Immune-mediated hemolytic anemia: Understanding and diagnosing a complex disease

Reddogs / stock.adobe.comImmune-mediated hemolytic anemia (IMHA), also known as autoimmune hemolytic anemia (AIHA), is a complex disease in which hemolysis occurs because of anti-erythrocyte antibody production. In this article, we will explore the pathophysiology and diagnosis of primary and secondary IMHA in dogs and cats. In a companion article, “Immune-mediated hemolytic anemia: Treating cats and dogs with a complex disease,” we review the treatment of and prognosis for patients diagnosed with IMHA.

In our review of the recent literature on IMHA in companion animals, we found primarily case series and a lack of randomized controlled clinical studies. However, this has recently been called to the attention of the veterinary profession, and IMHA is likely to be the focus of future research.1

REGENERATIVE ANEMIAS

Regenerative anemias occur primarily from hemolysis and blood loss. Although hemorrhage is typified by reduction in both red blood cells (RBCs) and total protein, these are not hard and fast rules. Acute hemorrhage can start with protein loss alone. Regardless, unless there is a concurrent condition causing hypoproteinemia (e.g. protein-losing nephropathy, protein-losing enteropathy, liver failure), a low hematocrit in the face of normal or elevated protein levels suggests hemolysis. Hyperbilirubinemia and hemoglobinemia are inconsistent findings, but they should increase your clinical suspicion for hemolysis. The rapid osmotic fragility test may help differentiate hemolysis from hemorrhage.2

HEMOLYTIC ANEMIA vs. IMHA

In dogs and cats, there are many causes of hemolysis, so a clear distinction should be drawn between hemolytic anemia and IMHA.

Hemolytic anemia

Hemolytic anemias are conditions in which RBCs are destroyed at an accelerated rate and, typically, the bone marrow mounts a normal regenerative response. In these non–immune-mediated conditions, immunoglobulins or complement do not mediate lysis. RBCs can be destroyed as a result of infectious organisms, inherited membrane and enzyme defects, increased membrane fragility or metabolic causes such as hypophosphatemia.3 Unlike with immune-mediated anemia, long-term immunosuppression is generally not indicated; in fact, it could make clearance of an infectious agent more difficult. Thus, when you suspect hemolysis, it is imperative that you consider other causes of hemolysis before assuming the condition is immune-mediated.

Below we discuss some of the common non–immune-mediated causes of hemolytic anemia. Table 1 below includes a more exhaustive list.

Zinc and copper toxicosis. In dogs, one of the most common causes of hemolytic anemia is the ingestion of zinc-containing objects. High zinc concentrations can be found in pennies minted since 1983, board game pieces, zippers, zinc oxide ointment and various other sources. Zinc toxicosis can cause a severe intravascular hemolysis that is associated with small amounts of Heinz body and spherocyte formation. The clinical presentation of zinc toxicosis can overlap with IMHA, so a thorough patient history and survey radiographic examination are recommended. Treatment consists of removing the foreign object and providing supportive care.

Copper toxicosis can also result in a marked intravascular hemolysis and methemoglobinemia.3

Heinz body anemia. Heinz bodies are the result of oxidative damage and are made of denatured and precipitated hemoglobin. Under the microscope, they appear as pericellular, dark-staining, refractile material. Because feline RBCs are especially sensitive to oxidative damage, Heinz body anemia is more commonly seen in cats and can be present during toxicosis, as well as in other diseases such as hyperthyroidism, diabetes mellitus and lymphoma.3 Heinz body anemia results most commonly when companion animals ingest plants in the allium family (onions, garlic, leeks) or when cats receive drugs such as propofol, propylene glycol, acetaminophen, methylene blue, vitamin K3 or DL-methionine.3 In some cases of hemolytic anemia, you may see eccentrocytes-cells in which the damaged hemoglobin is clustered together and shifted to one side of the RBC membrane, leaving a crescent-shaped clear region. Eccentrocytes and cells containing Heinz bodies have less deformability and more rigidity, making them more likely to be lysed or removed from circulation by the spleen. Treatment is focused on addressing the underlying condition.

Hypophosphatemia. Moderate to severe hypophosphatemia can also cause hemolytic anemia, especially in cats. Hypophosphatemia is a common abnormality in patients being treated for diabetes mellitus, hepatic lipidosis, starvation and other conditions.3 In these cases, depletion of RBC adenosine triphosphate (ATP), decreased 2,3-diphosphoglycerate concentrations and decreased glutathione concentrations lead to decreased membrane deformability, increased osmotic fragility and susceptibility to oxidative injury. Treatment of hypophosphatemia consists of phosphate supplementation and therapy for the underlying condition.3

Inherited defects. If a patient has persistent mild to moderate hemolytic anemia, despite receiving appropriate therapy, you should consider the possibility of inborn errors of metabolism. These are relatively uncommon, but they include pyruvate kinase (PK) and phosphofructokinase (PFK) deficiencies. A full explanation of these diseases is beyond the scope of this article, but you can consult additional resources for more information.5

Primary vs. secondary IMHA

IMHA has been described in people, dogs, cats and a wide range of other species. Specific causes of primary and secondary IMHA in dogs and cats are summarized below in Table 2.

In dogs, it is estimated that 70% to 80% of IMHA cases are considered primary, or idiopathic, meaning that no underlying cause can be found.3

In people, there appears to be a link between specific human leukocyte antigen (HLA) haplotypes and autoimmune disease, although the genes involved may differ for each disease. There are also studies that demonstrate an increased frequency of certain dog leukocyte antigen (DLA, an analog of HLA) haplotypes in dogs that develop such diseases. However, in one study,13 an allelic frequency of candidate gene DLA-79 was found in only about 20% of dogs with immune-mediated diseases, suggesting a multifactorial etiology. Specific candidate genes associated with immune-mediated disease in people have also been evaluated in cocker spaniels, but researchers did not identify a clear link between any haplotype and disease.14

In secondary IMHA, RBC destruction occurs as a consequence of the immune system reacting to an underlying condition or to the presence of an immune trigger.15 Broadly speaking, these underlying causes include infections, certain drugs and neoplasia. Although some consider vaccination a potential trigger for immune-mediated disease, this association is not robust. A recent case series of IMHA following elapid envenomation was reported in four dogs in Australia.16

PATHOPHYSIOLOGY

IMHA is typified by a type II hypersensitivity reaction in which RBCs are destroyed by the immune system. The process involves a breakdown of immune self-tolerance and the production of complement, anti-erythrocyte IgG and IgM antibodies or a combination of the three. Recent studies have shown a mixed cytokine response, with the variability depending on the severity of IMHA.17 These antibodies recognize and bind to RBC membrane glycoproteins.18 Erythrocyte destruction is then initiated by the reticuloendothelial system,19 or RBCs may lyse because of decreased membrane stability.

When RBCs are destroyed by macrophages in the liver or spleen, it is termed extravascular hemolysis and results in hyperbilirubinemia with uncommon hemoglobinemia. In this case, macrophages either consume the entire erythrocyte or remove a portion of the membrane, leaving smaller RBCs with no central pallor (spherocytes). These spherocytes are destroyed at an increased rate because of their membrane rigidity. When erythrocyte destruction occurs within vessels, it is termed intravascular hemolysis, and there is frequently both hyperbilirubinemia and hemoglobinemia (and subsequent hemoglobinuria and bilirubinuria).3

Intravascular hemolysis does not occur alone; there is typically concurrent extravascular destruction. In most cases of IMHA, there is destruction of circulating RBCs, but destruction can also occur within the bone marrow, causing maturation arrest of RBS precursors or destruction of all RBC precursors, a condition known as pure red cell aplasia (PRCA).20

Secondary IMHA is probably the result of several factors, but a common factor is loss of self-tolerance, resulting in an autoimmune attack. There are varied mechanisms by which self-tolerance is lost, but, as with primary IMHA, it is probably multifactorial.15 For example, in infectious diseases such as mycoplasmosis in cats and babesiosis in cats and dogs, there is hemolysis caused by both the organism itself and a secondary immunologic response.

Thrombosis is a commonly reported complication of IMHA, and a 2013 review detailed the prothrombotic mechanisms that are implicated in IMHA.21 There are cases of both arterial and venous thromboses, which purportedly occur as a result of excessive platelet activation, procoagulant microparticles in circulation and increased monocyte activation with associated increased cytokine production that induces increased tissue factor expression. The advent of the test method thromboelastography (TEG) has allowed researchers in numerous studies to demonstrate that a hypercoagulable state is common in IMHA.22–24

SIGNALMENT, HISTORY AND CLINICAL SIGNS IN DOGS

The age of onset for IMHA can vary, but the disease frequently occurs in young to middle-aged dogs. Although primary IMHA can affect any breed, certain breeds-cocker spaniels, poodles, Irish setters, and Old English sheepdogs-are overrepresented in studies (Table 2). Although female dogs are typically suspected to have a twofold increased risk of certain autoimmune diseases (including IMHA), a 2016 report from University of California-Davis showed that compared to intact dogs, neutered dogs had an increased risk for autoimmune diseases, and that female dogs had an increased risk compared to males, but not for IMHA.25

Historical findings of IMHA are highly variable. Patients can have an acute or a chronic history of malaise. Vomiting or diarrhea may occur before the classic signs of hemolytic anemia, which are lethargy, inappetence, pallor or icterus, tachypnea and changes in urine or feces color.

Clinical signs can include weakness, pale or icteric mucous membranes, bounding pulses, tachypnea, tachycardia, hepatosplenomegaly and a heart murmur. A patient's clinical signs and physical examination findings also may be related to an underlying disease process rather than to the anemia itself.3 For example, patients with concurrent severe thrombocytopenia (<50,000 platelets/µL) may also have clinical signs such as petechiation, ecchymosis, epistaxis or melena.

To investigate potential underlying causes of IMHA, you should collect a thorough and specific patient history. Ask the client questions regarding a wide range of environmental and historical circumstances to determine the following:

• The pet's propensity to ingest foreign objects (e.g. zinc, copper, etc.)

• Diet and treat history (e.g. recent ingestion of onions or garlic)

• Recent history of drug administration or vaccination

• Travel history and acquisition of the pet

• History of transfusions or recent dog fights

• Recent tick or flea exposure, bee stings, or elapid snake bites16

• Flea, tick and heartworm preventive status

• Reproductive status

• Other clinical signs that may be related to underlying diseases

DIAGNOSTIC TESTS IN DOGS

Rules of thumb for diagnosing IMHA in dogs and cats

These guidelines should help you avoid some of the common mistakes that occur in diagnosing IMHA.

Search for causes of nonimmune hemolytic anemia including inherited, infectious, metabolic, toxic and cancerous causes. The most common treatments for IMHA are at best ineffective; at worst, they may exacerbate illness.

Evaluate patients for underlying disease. Even with a diagnosis of IMHA, finding an underlying cause is vital for successful treatment. For example, if IMHA was secondary to long-term cephalosporin use, attaining remission while the patient is still receiving cephalosporin may be impossible.

While waiting for test results, treat the treatable, especially if the treatment is unlikely to cause harm. We routinely start patients with IMHA on doxycycline while test results for infectious diseases are pending. Similarly, consider discontinuing any medications that are implicated in the onset of signs.

Baseline tests recommended for diagnosing IMHA and investigating causes of secondary IMHA include a complete blood count, a reticulocyte count, a serum chemistry profile, a blood smear, a slide agglutination test, a Coombs (direct agglutination) test and abdominal and thoracic imaging. Classically, a diagnosis of IMHA in dogs requires two of these three criteria: spherocytosis, autoagglutination and a positive Coombs test. Although uncommon, there can be cases where IMHA is suspected but not proven by these criteria. Other tests may also be useful and are discussed below.

Complete blood count

Complete blood count abnormalities usually include a regenerative anemia, although as many as 33% to 50% of dogs with IMHA can have nonregenerative anemia.26 A mild to marked increase in the white blood cell count can be seen, occasionally with a left shift and toxic change in neutrophils. This leukocytosis can result from many factors, including glucocorticoid-induced leukocytosis, anemic hypoxia, thromboembolic disease and tissue necrosis.27  Low platelet counts are reported up to 67% of the time.4 Concurrent immune-mediated destruction of RBCs and platelets (Evans syndrome) has been reported, but thrombocytopenia due to hypercoagulability and resulting consumption is more common.

Serum chemistry profile

Abnormalities in the serum chemistry profile may reflect organ damage from hypoxia or indicate an underlying disease process. Elevated bilirubin concentrations are common.28 Even before glucocorticoid administration, many patients can have elevated liver enzyme activities from hepatic hypoxia, inflammation or necrosis.28 Bilirubin concentrations can also be normal if the hemolysis has been chronic and the liver has had time to metabolize the bilirubin.3 Other serum chemistry abnormalities may reflect underlying conditions or secondary effects of disease, such as acute kidney injury from antibody-antigen complex deposition, hemoglobin-induced renal tubular damage or infarction from thrombotic events.

Blood smear evaluation and slide agglutination

At the outset of every suspected case of IMHA, you should perform a slide agglutination test and blood smear. For a slide agglutination test, mix a drop of blood with a drop of saline solution on a glass slide (or up to nine drops of saline for cats because of their tendency to have rouleaux formations). Gently agitate the mixture and inspect it for macroagglutination (Figure 1).

Figure 1. A blood smear showing macroagglutination. All images courtesy of Drs. Zachary Kern and Karyn Harrell.

Figure 2. In this peripheral blood smear from an anemic dog, most of the RBCs are small and dark and lack central pallor (i.e. spherocytes). There are also several large polychromatophilic cells, which suggest a regenerative response to the anemia (Wright-Giemsa stain; 1000X).Evaluating this slide with a coverslip at 400X or 1000X magnification may reveal microagglutination. Use a routine blood smear to evaluate the blood for spherocytes (Figure 2), Heinz bodies, hemic parasites and microagglutination (Figure 3). Having these tests performed by a pathologist may increase sensitivity. As many as 89% of dogs with IMHA have spherocytes; however, dogs with secondary hemolysis from hypophosphatemia, zinc intoxication or splenic disease can also have them in small numbers.20,29

Coombs test

Figure 3. A peripheral blood smear from an anemic dog reveals that the RBCs are prominently clumped together, which indicates agglutination. A saline dispersion test should be performed for confirmation. Also shown: moderate polychromasia and rare nucleated RBCs, which indicate a regenerative response; small round cells lacking central pallor, suggestive of spherocytes; and a neutrophilic leukocytosis, which indicates inflammation (Wright-Giemsa stain; 500X). The Coombs test, also called the direct antiglobulin test (or DAT), can be used when IMHA is suspected but macroagglutination or microagglutination is not evident. This test is run on an EDTA blood sample and will identify antibodies or complement on a patient's RBC surface. A positive result does not distinguish between primary and secondary causes of IMHA.30

False-positive results are seen in both dogs and cats and can be a result of concurrent diseases such as neoplasia, infections, inflammatory conditions or recent drug administration.30 False-negative results are also common and can occur in up to 50% of all dogs with IMHA.31 Thus, you should interpret this test with caution.

Imaging

Obtain abdominal radiographs to evaluate the pet for zinc-containing foreign objects or abdominal neoplasia. If abdominal radiographs reveal suspicious findings, an abdominal ultrasonographic examination may be warranted. In middle-aged to older dogs, it is recommended that thoracic radiographs also be obtained to look for primary or metastatic neoplasia.

Other diagnostic tests

A thorough search for underlying infection is warranted in cases of IMHA. Chronic infections, such as pyometra, abscesses, urinary tract infections and discospondylitis, have all been associated with IMHA.3 Transmissible causes of hemolytic anemia, including the hemoprotozoans Babesia gibsoni and Babesia canis, are being diagnosed with increased frequency in the United States.32 Studies have described a breed predisposition to B. gibsoni infection in American pit bull terriers32 and B. canis infection in greyhounds.33 Babesia species can be transmitted through dog bites and blood transfusions.34-36 Other infectious organisms, such as Ehrlichia and Dirofilaria species, have also been associated with anemia in dogs.37,38 Hemotropic mycoplasma (Mycoplasma haemocanis) does not cause a clinically relevant anemia in dogs that have not undergone a splenectomy, but Mycoplasma haemofelis can cause a mild to severe anemia in cats.3

Although uncommon, Leptospira infection can cause hemolytic anemia,39 and clinical suspicion should increase based on other clinicopathologic changes (e.g. increased hepatic enzymes, glucosuria and cylindruria). In general, investigation for infectious disease should be tailored to the agents endemic to your practice area or regions where the patient has traveled. A combination of serology and PCR increases testing sensitivity for most diseases, and empiric therapy may be indicated out of precaution. Microscopy to directly visualize hemic parasites rarely detects infection and is not recommended as the sole means of investigation.40

A coagulation panel is justified as an indicator of disseminated intravascular coagulation (DIC).4 Prolonged coagulation times have not been shown to predict thromboembolism or mortality in patients with IMHA. However, the presence of DIC may indicate a more severe case of IMHA and prompt referral of the patient to a facility where transfusions and critical care can be provided.

Urinalysis may show the presence of hemoglobinuria, evidenced by a red supernatant after centrifugation of the urine sample. It may also reveal bilirubinuria, bacteriuria if a chronic UTI is present or evidence of tubular damage from the pigments in circulation (e.g. cylindruria, proteinuria).

An antinuclear antibody (ANA) test is occasionally performed in patients with IMHA to check for systemic lupus erythematosus (SLE). Your clinical suspicion of SLE should increase if you see two or more manifestations of autoimmunity (e.g. vasculitis, polyarthropathy, mucosal lesions, glomerulonephritis) in addition to IMHA.41 False-positive ANA results can occur with many underlying infections and neoplastic diseases, so results must be interpreted with caution.

A bone marrow aspirate or core biopsy may be helpful if, despite adequate time for a regenerative response, the patient continues to have nonregenerative anemia. These tests search for underlying diseases such as PRCA, concurrent immune-mediated destruction of RBC precursors or neoplastic infiltration of the bone marrow.4 A 2008 review of bone marrow cytology from dogs with nonregenerative hemolytic anemia revealed that the majority had erythroid hyperplasia in the bone marrow (i.e. a “preregenerative” state), though about 20% had maturation arrest due to destruction of erythroid precursors.42

Other tests that are not typically available in general practice may help characterize or diagnose IMHA. Flow cytometry can detect anti-erythrocyte antibodies and is more sensitive than the Coombs test for detecting IgG.43,44 Similarly, TEG is increasingly used to demonstrate the presence of hypercoagulability. TEG is typically available only in large referral practices or academic hospitals. Blood typing is indicated for patients that are likely to need transfusions.

CATS AND IMHA: PATHOGENESIS AND DIAGNOSIS

Whereas there is a growing body of research on IMHA in dogs, there is a dearth of research in cats. Therefore, many extrapolations are made regarding the diagnosis and treatment of the condition in cats without strong evidence to support the claims. Nevertheless, the pathogenesis of IMHA in cats is presumed to be identical to that in dogs, and here we will highlight only the pertinent differences between cats and dogs.

Given the difficulty of assessing cats for spherocytes, a diagnosis of IMHA in cats should include the presence of ghost cells, an absence of Heinz bodies and microagglutination or macroagglutination. When performing autoagglutination testing, a larger volume of saline solution is required (1:4 or 1:10) because of the tendency for feline RBCs to make rouleaux formations. Similarly, Coombs testing, while commonly positive in cats with IMHA, can also yield false-positive results, as seen with infectious feline anemias.45

A recent retrospective analysis of 107 cats with IMHA elaborated on the epidemiology of feline IMHA. As in dogs, young adult cats are more likely to develop IMHA than other age groups, and primary IMHA is more common than secondary conditions. This study, however, failed to demonstrate a sex predilection for neutered males, something that had been identified in previous (uncontrolled) studies.46

In cats, secondary IMHA is most often a result of feline leukemia virus (FeLV) or Mycoplasma haemofelis (formerly Haemobartonella felis) infection, although it can also be seen with other infections (feline infectious peritonitis, Cytauxzoon felis, Leishmania infantum), with drug therapy (methimazole, propylthiouracil) and with neoplasia (lymphoma).47-49 Although famotidine is anecdotally reported to cause immune-mediated hemolysis, a 2008 study documented no increased risk of IMHA in 56 hospitalized cats given subcutaneous or intravenous famotidine.50 A case of IMHA presumed secondary to pancreatitis was recently reported,51 which, if true, is a predisposing cause unique to cats.

Be sure to also read the companion article, “Immune-mediated hemolytic anemia: Treating cats and dogs with a complex disease,” in which we review the treatment of and prognosis for dogs and cats diagnosed with IMHA.

Acknowledgment

Special thanks to Jennifer Neel, DVM, DACVP, assistant professor of clinical pathology, Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, for providing the micrographs in this article.

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Dr. Zachary Kern is a small animal internal medicine resident at North Carolina State University. He arrived there by way of University of Pennsylvania School of Veterinary Medicine and internships at BluePearl Veterinary Partners in New York City and Veterinary Specialists and Emergency Services in Rochester, New York.

Dr. Karyn Harrell obtained her veterinary degree from Michigan State University, completed an internship at the University of Minnesota and a small animal internal medicine residency at North Carolina State University. After spending six years at the Animal Medical Center in New York City, she returned to North Carolina State University and is currently a clinical assistant professor.