Diagnosis of Immune-mediated Hemolytic Anemia

Kristin M. Hiers, DVM; Kenneth Latimer, DVM, PhD; Perry J. Bain, DVM, PhD; Paula M. Krimer, DVM, DVSc

Class of 2003 (Hiers) and Department of Pathology (Latimer, Bain, Krimer), College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7388

Introduction

Immune-mediated hemolytic anemia (IMHA) is an accelerated destruction of red blood cells due to the attachment of immunoglobulin and/or complement to the erythrocyte membrane. It is a common cause of severe anemia and hemolysis in dogs with a mortality rate of over 40%.^1,2

IMHA can be classified as primary or secondary depending on the cause of the red cell destruction. Secondary IMHA involves hemolysis due to an immunologic reaction to a "non-self" antigen. Possible causes include infection, chemical agents, drugs, or neoplastic disease.^3,4 Primary IMHA is synonymous with idiopathic and autoimmune-mediated hemolytic anemia (AIHA). There is a true autoantibody specific for the self-antigen of the red cell membrane. Normally the immune system recognizes self-antigen and does not respond to it. This is the principle of self-tolerance. It is thought that when something triggers a change in immunoregulation, such as infection, there may be a loss of self-tolerance and subsequent autoimmune disease. There is a genetic predisposition for the development of AIHA, with a greater prevalence in Cocker Spaniels (representing up to 40% of all dogs), Old English Sheepdogs, Poodles, and Irish Setters.^4,5

Diagnosis of IMHA

When a patient presents with a history and clinical signs that are consistent with IMHA, multiple clinicopathological parameters must be considered in the diagnosis of this disease. These include the presence of anemia, autoagglutination, spherocytes, positive direct antiglobulin (Coombs’) test, and the elimination of any other underlying cause of anemia. No single finding is pathognomonic for IMHA, so careful interpretation is important.

The anemia of IMHA is often severe, with a PCV of less than 20%. The anemia is usually regenerative, since erythropoeisis is not adversely affected unless the immune response affects hematopoietic cells. Therefore, reticulocytosis, polychromasia, anisocytosis, and nucleated erythrocytes may be present.³ One-third of all cases of IMHA present with a nonregenerative anemia, possibly due to acute disease onset (lack of time for a regenerative response) or antibodies directed against erythroid precursors may account for this finding.⁴

Microscopic evaluation of a blood smear can provide important evidence to support a diagnosis of IMHA. Autoagglutination and/or the presence of spherocytes are commonly described as hallmark laboratory findings of IMHA.^2,4 Autoagglutination may be seen grossly or microscopically (Figs. 1 & 2) and must be distinguished from rouleaux (Fig. 3). True agglutination persists after washing or dilution of the red cells with saline, while rouleaux will disperse. Persistent autoagglutination can confirm IMHA and eliminate the need for direct antiglobulin (Coombs’) testing, but is not a consistent finding in all cases. One study reported that only 10% of 42 cases evaluated demonstrated persistent autoagglutination.⁵ Spherocytes (Fig. 4) are small abnormal erythrocytes that have lost part of their cell membrane due to partial phagocytosis. Their spherical shape results in a small erythrocyte lacking central pallor. Spherocytosis was noted in 67% of dogs with IMHA.⁵ It has been proposed that the phagocyte and complement systems can clear or lyse spherocytes. Therefore, some dogs with IMHA may not have evidence of spherocytosis.^4,5 In addition, spherocytes can develop due to other secondary causes such as zinc toxicosis and rickettsial diseases that should be investigated. Autoagglutination and/or spherocytosis are highly supportive findings in the diagnosis of IMHA, but are not present in every case.

Figure 1. Autoagglutination is visible on the sides of the blood tube.	Figure 2. Microscopic agglutination of erythrocytes is visible in this blood smear (Dog, blood smear, Wright’s stain).

Figure 3. Rouleaux are linear, stacked arrangements of erythrocytes that occur on blood smears, often as a result of high serum fibrinogen or antibody concentrations. This phenomenon may resemble agglutination, but rouleaux can be dispersed by dilution of blood with saline (1:1) prior to preparation of the blood smear.	Figure 4. Typical spherocyte, appearing as a small erythrocyte that lacks central pallor because of its spherical (rather than biconcave) shape (Dog, blood smear, Wright's stain).

Direct Antiglobulin (Coombs’) Test

Understanding when direct antiglobulin (Coombs’) testing is necessary, and appropriate interpretation of the test results is important to an accurate diagnosis of IMHA. A direct antiglobulin test is used to detect the presence of antibody against red blood cells when the anti-erythrocyte concentration is too low to cause spontaneous autoagglutination.^4,9 There is a direct antiglobulin test and an indirect antiglobulin (Coombs') test. The direct antiglobulin test detects antibodies attached to red blood cells and is the most useful test in diagnosing IMHA. The indirect antiglobulin test detects antibodies to red blood cells that are present in the patient’s serum. Since most anti-erythrocyte antibody is found attached to the RBC’s the direct antiglobulin test is a more sensitive test and results in fewer false negatives. Test results of a direct antiglobulin test are reported simply as positive or negative. The actual interpretation of this result is not so simple and should be given more attention.

The principle of the direct antiglobulin test is illustrated in Figure 5. Washed patient erythrocytes are incubated with the Coombs’ reagent. The Coombs' serum may be monovalent (directed against one immunoglobulin class such as IgG or complement fragment) or may polyvalent (directed against IgG, IgM, and complement). The Coombs' serum must be species-specific for an accurate result.⁹ If autoantibody to immunoglobulins and/or complement are present on the patient’s RBCs, the Coombs’ reagent antibodies bind to them and crosslinking occurs. This is visible as gross or microscopic agglutination and represents a positive test result (Fig. 5).

Figures 5 A, B, C. Direct antiglobulin or Coombs’ test. A) Animal with IMHA has autoantibodies bound to the surface of its erythrocytes. B) Polyvalent Coombs’ reagent (with antibodies against IgG, IgM, and complement) is added. C) Agglutination occurs as Coombs’ antibodies bind to autoantibodies on the surface of the RBCs.

Many laboratories perform dilutions of the Coombs’ reagent to decrease the chance of false negative reactions due to the presence of very large amounts of antibody in undiluted samples. The phenomenon of a false negative test result is referred to as a prozone effect.⁹ Dilution of the Coombs' reagent allows the correct ratio between red cell antibody and reagent to allow agglutination. Titers are not commonly reported with test results since there is no clear association between the titer and disease severity or any further diagnostic information available from the titer value.^3,9

Most studies report some degree of false negative test results with use of the direct antiglobulin test to detect agglutination. Two studies noted a sensitivity of only 60%.^6,8 Other studies also noted the occurrence of negative test results in dogs with IMHA. For this reason, it is important to search for other possible causes of hemolysis. Furthermore, the possibility of IMHA should not be summarily dismissed because a negative test result. Possible causes for inaccurate results from the direct antiglobulin test are listed below:

Conclusion

Accurate evaluation of clinical, hematologic, and immunologic data is necessary to establish a diagnosis of IMHA.^4,5 Clinicians should remember the importance of blood smear findings (agglutination and spherocytes) and not rely solely on the results of the direct antiglobulin (Coombs’) test to diagnose IMHA.⁷ It also is important to submit both an EDTA blood sample and an air dried blood film for evaluation. To eliminate the prozone effect, direct antiglobulin testing should use species-specific Coombs' serum that is tested in serial dilutions.

References

1. Scott-Moncrieff JC, Treadwell NG, McCullough SM, Brooks MB: Hemostatic abnormalities in dogs with primary immune-mediated hemolytic anemia. J Am Anim Hosp Assoc 37:220-227, 2001.

2. Canine Immune-Mediated Hemolytic Anemia, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, Bellwether 49 – Spring, 2001

3. Day M J: Immune-mediated hemolytic anemia. In: BF Feldman, JG Zinkl, NC Jain (eds): Schalm's Veterinary Hematology. Philadelphia, Lippincott Williams and Wilkins, 2000, pp. 799-806.

4. Giger U: Regenerative anemias caused by blood loss or hemolysis. Ettinger SJ, Feldman EC (eds): Textbook of Veterinary Internal Medicine: Diseases of the

Dog and Cat, 5^th ed. Philadelphia, W. B. Saunders Co., 2000, pp. 1784-1804

5. Klag A, Giger U, Shofer F: Idiopathic immune-mediated hemolytic anemia in dogs: 42 cases (1986-1990). J Am Vet Med Assoc 202:783-787, 1993.

6. Reimer ME, Troy GC, Warnick LD: Immune-mediated hemolytic anemia: 70 Cases 1988-1996). J Am Anim Hosp Assoc 35:384-390, 1999.

7. Klag AR, Giger U, Shofer FS: Idiopathic immune-mediated hemolytic anemia in dogs: 42 cases (1986-1990). J Am Vet Med Assoc 202:783-788, 1993.

8. Wilkerson MJ: Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia. J Vet Intern Med 14:190-196, 2000.

9. Direct Coombs' test, Cornell University College of Veterinary Medicine, Ithaca, NY

Acknowledgement

"Irish Setter hiding behind a fleece lined coat" by Diana Hammond. The copyrighted image is used with permission of the artist and is from the website DHH Portfolio.

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