Canine , Clinical Medicine , Feline , Hematology , Uncategorized

Diagnosis of Immune-Mediated Hemolytic Anemia

Diagnosis of Immune-Mediated Hemolytic Anemia

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Todd Archer, DVM, Diplomate ACVIM, and Andrew Mackin, BSc, BVMS, Diplomate ACVIM

Immune-mediated hemolytic anemia is one of the most common immune-mediated hematologic disorders in dogs and cats. Part 1 of this 2-article series discusses pathophysiology and diagnosis of IMHA, including patient predilection, history and clinical signs, physical examination, diagnostics, and differential diagnoses.


Immune-mediated hemolytic anemia (IMHA) is one of the most common immune-mediated hematologic disorders in dogs and cats.

In dogs, immune-mediated hemolytic anemia:

  • Is commonly primary or idiopathic in origin
  • Often affects particular breeds, including cocker spaniels, English springer spaniels, collies, poodles, and Irish setters1,2
  • Most commonly affects middle-aged female dogs
  • Also occurs secondary to triggers, such as infectious, inflammatory, and neoplastic diseases; drugs; and vaccines (Table 1).

In cats, there is no breed predilection for IMHA, and the condition is usually secondary to an underlying cause.3

Pathophysiology of IMHA

IMHA is a type II immune reaction, where antibody and/or complement formation against RBCs causes accelerated cell destruction and subsequent anemia. The anti-RBC antibodies can be either immunoglobulin G or M (IgG or IgM).4When the body is correctly responding to an immune reaction, antibodies ensure an appropriate immune response while the complement system enhances the ability of antibodies and phagocytic cells to clear pathogens. However, in cases of IMHA:

  • High levels of antibodies induce activation of the complement system and formation of the membrane attack complex; RBC destruction tends to be intravascular due to osmotic lysis.1
  • Macrophages within the spleen, liver, and other organs recognize the Fc portion of antibodies and/or the C3b portion of complement bound to RBCs and prematurely remove cells from circulation and destroy them (extravascular hemolysis).1,4

Classically, the bone marrow mounts an appropriate and strongly regenerative response. Less commonly, antibodies are also directed against marrow RBC precursors, resulting in nonregenerative anemia.

Table 1. Important Differential Diagnoses for Hemolytic Anemia
CHEMICAL/TOXIN INJURY
Castor bean
Copper
Garlic/onion
Methylene blue
Propylene glycol
Zinc
IMMUNE-MEDIATED
Disseminated intravascular coagulation*
Glomerulonephritis*
Incompatible transfusions*
Neonatal isoerythrolysis*
Primary IMHA*
Systemic lupus erythematosus*
INFECTIOUS
Anaplasma species*
Ancylostoma caninum*
Babesia canis*
Babesia gibsoni*
Cytauxzoon felis*
Dirofilaria immitis*
Ehrlichia species*
FeLV*, FIP*, FIV*
Histoplasmosis*
Leishmaniasis*
Leptospirosis*
Mycoplasma haemocanis*
Mycoplasma haemofelis*
INTRINSIC/INHERITED RBC DEFECTS
Chondrodysplasia (in malamutes)
Hereditary osmotic fragility
Idiopathic Heinz body anemia
Methemoglobin-reductase deficiency
Phosphofructokinase deficiency
Pyruvate kinase deficiency
MEDICATIONS
Acetaminophen*
Cephalosporins*
Chlorpromazine*
Dipyrone*
Heparin*
Levamisole*
Methimazole/propylthiouracil*
Penicillins*
Phenazopyridine hydrochloride*
Procainamide*
Sulfonamides*
Topical benzocaine*
Vitamin K*
Quinidine*
NEOPLASIA
Diffuse sarcoma*
Hemangiosarcoma*
Lymphocytic leukemia*
Lymphoma*
Multiple myeloma*
Other solid tumors*
OTHER
Bee-sting envenomation*
Recent vaccination*
Severe hypophosphatemia
Snake-bite envenomation
*Denotes conditions that have been suggested to induce immunologic destruction of RBCs
FeLV = feline leukemia virus; FIP = feline infectious peritonitis; FIV = feline immunodeficiency virus; IMHA = immune-mediated hemolytic anemia; RBC = red blood cell

PATIENT EVALUATION

History

When a patient presents with possible IMHA, history should include a detailed account of any recent medications or vaccines. Historical clues that suggest a possible underlying or triggering disease process should also be investigated (Table 1).

Clinical Signs

Clinical signs seen in IMHA patients often include those associated with anemia and tissue hypoxia, including:

  • Lethargy
  • Weakness
  • Tachypnea.

When anemia is severe and acute in onset, patients tend to be the most significantly affected. When red blood cell (RBC) destruction is more chronic, patients may only be mildly affected despite marked anemia.

Physical Examination

Physical examination may reveal:

  • Pale mucous membranes
  • Tachycardia
  • Bounding pulses
  • Less commonly: Splenomegaly, hepatomegaly, lymph node enlargement, and fever.

Other physical examination findings may include:

  • Hemic murmur, which should resolve once anemia is corrected
  • Jaundiced mucous membranes and tissues if there is acute severe hemolysis (Figure 1)
  • Hemoglobinuria (“port wine” urine) in patients with intravascular hemolysis
  • Petechiae, ecchymoses, and melena in patients with concurrent immune-mediated thrombocytopenia (Evan’s Syndrome)
  • Signs that reveal the underlying cause of IMHA.

Figure 1. Jaundice in a dog with severe IMHA

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DIAGNOSTICS

Initial diagnostics in an anemic patient should focus on identifying the cause of the anemia. A final diagnosis of IMHA is based on evidence of accelerated RBC destruction, with an underlying immune-mediated pathogenesis.

There is no single test that is definitively diagnostic for IMHA. Instead, evidence from various analyses is used to determine the diagnosis (Table 2). The following signs and results support a diagnosis of primary IMHA:

  • Anemia
  • Evidence of accelerated RBC lysis, including, but not limited to, hemoglobinemia/hemoglobinuria (intravascular hemolysis) or bilirubinemia/bilirubinuria
  • Evidence of an immune-mediated process, such as autoagglutination, a positive Coombs’ test, or increased numbers of circulating spherocytes
  • Lack of other identifiable causes of anemia.
Table 2. Diagnostic Overview for Immune-Mediated Hemolytic Anemia
No single test is definitively diagnostic for IMHA. Instead, evidence from various analyses is used to determine the diagnosis.
Diagnostic Process Results That May Indicate IMHA
Predilection
  • Often affects particular breeds*
  • Commonly affects middle-aged female dogs
  • In cats, no breed predilection
History
  • Infectious, inflammatory, or neoplastic disease
  • Drug or vaccine administration
  • In cats, IMHA usually secondary to underlying cause
Clinical Signs
  • Lethargy
  • Weakness
  • Tachypnea
Physical Examination
  • Pale mucous membranes, tachycardia, bounding pulses, and hemic murmur
  • Splenomegaly, hepatomegaly, enlarged lymph nodes, and fever
  • Signs related to the underlying cause
  • Acute severe hemolysis: Jaundiced mucous membranes and tissues
  • Intravascular hemolysis: Hemoglobinuria (“port wine” urine)
  • Immune-mediated thrombocytopenia: Petechiae, ecchymoses, and melena
Blood Analysis
  • CBC/blood smear: Anemia, accelerated RBC lysis, spherocytosis, neutrophilia
  • Agglutination (anti-RBC antibodies): Macroagglutination, microagglutination
  • Direct Coombs’ test: Positive result
  • Serum biochemistry: Hyperbilirubinemia, increased liver enzymes
Coagulation Tests
  • PT & aPTT: Disseminated intravascular coagulation, thromboembolic disease
Urinalysis
  • Bilirubinuria
  • Hemoglobinuria (intravascular hemolysis)
Additional Testing
  • Bone marrow evaluation: Indicated if persistant nonregenerative anemia or pancytopenia is present on blood analysis.
  • Infectious disease identification: Indicated if infectious cause of hemolysis is suspected based on signalment, clinical signs, and geographic location.
  • Imaging: Indicated when identifying underlying causes of IMHA
* Cocker spaniels, English springer spaniels, collies, poodles, and Irish setters
aPTT = activated partial thromboplastin time; CBC = complete blood count; IMHA = immune-mediated hemolytic anemia; PT = prothrombin time; RBC = red blood cell

 

Complete Blood Count & Blood Smear Analysis

In IMHA patients, complete blood count (CBC) with blood smear analysis often reveals anemia and RBC changes, which are suggestive of a regenerative response, such as polychromasia, anisocytosis, and nucleated RBCs.

Reticulocytes

  • An increased absolute reticulocyte count (> 60,000/mcL, dogs; > 50,000/mcL aggregate reticulocytes, cats) or corrected reticulocyte percentage (> 1%, dogs; > 0.5%, cats) documents a regenerative marrow response.5
  • Since a regenerative response takes approximately 3 to 5 days to mount, acute cases may initially appear poorly regenerative.
  • A nonregenerative response may also suggest the presence of antibodies directed against marrow precursors.

Spherocytes

Spherocytes may also be seen on blood smears. Spherocytes are small RBCs with a loss of central pallor produced by incomplete destruction of RBCs by macrophages (Figure 2). Spherocytosis is very suggestive of IMHA.

Archer_fig02

Figure 2. Marked spherocytosis observed on a Wright’s-stained blood smear from a dog with IMHA; spherocytes are recognized as smaller RBCs that lack central pallor. They form when macrophages remove a segment of RBC membrane without loss of intracellular contents; the remaining cell membrane must form a globoid sphere in order to stretch over and contain the cytoplasm.

Other Findings

The blood smear should also be carefully evaluated by an experienced clinical pathologist for:

  • Presence of RBC parasites, such as Mycoplasma haemofelis (formerly Haemobartonella) and Babesia
  • Neutrophilia, often with a left shift, is commonly seen in IMHA patients
  • Extreme leukocytosis (“leukemoid response”) occurs in some dogs with IMHA and has been associated with severe tissue injury6
  • Thrombocytopenia will be observed in animals with Evan’s Syndrome.

Anti-RBC Antibodies

Spontaneous Autoagglutination

High levels of anti-RBC antibodies sometimes result in their attachment to more than one cell, causing spontaneous RBC agglutination. Agglutination may be appreciated as red speckles when blood is placed in an EDTA tube (Figure 3) or onto a microscope slide.

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Figure 3. Autoagglutination observed as red speckles in EDTA-anticoagulated blood from a dog with severe IMHA.

Slide Agglutination Test

The slide agglutination test can be easily performed in practice, and is used to differentiate true autoagglutination from rouleaux formation (nonimmune RBC adhesion).

  • A single drop of EDTA-anticoagulated blood is placed onto a microscope slide and mixed with saline (1–2 drops in dogs; 3–4 drops in cats due to their greater propensity to develop rouleaux).
  • The slide is rocked back and forth; then evaluated for the formation of macroagglutination (obvious agglutination to the naked eye) (Figure 4).
  • A coverslip can then be placed on the mixture, and the slide evaluated under a microscope for microagglutination (4 or more RBCs in a cluster) (Figure 5).
  • True agglutination appears as “clusters of grapes” while rouleaux appear as “stacks of coins” (Figure 6).
  • Rouleaux can further be differentiated from autoagglutination by adding additional saline and by RBC washing techniques; extra saline often disperses rouleaux but will not disperse true autoagglutination.

Since autoagglutination is only seen with high antibody levels, a negative slide agglutination test does not rule out IMHA. 

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Figure 4. Positive slide agglutination test in a dog with IMHA, demonstrating obvious macroagglutination. Since false positives can also occur, the Coombs’ test should be carefully interpreted in each individual patient.

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Figure 5. Microagglutination observed on a standard Wright’s-stained blood smear from a dog with IMHA; RBCs are strongly adhered to one another by a high level of anti-RBC antibodies that are attached to more than one cell.

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Figure 6. Rouleaux observed on a Wright’s-stained blood smear from a cat with a serious systemic illness. RBCs can form stacks in conditions associated with high plasma protein levels; while these stacks can appear as speckles to the naked eye, cell-to-cell bonding is weak and easily dispersed by saline.

Direct Coombs’ Test

The direct Coombs’ test is also known as the direct antiglobulin test (DAT) and identifies antibodies or complement adhered to RBCs. IMHA patients that do not demonstrate autoagglutination may still test positive on the Coombs’ test. However, diagnostic sensitivity of the Coombs’ test ranges from 60% to 89%, so a negative test does not exclude IMHA.

Additional Diagnostics
Serum Biochemical Profile

Common serum biochemistry changes in IMHA patients include hyperbilirubinemia and increased liver enzymes.

Hyperbilirubinemia

  • With accelerated RBC destruction, increased bilirubin production by macrophages can overwhelm hepatic processing capacity, resulting in hyperbilirubinemia. However, it may also be due to concurrent hepatobiliary disease.
  • Normal bilirubin levels are often seen in mild or chronic cases of IMHA because a healthy liver can still handle the extra bilirubin.

Increased Liver Enzymes

  • Liver enzyme elevation, especially alanine aminotransferase, may be present due to hypoxic liver damage.
  • Azotemia may sometimes occur due to either prerenal causes (dehydration) or renal causes (hemoglobin-induced renal damage).

Coagulation Tests

Coagulation tests, such as the 1-stage prothrombin time (PT) or activated partial thromboplastin time (aPTT), are indicated to assess for hemostatic disorders, such as:

  • Disseminated intravascular coagulation (DIC)
  • Thromboembolic disease.

D-dimer concentration or antithrombin activity may be needed to characterize DIC or thrombotic diseases. Both conditions can be common and serious complications of IMHA.

Bone Marrow Evaluation

Marrow evaluation is indicated if there is a persistent (beyond 3–5 days) nonregenerative anemia or pancytopenia is present on blood analysis.

Infectious Disease

Testing for infectious causes of hemolysis, such as Mycoplasma haemofelis, Babesia canis, or Babesia gibsoni should be considered in individual patients based on signalment, clinical signs, and geographic location.

Urinalysis

Urinalysis often reveals bilirubinuria or, with intravascular hemolysis, hemoglobinuria.

Imaging

Diagnostic imaging is indicated to identify underlying causes of IMHA, such as neoplasia.

  • Thoracic radiographs, abdominal radiographs, and abdominal ultrasound should be considered.
  • Aspirates and histologic biopsies should be performed on any masses/abnormal-appearing organs found on imaging.
  • Abdominal radiographs are also indicated to exclude zinc foreign bodies, since zinc toxicosis can mimic IMHA.

Part 2 of this series—Management of Immune-Mediated Hemolytic Anemia—will be published in the September/October 2013 issue of Today’s Veterinary Practice.

aPPT = activated partial thromboplastin time; CBC = complete blood count; DAT = direct antiglobulin test; DIC = disseminated intravascular coagulation; IgG = immunoglobulin G; IgM = immunoglobulin M; IMHA = immune-mediated hemolytic anemia; PT = prothrombin time; RBC = red blood cell

References

  1. Balch A, Mackin A. Canine immune-mediated hemolytic anemia: Pathophysiology, clinical signs, and diagnosis. Compend Contin Educ Pract Vet 2007; 29:217-225.
  2. Carr AP, Panciera DL, Kidd L. Prognostic factors for mortality and thromboembolism in canine immune-mediated hemolytic anemia: A retrospective study of 72 dogs. J Vet Intern Med 2002; 16:504-509.
  3. August J. Immune-mediated hemolytic anemia. Consultations in Feline Internal Medicine, vol 6. St. Louis: Saunders, 2006, pp 617-627.
  4. McCullough S. Immune-mediated hemolytic anemia: Understanding the nemesis. Vet Clin North Am Small Anim Pract 2003; 33:1295-1315.
  5. BF Feldman, Zinkl JZ, NC Jain. Schalm’s Veterinary Hematology, 5th ed. Baltimore: Lippincott Williams & Wilkins, 2000, pp 110-116.
  6. McManus PM, Craig LE. Correlation between leukocytosis and necropsy findings in dogs with immune-mediated hemolytic anemia: 34 cases (1994-1999). JAVMA 2001; 218(8):1308-1313.

f04_ArcherTodd M. Archer, DVM, Diplomate ACVIM, is an assistant professor of small animal medicine in the Department of Clinical Sciences at Mississippi State University College of Veterinary Medicine. Dr. Archer’s clinical interests include hematology, immunology, and endocrine disorders as well as interventional radiologic procedures. His research has primarily focused on T-cell responses in dogs to cyclosporine using both flow cytometry and qRT-PCR. Dr. Archer has spoken at national, state, and local meetings and also published research articles regarding his work with cyclosporine. He received his DVM and completed an internship and residency at Mississippi State University.

 

f04_MackinAndrew Mackin, BVMS, DVSc, Diplomate ACVIM, is currently professor and Ward Chair of Medicine at Mississippi State University College of Veterinary Medicine. His clinical and research interests focus on hematology, hemostasis, immunosuppressive therapy, and transfusion medicine. Dr. Mackin received the 2006 Carl Norden-Pfizer Distinguished Teacher Award. He received his veterinary degree from Murdoch University in Western Australia; then completed an internship and residency in small animal medicine at University of Melbourne as well as an internal medicine residency at Ontario Veterinary College.

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