Immune Mediated Hemolytic Anemia in a German Shepherd : Diagnosis and Successful Long Term Treatment

Immune-Mediated Hemolytic Anemia
in a German Shepherd:
Diagnosis and Successful Long -Term
Treatment







Senior Seminar Paper by Emily M. Walker
Cornell University College of Veterinary Medicine
Advisor: Dr. John Randolph
Clinician: Dr. Alice Hadden
Presentation Date: February 7, 2007


Key Words: Immune-Mediated Hemolytic Anemia, Azathioprine, Corticosteroids,
Mycophenylate, Pulmonary Thromboembolism

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Summary


“Darby,” a three year old female spayed German Shepherd, presented to the
Cornell University Hospital for Animals (CUHA) with previously diagnosed immune-
mediated hemolytic anemia (IMHA) that had been treated with dexamethasone and
penicillin. Initial evaluation of Darby at the referring veterinarian’s hospital disclosed
lethargy, anorexia, brown-tinged urine, fever, splenomegaly, and icteric, pale mucous
membranes. Despite increasing doses of dexamethasone and a blood transfusion, Darby’s
anemia and clinical condition worsened over the one week period of management.
At CUHA, in addition to previously noted clinical signs, Darby had muscle
wasting, skin fragility, gastrointestinal hemorrhage, urinary tract infection, and propensity
to bleeding and clot formation. These changes were attributable to complications of
IMHA and corticosteroid therapy. A specific cause of IMHA was never detected. During
two weeks of hospitalization, Darby slowly improved with red blood cell and plasma
transfusions, expanded immunosuppressive therapy, antibiotics, anti-emetics, an anti-
thrombotic agent, gastroprotectant medications, nasal oxygen, and extensive nursing care.
Seven months after hospital discharge, Darby remains healthy with a PCV of 45%, and
continues to be slowly tapered off medications.

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Case Report
A three year old female spayed German Shepherd, “Darby,” presented to the
Cornell University Hospital for Animals (CUHA) with previously diagnosed immune-
mediated hemolytic anemia (IMHA). Darby first presented to her referring veterinarian
for lethargy, anorexia, and brown urine. Historically, Darby had no administration of
medications other than seasonal Heartgard and Frontline, and no vaccinations had been
given recently. There was no exposure to toxins in the home environment, and a
companion dog at home remained healthy. Darby’s initial physical examination at the
referring veterinarian’s hospital revealed pale, slightly icteric mucous membranes,
splenomegaly, and fever. Clinicopathologic findings of regenerative anemia (PCV 19%,
reference range 42-57%) with spherocytes, agglutination, and hyperbilirubinemia were
consistent with IMHA. Subsequently, Darby was treated with penicillin and
dexamethasone (2-4.5 mg/kg/day). However, despite these increasing doses of
dexamethasone and a blood transfusion, Darby’s anemia and clinical condition worsened
over the one week period of management. (Graph 1)
At the CUHA, Darby was recumbent and vomiting. In addition to the previously
noted clinical signs, the dog showed severe jaundice, dramatic muscle wasting, skin
fragility with crusts over pressure points, gastrointestinal hemorrhage (melena on rectal
examination and frank blood in vomitus), tachycardia, increased respiratory effort, and a
jugular thrombus at the site of a prior intravenous catheter.
Additional tests were run to further evaluate her clinical status. Complete blood count
revealed a regenerative anemia (PCV 14%, reference range 42-57%) with spherocytes,
nucleated red blood cells, and macroscopic and microscopic auto-agglutination; a

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leukocytosis consisting of a neutrophilia (47 thou/uL, reference range 6-14 thou/uL) with
a left shift was also noted. Coagulation profile revealed hyperfibrinogenemia (928
mg/dL, reference range 150-480 mg/dL), increased prothrombin time [PT] and activated
partial thromboplastin time [aPTT] (21 and 24 seconds respectively, reference ranges 13-
18 and 10-17 seconds, respectively), increased D-dimers (>2000 ng/mL, reference range
<250 ng/mL), and decreased antithrombin levels (61%, reference range 75-120%).
Abnormalities in her serum biochemical profile revealed azotemia (BUN 256 mg/dL,
reference range 8-30 mg/dL and creatinine 5.3 mg/dL, reference range 0.5-1.3 mg/dL),
increased liver enzyme activities (alanine aminotransferase [ALT] 470 U/L, reference
range 25-106 U/L; alkaline phosphatase [ALP] 518 U/L, reference range 12-122 U/L;
gamma glutamyl transferase [GGT] 140 U/L, reference range 0-10 U/L; aspartate
aminotransferase [AST] 383 U/L, reference range 16-50 U/L), evidence of muscle
damage (AST 383 U/L, reference range 16-50 U/L; creatine kinase 2085 U/L, reference
range 58-241 U/L), hypoproteinemia (3.7 g/dL, reference range 5.6-7.1 g/dL), and
hyperbilirubinemia (19.5 mg/dL, reference range 0-0.3 mg/dL). A urine culture and
sensitivity revealed a urinary tract infection consisting of two strains of E. coli sensitive to
cefazolin, cefpodoxime, and imipenem.
Thoracic radiographs and abdominal ultrasound were performed, and no sign of
neoplasia was found. However, changes consistent with possible pulmonary
thromboembolism were detected. Serologic tests for Dirofilaria immitis, Ehrlichia canis,
Ehrlichia risticii, Anaplasma phagocytophilum, Babesia canis, and Borrelia burgdorferi
were negative.

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Darby was immediately blood typed and given a packed red blood cell transfusion.
Due to concerns about hypercoaguability and potential disseminated intravascular
coagulation (DIC), a plasma transfusion also was given within 24 hours of her
presentation. Additionally, Darby was treated with an expanded immunosuppressive
therapy protocol including dexamethasone (0.1 mg/kg/day), azathioprine (2 mg/kg/day
SID for one week as a loading dose followed by 2 mg/kg every other day), and
mycophenylate (9 mg/kg/day). Later, the dexamethasone was changed to prednisone (1
mg/kg/day). Furthermore, ultra low-dose aspirin therapy (0.5 mg/kg/day) was begun for
its antithrombotic effects. Anti-emetics (dolasetron and metoclopramide) and
gastroprotectants (famotidine and sucralfate) were given, as were antibacterial drugs
(doxycycline, enrofloxacin, metronidazole, and cefpodoxime).
Being recumbent, Darby was maintained on a waterbed, and every 6 hours she was
turned, her bladder was expressed, range-of-motion exercises with massage were
administered, and her skin lesions were cleaned and dried. Her PCV was monitored twice
daily, and every other day blood was drawn to assess her status via a complete blood
count and serum biochemical profile. Oxygen therapy was administered through a nasal
canula when her oxygenation appeared to be impaired.
Although Darby continued to remain anemic throughout her two week stay at CUHA,
her PCV remained steady at 20%. This finding suggested that the immunosuppressive
therapy had slowed any on-going RBC destruction by her immune system, allowing her
regenerative capacity to stabilize her PCV. Additionally, gastroprotectants were likely
abating her gastrointestinal bleeding, as further demonstrated by resolution of her melena.
Fluid therapy resulted in a rapid normalization of her creatinine and dramatic decrease in

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her BUN, suggesting that the initial azotemia was a result of dehydration. The persistent
milder increase in BUN is likely attributable to digestion of blood from the
gastrointestinal bleeding.
Darby slowly recovered from her gastrointestinal bleeding, muscle wasting, and
pulmonary thromboemboli. Although weakened from her disease, Darby did regain
enough strength to begin to walk outside and urinate on her own. Although she did have
dramatic skin and haircoat damage from being recumbent and receiving high-dose
corticosteroid therapy, her wounds did not become infected and over time she grew fur
over these areas. Her coagulation profile eventually normalized, as did the parameters on
her biochemical profile. Within two months of the initial onset of disease, Darby’s PCV
returned to within the reference range and has remained there ever since, even as the
immunosuppressive drugs are tapered. (Graph 2)

Discussion
Darby was diagnosed with primary, or idiopathic, immune-mediated hemolytic
anemia. Primary IMHA develops when the body fails to recognize red blood cells as self,1
but rather identifies them as foreign, opsonizes them with IgG, IgM, or complement, and
allows phagocytes in the spleen, liver, or bone marrow to partially or completely destroy
them.2,3 Secondary IMHA, on the other hand, is triggered by antibodies or complement
that respond to altered red blood cell surface antigens induced by a microorganism, drug,
previous transfusion, or toxic agent.1,3,4 Treatment for primary IMHA principally
involves immunosuppression and transfusion therapy as necessary. General support of
the patient through the ensuing complications of the disease and its treatment is essential.

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Throughout the first week of management, Darby received repeated high daily doses
of dexamethasone (2- 4.5 mg/kg/day) that failed to control the immune-mediated disease.
Unfortunately, these high doses of dexamethasone likely contributed to complications
Darby subsequently faced including muscle wasting, gastrointestinal bleeding, skin
lesions, and urinary tract infection. Dexamethasone is a corticosteroid considered to be
seven to ten times as potent as the corticosteroid prednisone. Prednisone, typically given
at 2-4 mg/kg/day as an immunosuppressive dose in the dog, is known to decrease
circulating levels of T-lymphocytes, inhibit phagocytosis and chemotaxis, and antagonize
the complement cascade.5 Side effects of gastrointestinal ulceration and bleeding are often
related to the fact that it increases gastric acid, pepsin and trypsin levels, changes the
structure of mucin, and decreases mucosal cell proliferation.5 Prednisone and
dexamethasone also have catabolic effects that may result in muscle and skin atrophy, and
their immunosuppressive effects may lead to opportunistic infections. Additionally
corticosteroid treatment is known to increase serum activities of ALT, GGT, and ALP.5
To minimize the deleterious side effects of prednisone or dexamethasone, management of
canine IMHA frequently incorporates use of other immunosuppressive agents to allow for
lower dose or more rapid taper of corticosteroids.6
Azathioprine is a drug that antagonizes purine metabolism, thereby inhibiting RNA
and DNA synthesis and mitosis. It works best on delayed hypersensitivity and cellular
immunity.5 The drug is typically given at a dose of 2mg/kg/day by mouth as a loading
dose for 5-7 days, and then reduced to 1-2 mg/kg by mouth every other day. It often takes
several weeks to become fully effective. Azathioprine can be further tapered or removed
from immunosuppressive treatment plans, although the tapering process may take months.

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Side effects of azathioprine include bone marrow suppression, hepatotoxicity, and
pancreatitis.5

Darby’s third immunosuppressive drug, mycophenylate, restrains proliferation of B
and T lymphocytes and reduces production of cytotoxic T cells by inhibiting an enzyme
crucial for their biosynthesis.7 Initial drug dose is approximately 10 mg/kg/day, and the
main side effect is myelosuppression.
Other immunosuppressive drugs that have been used in treatment of canine IMHA
include danazol, cyclosporine, cyclophosphamide, and human immunoglobulin; bovine
hemoglobin has been used as an alternative to red blood cell transfusion. Each of these
therapies has been evaluated as to its risk or benefit in canine IMHA.8,9 However,
because these drugs were not used in Darby, they will not be discussed in this manuscript.
Hypercoaguability, a common finding in dogs with IMHA, is caused by a
combination of factors. Red blood cells are destroyed which results in circulating
breakdown products like hemoglobin or bilirubin that cause inflammation and injury to
vascular endothelium.6 With red blood cells that are agglutinating, decreased blood flow,
and activated hyperexcitable platelets in this disease, it is no surprise that an irritated and
inflamed endothelium lays the foundation for excessive clot formation.6,10 The hyper-
coaguability predisposes these patients to pulmonary thromboemboli, 6,11,12 as well as
more generalized thromboembolic phenomenon and disseminated intravascular
coagulation (DIC). Unfortunately, the hypercoaguable state and consequent pulmonary
thromboembolism may be aggravated by concurrent corticosteroid treatment,11 although
the mechanism is not fully understood.

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Aspirin causes irreversible acetylation of platelet cyclooxygenase sites, thereby
inhibiting activation of platelets and initiation of clot formation.5 When given at an ultra
low-dose (0.5 mg/kg/day), aspirin can provide thromboprophylaxis in a dog with IMHA
that has or will likely become hypercoaguable.1,6 Mixed or low molecular weight heparin
has also been used historically to achieve similar thromboprophylaxis, although several
studies find a poor response.4,6
Fresh-frozen plasma transfusion is frequently used in dogs with evidence of
hemostatic abnormalities in an attempt to correct prolonged PT and aPTT or reduced
antithrombin activity. However, administration of fresh-frozen plasma has not been
shown to be effective in preventing thromboembolic complications in dogs with primary
IMHA.12 In fact, one study demonstrated that antithrombin activity 30 minutes following
fresh-frozen plasma transfusion was not increased, the incidence of thromboembolism
was not decreased, and short term survival was not improved.12
Darby faced many complications associated with IMHA and its treatment. Having
specific reliable prognostic indicators that would help clinicians and owners assess a dog
with IMHA might be beneficial. Historically, studies have suggested that poor prognostic
factors in canine IMHA include the severity of the anemia, absence of regeneration,
presence of agglutination, thrombocytopenia, hyperbilirubinemia, increased serum ALP
activity, and hypoalbuminemia. A more recent study at the Cornell University College of
Veterinary Medicine6 demonstrated that only thrombocytopenia and hyperbilirubinemia
were truly negative prognostic indicators. The Cummings School of Veterinary Medicine
at Tufts University13 currently is developing a canine hemolytic anemia objective score
(CHAOS) based on age, body temperature, agglutination, hyperbilirubinemia, and

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hypoalbuminemia to predict outcome. Clearly, more work is needed to fully understand
the complexities of this disease process and to be able to more accurately predict the
outcome of a given case. In retrospect, Darby would likely have been given a poor
prognosis based on most prognostic schemes. Yet, with extensive care and intense
therapies, she has survived eight months.
Recurrence of disease in dogs with IMHA is known to occur. In one study, as many as
15% dogs with IMHA surviving greater than 60 days had recurrence of disease that
tended to coincide with tapering or cessation of immunosuppressive medications.6
Despite slowly tapering Darby’s immunosuppressive medications, there has been no
recurrence of her anemia.

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