Acad Sci U S A 2009;106:17413–17418.
31. Bony V, Gane P, Bailly P, et al. Time-course expression of polypeptides carrying blood group antigens during human erythroid differentiation. Br J Haematol 1999;107:263–274.
32. Blacklock HA, Katz F, Michalevicz R, et al. A and B blood group antigen expression on mixed colony cells and erythroid precursors: relevance for human allogeneic bone marrow transplantation. Br J Haematol 1984;58:267–276.
33. Wada H, Suda T, Miura Y, et al. Expression of major blood group antigens on human erythroid cells in a two phase liquid culture system. Blood 1990;75:505–511.
34. Rowley SD, Donato ML, Bhattacharyya P. Red blood cell-incompatible allogeneic hematopoietic progenitor cell transplantation. Bone Marrow Transplant 2011;46:1167–1185.