Clinical approach to anemia (Proceedings)

Anemia is commonly seen in veterinary emergency and critical care medicine. Patients may be brought in with the presenting complaint of anemia or may develop anemia during hospitalization as a result of their underlying disease or treatment. Anemia may contribute to patient morbidity, cost of treatment, and length of stay, frequently necessitating expensive interventions such as blood transfusion while the underlying disease is being treated.

Classification of anemia

Anemia seen in veterinary patients may be classified into three broad categories that relate to cause; blood loss, hemolysis, and decreased production. Classification of anemia in this way is not merely academic but is crucial to the workup of anemic patients. Because regenerative and non-regenerative anemias have different sets of differentials and diagnostics, this classification will guide further testing and provide useful prognostic information.

Three simple, in-house diagnostic tests can be performed in all anemic patients to help classify their anemia. These tests are inexpensive, easy to perform, and will frequently provide a great deal of information about the cause of the anemia. They can all be performed in approximately 5 minutes, allowing the clinician to classify the anemia and provide an appropriate diagnostic plan while the owner is still present.

The first test is the packed cell volume (PCV) and total solids (TS). The importance of interpreting the PCV in conjunction with the total solids cannot be overemphasized. If the PCV and TS are both low, acute blood loss should be suspected. In contrast, a low PCV with normal total solids would be consistent with hemolysis or decreased red blood cell production. To differentiate these two clinical entities, the plasma of the spun sample should be carefully evaluated for the presence of hemoglobin or bilirubin that may suggest intravascular or extravascular hemolysis respectively.

The second test that should be performed is the blood smear. Blood smears are useful for differentiating hemolysis from decreased production anemia as the presence of significant polychromasia and anisocytosis may indicate the presence of a regenerative response. Blood smears should also be evaluated for blood parasites and telltale alterations in red blood cell morphology. Heinz bodies are characterized by bulging of the red blood cell membranes and indicate oxidative red blood cell damage secondary to toxins such as onions, garlic, or propylene glycol. Spherocytes are small, round erythrocytes with loss of central pallor that result when antibodies bound to red blood cell membranes lead to a portion of the membrane being phagocytized or "pinched off" by macrophages. Large numbers of these cells are typically seen in dogs with immune-mediated hemolysis. Schistocytes are erythrocytes that have become fragmented as a result of passage through narrowed microvasculature. Schistocytes typically reflect microangiopathic causes of hemolysis such as caval syndrome, disseminated intravascular coagulation, hemangiosarcoma, or splenic torsion. Acanthocytes are red blood cells with long spiny projections that are frequently seen in patients with hepatic or splenic neoplasia, though they may also be seen in animals with disorders of lipid metabolism as well.

Finally, a slide agglutination test should be performed when hemolysis is suspected. In this test, a drop of anticoagulated blood from a purple top tube or capillary tube is mixed with several drops of saline. Autoagglutination may be evidenced by the development of obvious flecks within the drop of blood. The saline is used to disperse rouleaux that may mimic agglutination. Autoagglutination is caused by cross-linking of antibodies bound to the erythrocyte membranes, and as such is diagnostic for an immune-mediated component to the hemolysis.

Formulating a list of differential diagnoses

Once the anemia has been classified as blood loss, hemolysis, or decreased production, a list of differentials may be formulated. (see table 1)

Table 1. Some common differentials for anemia

Acute blood loss

Diagnosing acute blood loss is simple when an external source of bleeding is present. However, cavity bleeding, gastrointestinal losses, coagulopathies, and chronic blood loss may be more challenging clinical entities. History and physical exam are usually the key elements in identifying a source of acute blood loss. Additional diagnostic testing generally includes minimum database (complete blood count, serum biochemistry panel, and urinalysis) and imaging studies such as radiographs and/or ultrasound to identify the source of bleeding. Platelet counts and coagulation testing should be performed any time hemostatic defects are suspected. Gastrointestinal blood loss should be suspected when external or cavitary bleeding is not identified. Significant gastrointestinal blood loss may occur before signs of melena, hematemesis, or hematochezia are noted.

Treatment of acute blood loss consists of providing hemostasis, administering fluids for volume replacement, and considering blood transfusion if volume support alone is insufficient to provide for tissue oxygen delivery and clinical signs of anemia are present.

Hemolysis

In patients presenting with hemolysis, CBC, chemistry, and urinalysis should be run as part of a minimum database. The presence of hemoglobinemia/hemoglobinuria or bilirubinemia/bilirubinuria may suggest intravascular or extravascular hemolysis, respectively. Leukocytosis is frequently noted on the CBC from patients with IMHA and may result from non-specific "gearing up" of the bone marrow, or more likely, from tissue damage secondary to hypoxia and thrombosis. White blood cell counts in excess of 40,000/μl have been associated with a poorer prognosis in dogs with IMHA.1 Platelet counts should also be evaluated. Moderate thrombocytopenias may suggest consumptive coagulopathy or tick-borne illness, while severe thrombocytopenias (<50,000/μl) should prompt consideration of a concurrent immune-mediated thrombocytopenia. A Coombs test is indicated if hemolysis is suspected but autoagglutination is not present. The Coombs test, or direct antiglobulin test, is essentially a test for the presence of antibodies or complement bound to erythrocyte membranes. It is performed by adding anti-dog antibodies (immunoglobulins directed against canine IgG, IgM, or complement) to a sample of the patient's red blood cells. If autoantibodies are present on the patient's blood cells, the antiserum binds to them and cross-linking occurs. Because the end (positive) result of this test is agglutination, the Coombs test need not be run if the patient is already autoagglutinating. Note also that the Coombs test is not highly sensitive. Review of cases seen at our hospital (unpublished data) identified a sensitivity of only 66%, comparable to other reports in the veterinary literature.

A search should also be conducted for possible trigger factors. History taking should include questioning about recent vaccinations or medications. Recent vaccination (ie. within 4 weeks) has been associated with the development of IMHA in retrospective studies.2 Sulfa drugs, penicillins, and cephalosporins may also cause IMHA by acting as haptens, substances that become adsorbed to erythrocyte membranes and subsequently are able to stimulate an immune response. Neoplastic processes such as hemangiosarcoma, lymphoma, leukemia, and histiocytic sarcoma are another common trigger factor, and chest radiographs and abdominal ultrasound are frequently performed to rule out these entities. Testing should also be performed for vector-borne illnesses such as Ehrlichiosis, Babesiosis, Bartonellosis, and Hemoplasmosis.3-5 FeLV and FIV testing should not be overlooked in the cat.

Non-immunologic causes for hemolysis should also be considered. In addition to the oxidative toxins such as onions described above, ingestion of zinc may result in fulminant intravascular hemolysis, hemoglobinuria, multi-organ dysfunction, and DIC. Hereditary diseases such as phosphofrucktokinase deficiency seen in English Springer Spaniels may result in episodic hemolytic anemia, easily confused with IMHA because of its "apparent" response to steroids.

Reticulocyte count should always be performed to assess regenerative response. Hemolytic anemias are typically strongly regenerative, though it may take three days for regenerative response to be noted. The presence of a non-regenerative anemia (absolute reticulocyte count < 60,000/μl) should prompt suspicion of non-regenerative immune-mediated anemia (NRIMA), bone marrow disease, or other forms of decreased production anemia described below.6-8

Immunosuppressive therapies like prednisone ideally should not be initiated until neoplastic and non-immunologic causes of hemolysis have been ruled out, as the use of these drugs may interfere with accurate diagnosis and subsequent therapies. However, in cases where IMHA is strongly suspected and clinical signs are severe, prednisone is typically started pending labwork to avoid excessive delays in therapy.

Decreased production

An anemia should be considered non-regenerative when the reticulocyte count is less than 60,000/μL (corresponding to a corrected reticulocyte count of less than 1%). However, it should be noted that a regenerative response usually becomes apparent after a minimum of 2-3 days, so acute blood loss or hemolysis may initially appear to be non-regenerative. Once a non-regenerative anemia is identified, bone marrow aspiration is generally indicated. Differentials for decreased production anemias may be grouped according to bone marrow histopathology. Some diseases cause a selective hypoplasia of red cell lines, while others affect all cell lines within the bone marrow (see table 1 above).

Selective erythroid hypoplasia

Anemia of chronic disease

Also termed anemia of inflammation, is immune driven. Cytokines and cells of the reticuloendothelial system (RES) induce changes in iron homeostasis, erythrocyte lifespan, production of erythropoietin, and proliferation of erythroid lines. Iron is diverted from circulation to storage sites within the RES, limiting availability for erythroid progenitors. Inflammatory mediators (TNF, IL-1, IFN) suppress activity of erythroid precursors and decrease their responsiveness to erythropoietin. Release of erythropoietin is also inhibited. Finally, erythrophagocytosis and free radical mediated erythrocyte damage shorten RBC survival. In contrast to iron deficiency anemia, anemia of chronic disease tends to be normocytic, normochromic, rather than microcytic, hypochromic. Serum iron tends to be low, but bone marrow iron stores are adequate. Anemia of chronic disease is generally mild to moderate unless complicated by other factors such as blood loss or hemolysis. Treatment is therefore directed at correcting the underlying disease. Transfusion may be considered if anemia is associated with clinical signs. Iron supplementation for anemia of chronic disease is controversial, and indications for its use in veterinary patients with chronic disease is unclear.

Chronic renal failure

Is typically associated with mild to moderate anemia. Because of the gradual and chronic nature of this type of anemia, it tends to be well compensated until very advanced stages. Anemia in renal failure is multifactorial and results from decreased erythropoietin production by the kidney, impaired responsiveness of bone marrow precursors, shortened RBC lifespan due to uremia, and GI blood loss resulting from uremic ulcers. Anemia of renal failure is typically well compensated, though transfusions may be indicated in the event of concurrent losses or surgery. Human recombinant erythropoietin has been used to stimulate RBC production in veterinary patients with renal failure, but is increasingly being used only as a "last ditch effort" as antibody production against epogen may lead to antibodies being directed against the patient's own erythropoietin as well. Canine recombinant erythropoietin has not been associated with antibody production in dogs with renal failure, but is unfortunately not commercially available. Anabolic steroids (Winstrol-V) have been used in patients with renal failure based on the observations that they increase RBC mass in healthy animals. A benefit in these cases has not been clearly identified.

Pure red cell aplasia (PRCA)

Is an immune mediated disease directed against erythrocyte precursors. The anemia is non-regenerative, normocytic-normochromic, with normal leukocyte and platelet counts. Animals tend to present with marked anemia, as the progression of the disease is typically slow and there is adequate time to mount a compensatory response. Diagnosis is made on the basis of bone marrow aspiration or biopsy, with few to no erythroid precursors seen. In cases of non-regenerative immune-mediated anemia (NRIMA), left shifted erythroid hyperplasia may be seen, with maturation arrest at the level of the metarubricytes or rubricytes. Some animals with NRIMA will also have immune mediated destruction of mature erythrocytes, resulting in concurrent hemolysis. Cats with PRCA should always have PCR or IFA performed on the bone marrow to rule out feline leukemia C associated attack on erythroid progenitors, as this form of PRCA is typically fatal. Treatment for immune-mediated PRCA and NRIMA relies on immunosuppressive therapies similarly to IMHA. Prednisone (2-4 mg/kg/day) and cyclosporine (5-10 mg/kg/day) is the protocol most widely used at this time. Periodic transfusions may be needed until regeneration occurs. This may take weeks to several months. Clinical signs and progression tend to be less severe than IMHA, as the anemia results from decreased production, rather than hemolysis.

Endocrine diseases

Such as hypothyroidism and hypoadrenocorticism may also result in a decreased production anemia. Both cortisol and thyroid hormone have a permissive role in the response of red blood cell precursors to erythropoietin. These anemias are generally mild unless complicated by concurrent blood loss and resolve with hormone replacement therapy.

Generalized bone marrow hypoplasia

Generalized bone marrow hypoplasia

May result from radiation, toxic, or infectious insults to the bone marrow. Common toxins include estrogen, chloramphenicol, phenylbutazone, antifungals, and chemotherapeutic drugs. Infectious diseases resulting in bone marrow hypoplasia include feline leukemia and chronic Ehrlichiosis. Generalized bone marrow hypoplasia may also result from the crowding out of normal bone marrow precursors by neoplastic cells, a process termed myelopthisis. The most common neoplastic causes are the hematopoietic and lymphoid neoplasms including lymphosarcoma, granulocytic leukemia, and lymphoid leukemias. Myelofibrosis, the replacement of marrow spaces by connective/scar tissue, usually represents the endpoint of previous severe marrow injury (as in the case of estrogen toxicity and ionizing radiation) or it may occur spontaneously. Peripheral blood features of myelofibrosis usually include severe nonregenerative anemia, severe leukopenia, and a variable platelet response. Confirmation of the diagnosis depends on marrow core biopsy with a demonstration of connective tissue filling the marrow space.

Normal to hypercellular bone marrow

Iron deficiency anemia

Results from chronic blood loss. In young animals, parasitic infection is the primary ruleout for iron deficiency anemia, while in older animals, gastrointestinal masses or ulcers are generally implicated. Chronic blood loss leads to depletion of bone marrow iron stores over time, resulting in inability to form hemoglobin. Nuclear maturation of RBC precursors is normal however. Precursors continue to divide, getting smaller in size because they never acquire a complete amount of hemoglobin. This results in a hypercellular bone marrow with a build up of metarubricytes. Diagnosis is based on the presence of microcytic, hypochromic anemia, thrombocytosis, source of blood loss, and a bone marrow smear containing no stainable iron. Low serum iron is not diagnostic as it may rapidly decrease with inflammatory disease as a result of tissue sequestration. Treatment is aimed at removal of the source of blood loss. Ferrous sulfate may be administered at a dose of 100-300 mg per day in dogs and 50-100 mg per day in cats if needed. Note that this dose refers to ferrous sulfate, not elemental iron. Reticulocytosis should develop within 3-4 days of supplementation.

Myelodysplasia

Refers to a poorly understood group of diseases characterized by non-regenerative anemia or pancytopenia and prominent dysplastic changes in the bone marrow. Abnormal erythrocytes are generally unable to completely differentiate and early cell death results. Myelodysplasia may result from idiopathic (primary), neoplastic, toxic, immune-mediated, or infectious (FeLV) causes. The myelodysplasias tend to carry a very guarded prognosis, with treatment aimed at immunosuppression, chemotherapy, and/or erythropoietin depending on the suspected cause.

General comments on the treatment of non-regenerative anemias

Treatment of decreased production anemia is best aimed at identifying and eliminating any underlying disease processes or myelosuppressive drugs. Once this is done, clinical experience suggests that the most important thing we can do for patient is to buy time for the bone marrow to repopulate with normal precursor cells. Blood transfusions should be provided as needed until the patient is able to mount a regenerative response of their own. Most patients with non-regenerative anemia require transfusions every 4-6 weeks until their disease is well controlled. Broad-spectrum antibiotics are indicated in the event of severe neutropenia to prevent secondary infections. Immunosuppressive agents may be indicated if an immune-mediated disease (eg. red cell aplasia) is identified or strongly suspected. Myeloproliferative diseases and myelodysplasia tend to carry a poor prognosis, but other forms of decreased production anemia may respond well if the underlying disease or insult is eliminated and adequate time is provided for recovery.

Conclusion

A variety of diseases, both immunologic and non-immunologic in nature, may result in anemia and/or hemolysis in veterinary patients. Successful management relies upon accurate diagnosis and treatment of the underlying disease process. Simple test to help classify a patient's anemia as blood loss, hemolysis, or decreased production may facilitate correct diagnosis. Initiation of immunosuppressive therapy prior to performing a methodical search for infectious, neoplastic, or other causes of anemia may result in therapeutic "missteps" and treatment failure.

References

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2. Duval D, Giger U. Vaccine-associated immune-mediated hemolytic anemia in the dog. J Vet Intern Med 1996;10:290-295.

3. Stegman JR, Birkenheuer AJ, Kruger JM, et al. Transfusion-associated Babesia gibsoni infection in a dog. J Am Vet Med Assoc 2003;222:959-963.

4. Birkenheuer AJ, Levy MG, Breitschwerdt EB. Efficacy of combined atovaquone and azithromycin for therapy of chronic Babesia gibsoni infections in dogs. J Vet Intern Med 2004;18:494-498.

5. Van Audenhove A, Verhoef G, Peetermans WE, et al. Autoimmune haemolytic anaemia triggered by Bartonella henselae infection: a case report. Br J Haematology 2001;115:924-925.

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8. Weiss DJ. Primary pure red cell aplasia in dogs: 13 cases (1996-2000). J Am Vet Med Assoc 2002;221:93-95.