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Vol.18, No.

2 February 1996

Continuing Education Article

Diagnosis of Canine
★ Secondary causes of immune-
mediated disease must be ruled
Hematologic Disease
out because they affect treatment
decisions and prognosis.
Purdue University
Adam L. Honeckman, DVM
KEY FACTS Deborah W. Knapp, DVM, MS
William J. Reagan, DVM, PhD
■ Immunologic tests cannot
differentiate primary
(autoimmune) from secondary
immune-mediated hematologic
disease. I mmune-mediated hemolytic anemia and immune-mediated thrombocy-
topenia are the most common immune-mediated hematologic disorders in
dogs.1 In immune-mediated hemolytic anemia, destruction of erythrocytes
is accelerated because of antibodies and/or complement attached to erythrocyte
membrane.1 In immune-mediated thrombocytopenia, increased destruction or
■ Samples for immunologic
testing should be collected decreased production of platelets is mediated by antibodies and/or comple-
before immunosuppressive ment.1 Immune-mediated anemia or thrombocytopenia can occur alone or
therapy is begun. concurrently or in conjunction with systemic lupus erythematosus (SLE).2,3
This article describes the pathophysiology, clinical signs, and differential diag-
■ The direct immunoglobulin nosis of canine immune-mediated hematologic disease and presents a classifica-
(Coomb’s) test cannot detect tion scheme and diagnostic plan.
which antigen the antibodies are
directed against, nor does a PATHOPHYSIOLOGY
negative Coomb’s test rule out The immune-mediated disease process against erythrocytes or platelets may
immune-mediated hemolytic be primary (autoimmune) or secondary. The terms autoimmune and immune-
anemia. mediated are not synonymous. In an autoimmune response, the immune sys-
tem recognizes and attacks self antigen. A secondary immune response is di-
■ The direct immunofluorescence rected at a foreign antigen but leads to inadvertent damage to normal host
test is more sensitive than the tissue and cells. The frequency of primary and secondary immune-mediated
platelet factor 3 test, but a hemolytic anemia was similar in a recent study (42.9% and 57.1%, respective-
negative result does not rule ly).4 Causes of secondary immune-mediated anemia or thrombocytopenia in-
out immune-mediated clude neoplasia, infectious disease (parasitic, viral, bacterial, rickettsial, or fun-
thrombocytopenia. gal), and drug therapy.1 Neoplasia is reportedly the most frequent cause of
secondary immune-mediated hemolytic anemia.4
The immune-mediated destruction of erythrocytes involves antibodies bind-
ing to the cell membrane. This binding can result in any or all of the following:

■ Complement-mediated lysis (intravascular hemolysis), which is more likely

to occur with IgM than IgG antibodies.
Small Animal The Compendium February 1996

■ Complete phagocytosis and destruction by a macro- Diagnostic Workup

phage (extravascular hemolysis). Nonimmunologic Tests
■ Partial phagocytosis by a macrophage; only part of The diagnostic workup of a dog with suspected im-
the cell membrane and cytoplasm is removed, thus mune-mediated hemolytic anemia should include a
producing a spherocyte.5 complete blood count, reticulocyte count, blood smear
evaluation, serum chemistry profile, and urinalysis. A
In addition to being categorized as primary or sec- presumptive diagnosis of immune-mediated hemolytic
ondary, immune-mediated hemolytic anemia can also anemia is made when the blood smear shows appropri-
be classified according to the thermal dependency of ate morphologic features and other causes of anemia
antibody, presence or absence of agglutination, and have been ruled out. Immunologic tests help confirm
presence or absence of hemolysis. The box summarizes the diagnosis. It is important to distinguish primary
this classification scheme1,5: from secondary immune-mediated hemolytic anemia
The mechanisms of immune-mediated platelet de- (Figure 1).
struction are similar. Antibody binding to platelets can Because immune-mediated hemolytic anemia and
cause complement-mediated lysis or phagocytosis by immune-mediated thrombocytopenia can be concur-
macrophages in the spleen or liver.1,3,5 Antibodies bind- rent, a platelet count should also be performed. If
ing to megakaryocytes can cause thrombocytopenia by thrombocytopenia is present, then a diagnostic workup
interfering with the production of platelets.1,3,6,7 for immune-mediated thrombocytopenia should also
be performed.
ANEMIA In cases of immune-mediated hemolytic anemia, a
Signalment complete blood count confirms the anemia and often
Immune-mediated hemolytic anemia has been re- reveals leukocytosis (characterized by neutrophilia with
ported to occur more commonly in middle-aged female or without a left shift) due to complement activation
dogs, with an increased incidence in
cocker spaniels, poodles, and Old
English sheepdogs.5,8 Other investi- Antibodies Classified by
gators, however, have not noted a
gender or breed predisposition.2,5
Thermal Dependence
I. Warm antibody (i.e., optimally reactive at 37˚C)
Clinical Signs and
Differential Diagnosis A. Incomplete antibody
Dogs with immune-mediated ■ Does not by itself agglutinate erythrocytes suspended in
hemolytic anemia are often present- saline
ed because of lethargy, weakness, de- ■ Is the most common form of antibody in immune-
pression, or anorexia. The clinical mediated hemolytic anemia
signs usually have an insidious onset ■ Results in clinical disease with an insidious onset and
because most of the antibodies are chronic course
incomplete. Incomplete antibodies B. Warm agglutinins
cause slowly developing anemia ■ Cause in vivo agglutination
more often than they cause rapid au- ■ Result in clinical disease with severe, sudden onset and
toagglutination. Sudden onset of se- guarded prognosis
vere signs can occur, especially if the C. In vivo hemolysins
antibodies are of the warm agglu- ■ Antibody causes massive complement fixation
tinin type. Physical examination ■ Lead to in vivo hemolysis and hemoglobinuria
may reveal pale mucous membranes, II. Cold antibody (i.e., optimally reactive at <37˚C)
tachycardia, a heart murmur (sec- A. Cold agglutinins
ondary to decreased blood viscosity ■ Are usually mediated by IgM
from anemia), splenomegaly, hep- ■ Cause ischemic necrosis of extremities
atomegaly, icterus, and fever. 1,2,5,8 B. Nonagglutinating hemolytic
The major diagnostic differentials ■ Are rare in veterinary medicine
(see the box) for anemia in dogs ■ Causes paroxysmal cold hemoglobinuria.
must be considered for each pa-


The Compendium February 1996 Small Animal

History and physical examination

Key Anemia
Test or procedure
Findings low Complete blood count

Conclusion Anemia
Clinical suspicion
Diagnostic differentials Reticulocyte count
Blood smear for erythrocyte morphology
Serum biochemistry profile

no yes
Reticulocyte count > 60,000 cells/µl?
Corrected reticulocyte percentage > 1
Nonregenerative anemia Regenerative anemia
decreased normal
Acute blood loss or hemolysis Total protein

Extramarrow disease History of trauma With or without bilirubinuria,

• Renal disease Evidence of blood loss hemoglobinuria,
• Liver disease hyperbilirubinemia
• Inflammation
• Iron deficiency Blood loss
Intramarrow disease Acute blood loss
• Neoplastic infiltration Chronic blood loss Erythrocyte morphology
• Myelosuppressive drugs Iron deficiency
• Rickettsial disease
Spherocytes with or Heinz bodies
without agglutination Parasites
Bone marrow aspirate with or Schistocytes
yes Acanthocytes
without core biopsy

negative Direct antiglobulin positive Other causes

(Coomb’s) test
of hemolysis

False-negative Immune-mediated disease


Thoracic radiographs
Abdominal radiographs
Infection (e.g., rickettsial) negative Ultrasonography
Primary immune-mediated anemia
Drug reaction Ophthalmologic examination

Figure 1—Diagnostic approach to anemia in dogs.

Small Animal The Compendium February 1996

and neutrophil chemotaxis.5 phology and to estimate platelet numbers. Spherocyto-

Differential Diagnosis Total protein should be eval- sis or agglutination strongly suggests immune-mediated
uated carefully. In cases of hemolytic anemia. Agglutination appears as a grapelike
of Anemia in Dogs
anemia secondary to de- clustering of erythrocytes in the blood smear. It must
creased production or in- be distinguished from rouleaux (erythrocyte clusters re-
Decreased creased destruction of eryth- sembling stacks of coins), which can occur in cases of
erythrocyte production rocytes, total protein is usually inflammatory disease.
(nonregenerative anemia) normal. When anemia is sec- This distinction can sometimes be made by examin-
■ Rickettsial disease ondary to blood loss, the ing a wet-mount preparation of the blood sample.
■ Lymphoma or other packed cell volume and total One small drop of blood should be mixed with one
neoplasia protein may be decreased be- large drop of isotonic saline, put on a clean micro-
■ Chemotherapy, cause of loss of erythrocytes scope slide, covered with a coverslip, and examined
and plasma proteins, with a under a microscope. Rouleaux, unlike agglutination,
estrogens, or other
compensatory shift of fluid should be dispersed by the addition of saline.1,9 This
drugs from the interstitial space to test, however, is not infallible; false-negative results
■ Immune-mediated the intravascular compart- may result from weak agglutinins. Other morphologic
disease ment.10 features that may be present in the blood smear in-
■ Chronic renal disease An absolute reticulocyte clude polychromasia, anisocytosis, and metarubricyto-
■ Chronic inflammatory count or corrected reticulo- sis.1,5,8,9
cyte percentage should be Bone marrow aspiration or biopsy is indicated in cas-
used to determine whether es of nonregenerative anemia (or thrombocytopenia) to
the anemia is regenerative or evaluate the cause. If nonregenerative anemia is im-
Blood loss nonregenerative. An absolute mune mediated, erythroid hyperplasia is often present.
(regenerative anemia) number of reticulocytes A serum biochemistry profile and urinalysis should
■ Trauma greater than 60,000 per mi- be performed if immune-mediated hemolytic anemia is
■ Disseminated croliter or a corrected reticu- possible. Dogs with immune-mediated hemolytic ane-
intravascular locyte percentage (reticulo- mia often have hyperbilirubinemia or bilirubinuria.12
coagulopathy cyte percentage × observed The serum biochemistry profile and urinalysis are also
packed cell volume ÷ normal useful in ruling out other systemic diseases.
■ Gastrointestinal ulcers
packed cell volume) greater
■ Anticoagulant than one represents a regen- Immunologic Tests
rodenticide erative response.9,10 The direct antiglobulin test (also known as the direct
■ Neoplasia Typical cases of immune- Coomb’s test) is a useful additional test to support the
■ Parasitic (fleas, mediated hemolytic anemia diagnosis of immune-mediated hemolytic anemia. It
hookworms) are regenerative because ma- detects antibodies or complement on erythrocytes. The
ture erythrocytes are being Coomb’s reagent, which contains species-specific anti-
Hemolysis hemolyzed but erythrocyte bodies against various classes of antibodies and comple-
(regenerative anemia) precursors are unaffected.5,8,12 ment, is added to washed erythrocytes from the patient.
Although immune-mediated Agglutination occurs if the ratio between the antibodies
■ Immune-mediated
hemolytic anemia is usually or complement on the erythrocytes and the antiglobu-
disease regenerative, immune-medi- lins in the reagent is proper.5,9
■ Disseminated ated anemia may be nonre- The direct antiglobulin (Coomb’s) test is positive in
intravascular generative if antibodies and/ 60% to 70% of cases of immune-mediated hemolytic
coagulopathy or complement are directed anemia.2,8,9 As the following list indicates, however,
■ Microangiopathy (e.g., against erythrocyte precur- there are many possible causes of false-negative results
sors in the bone marrow or if and a negative Coomb’s test does not rule out immune-
the onset is acute.1,5,8,12 There- mediated hemolytic anemia5,8,9,13:
■ Heinz body anemia fore, lack of reticulocytosis
■ Pyruvate kinase does not rule out immune- ■ Insufficient quantity of antibody or complement on
deficiency mediated hemolytic anemia. erythrocytes
■ Babesiosis A blood smear should be
examined microscopically to ■ Improper antiglobulin-to-antibody ratio
evaluate erythrocyte mor- ■ Test performed at improper temperature


Small Animal The Compendium February 1996

■ Corticosteroid therapy (usually >1 week’s duration) THROMBOCYTOPENIA

Signalment Differential Diagnosis
■ Incorrect species-specific reagent
Immune-mediated throm-
of Bleeding
■ Drug-induced immune-mediated hemolytic anemia bocytopenia is reportedly
more common in middle-aged Disorders in Dogs
(if drug was not incorporated into test).
female dogs, especially Ger-
Vascular disorder
The direct antiglobulin (Coomb’s) test is fairly specif- man shepherds, standard poo-
dles, Old English sheepdogs, ■ Considered rare in
ic, but the following can cause false-positive results5,8,9,13:
and cocker spaniels.3,17–19 veterinary medicine
■ Infectious, inflammatory, neoplastic, and immune-
Clinical Signs and Qualitative platelet
mediated diseases that cause excess plasma antibody
Diagnostic Differentials dysfunction
that coats erythrocytes but does not cause immune-
mediated hemolytic anemia Dogs with immune-mediat- ■ Renal disease
ed thrombocytopenia may ■ Aspirin and other
■ Previous blood transfusion (especially 3 to 21 days be presented with lethargy, nonsteroidal
ago) weakness, epistaxis, melena, antiinflammatory
hematemesis, hematochezia,
■ Storage of clotted blood samples at 4˚C (thus caus- drugs
hematuria, petechiae, and/or
ing in vitro binding of complement to erythrocytes) ■ Von Willebrand’s
ecchymoses. It is important to
■ Titers (up to 1:8) to cold agglutinins not causing consider the diagnostic differ- disease
clinical disease. entials (see the box) for bleed-
ing disorders in dogs.3,7,17,20–22 Quantitative
The direct antiglobulin test only detects the presence platelet disorder
of antibody or complement on the surface of the blood Diagnostic Workup (thrombocytopenia)
cell. The test cannot determine which antigen the anti- Nonimmunologic Tests Decreased platelet
body is directed against and thus does not distinguish The diagnostic workup for production
primary from secondary immune-mediated hemolytic animals that have possible im- ■ Rickettsial disease
anemia. mune-mediated thrombocy- ■ Lymphoma and other
The test that uses a polyspecific reagent that detects topenia or other bleeding
IgG, IgM, or C3 on the erythrocyte is the most widely disorder should include a com-
used and available direct antiglobulin diagnostic test. plete blood count, blood-smear ■ Immune-mediated
The following tests are more sensitive: evaluation, platelet count, eval- ■ Estrogen toxicity
uation of platelet size (if avail- ■ Chemotherapy
■ A Coomb’s test using separate, specific tests for IgG, able), prothrombin time and Increased platelet
IgM, and C32,13 activated partial thromboplas- destruction or utilization
tin time, fibrin degradation
■ A direct enzyme-linked antiglobulin test that mea- ■ Immune-mediated
products, and examination of
sures the amount of IgG, IgM, and C3 on the eryth- bone marrow aspirate. ■ Disseminated
rocytes4,14 The presumptive diagnosis intravascular
■ A papain test that modifies erythrocyte membranes of immune-mediated throm- coagulopathy
to make them more susceptible to agglutination and bocytopenia is based on find- ■ Rickettsial disease
thus better able to detect incomplete antibodies15 ing the appropriate changes in Coagulation factor
the laboratory parameters as
■ Radioimmunoassays that measure the amount of well as ruling out other causes
IgG bound to erythrocytes.16 of thrombocytopenia, includ- ■ Anticoagulant
ing increased sequestration rodenticide toxicity
A recent study using the direct enzyme-linked and utilization of platelets. ■ Disseminated
antiglobulin test showed a correlation between the The diagnosis is confirmed intravascular
severity of anemia and the amount of IgG bound to the with immunologic tests. Once coagulopathy
erythrocytes. 4 Another study determined that the immune-mediated thrombo-
■ Hereditary coagulation
amount of erythrocyte-bound immunoglobulin fell as cytopenia has been diagnosed,
anemia improved during treatment.14 Some of these it must be classified as primary factor deficiency
tests are likely to gain more widespread use. or secondary (Figure 2).


Small Animal The Compendium February 1996

History and physical examination

Bleeding disorder

Complete blood count

decreased normal
Platelet count

abnormal normal abnormal normal

Coagulation profile Coagulation profile

Disseminated Bone marrow aspirate Acquired or hereditary Vascular disorder or

intravascular cytology with or coagulation factor qualitative platelet
without bone marrow
coagulopathy core biopsy deficiency disorder

decreased normal to increased


Decreased platelet Increased platelet

production destruction

Neoplastic infiltration, Immune-mediated

myelosuppressive thrombocytopenia
drugs, rickettsial
disease, or immune-
mediated disease

negative Antimegakaryocyte antibody test positive

Antiplatelet antibody test

False-negative Immune-mediated

Test or procedure Thoracic radiographs
Primary immune-
Findings mediated Abdominal radiographs
thrombocytopenia Ultrasonography
Ophthalmologic examination
Clinical suspicion
Diagnostic differentials

Figure 2—Diagnostic approach to bleeding disorders in dogs.

The Compendium February 1996 Small Animal

A complete blood count may be useful for detecting Immunologic Tests

concurrent cases of immune-mediated hemolytic ane- The platelet factor 3 test and antimegakaryocyte anti-
mia or severe anemia secondary to blood loss. The body test (detected by direct immunofluorescence) are
blood smear can be used to estimate the number of two ancillary tests that help confirm the diagnosis of
circulating platelets. Six or seven platelets per oil-im- immune-mediated thrombocytopenia. The platelet fac-
mersion field (×1000) in the monolayer of the blood tor 3 test is designed to detect antiplatelet antibodies in
smear corresponds to approximately 100,000 per mi- the patient’s serum. The patient’s serum is mixed with
croliter. It is generally believed that the platelet count normal washed platelets, coagulation factors XI and
must be below approximately 50,000 per microliter XII, and calcium chloride. A control serum sample is
(assuming that the platelets are functional and clot- tested at the same time. If the patient’s serum contains
ting factors are normal) before clinical bleeding prob- adequate antiplatelet antibodies, these bind to the nor-
lems occur. Therefore, fewer than three or four mal platelets and cause the release of membrane phos-
platelets per oil-immersion field (×1000) represents pholipid (platelet factor 3). This process results in
clinically significant thrombocytopenia (i.e., throm- accelerated clotting (as measured by partial thrombo-
bocytopenia severe enough to cause a bleeding disor- plastin time) compared with that of the control. The
der by itself ).23 platelet factor 3 test can also be run in the presence of a
The severity of thrombocytopenia and size of the drug if drug-induced immune-mediated thrombocy-
platelets may also help suggest a cause. In a retrospec- topenia is suspected.3,5
tive study, 21 out of 22 dogs with platelet counts below The platelet factor 3 test is considered specific but
20,000 per microliter were given the diagnosis of im- has relatively low sensitivity (15% to 70%) for im-
mune-mediated thrombocytopenia. Microthrombocy- mune-mediated thrombocytopenia.1,6 Therefore, a neg-
tosis (mean platelet volume below 5.4 femtoliters) has ative test does not rule out immune-mediated thrombo-
recently been shown to occur in dogs with immune- cytopenia. Because of its low sensitivity, the platelet
mediated thrombocytopenia.24 factor 3 test is seldom used. False-negative findings may
Seventeen out of 18 dogs that had mean platelet vol- result from an inadequate amount of antibody in
umes below 5.4 femtoliters were given the diagnosis of serum, drug-induced immune-mediated thrombocy-
immune-mediated thrombocytopenia. When all of the
dogs with immune-mediated thrombocytopenia were
evaluated, however, only 55% had microthrombocyto- Raise the Standard of Practice at Your Hospital with
sis. This probably reflects the variability in time of pre-
sentation of these dogs (early in the process, damage to

’S •


the platelets may lead to formation of small platelets).




R D S of
After a while, the bone marrow responds to decreased EMERGENCY AND CRITICAL CARE MEDICINE
platelet mass and larger platelets are produced.
Prothrombin time and activated partial thrombo- ...An exciting new publication

plastin time should be measured to help rule out dis-

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seminated intravascular coagulopathy, anticoagulant EMERGENCY AND CRITICAL CARE MEDICINE®

Feline Hepatic Lipidosis ■ Other abnormalities may be

found depending on the primary
rodenticide toxicity, and other coagulation-factor defi- Sharon A. Center, DVM, DACVIM disease.
Professor, Internal Medicine
College of Veterinary Medicine Laboratory Findings
Cornell University ■ CBC.

epatic lipidosis (HL) is the most common cause of jaundice in cats
• Poikilocytosis (i.e., irregular
RBC shapes) is common. Expert help fast in a
ciencies. Serial evaluations of the clotting profile may
in North America. It develops primarily in obese cats that have • A mild nonregenerative anemia
recently been anorectic. By definition, HL occurs when >50% of accompanies primary
hepatocytes accumulate excessive triglycerides (TGs), resulting in severe cell

practical and readable format

underlying chronic
vacuolation, cholestasis, and liver dysfunction. Left untreated, HL progresses inflammatory disorders.
to metabolic dysregulation and death. Although HL was initially considered • Hemolytic anemia may
an idiopathic disorder, it is now known to more commonly occur secondary be severe and related to

be most helpful in detecting these disorders. to other disease conditions.

Historical Information
■ Age/gender/breed
predispositions. None.
constipation may occur as part of
primary disease, but these signs are
highly variable among cats.
hypophosphatemia or Heinz
body formation at presentation
and/or during treatment.
• Leukogram reflects underlying
disorders; HL is not a reactive

Bone marrow samples should be aspirated in cases of Each monthly* issue

■ Other historical considerations. Physical Examination process (no necrosis,
• Most commonly noted in Findings inflammation, fibrosis).
indoor, obese cats; most cats ■ Unkempt and in poor condition, • Icteric plasma.
have been anorectic for several jaundice, and weakness. ■ Biochemistry.
days or longer. ■ May have ptyalism without • Hyperbilirubinemia.
• On presentation, many have provocation or on oropharyngeal • ↑↑↑ALP, ↑↑ALT, ↑↑AST,
is peer-reviewed
thrombocytopenia. Cytologic examination of bone lost at least 25% of pre-illness examination. normal or mildly ↑γGT.
body mass. ■ Variable dehydration attributed Discordance between
• Reclusiveness, affection, and to anorexia, vomiting, and ALP–γGT is an important
lethargy are typical owner diarrhea. diagnostic feature. The ↑γGT
observations. ■ Head and neck ventroflexion. indicates necroinflammatory
• Jaundiced mucous membranes, ■ Cats show some signs

marrow aspirate can reveal myelophthisic disease and nonpigmented skin or sclera, and
hyperbilirubinuria may be noted.
• Ptyalism, vomiting, diarrhea, or
Articles with this symbol provide
standards for canine patients.
consistent with severe hepatic
encephalopathy (HE), such as
lethargy, collapse, obtundation,
and seizure activity.
■ Abdominal palpation discloses

Peer-Reviewed Articles on
In each article you will find:
nonpainful hepatomegaly.

an estimate of the number of megakaryocytes. Bone Articles with this symbol provide
standards for feline patients.
Articles with both symbols cover canine
and feline topics.
■ Bleeding tendencies may be
evidenced by bruising around
venipuncture, palpation, or
ultrasound probe sites.
1 Feline Hepatic Lipidosis
6 Congenital Portosystemic Shunts
10 Correction
■ Danger signs
marrow aspirate should also be submitted for an- STANDARDS of CARE: EMERGENCY AND CRITICAL CARE MEDICINE
■ Guidelines
■ Step-by-step tips
timegakaryocyte antibody testing in possible cases of
immune-mediated thrombocytopenia. Subscribe today!
Standards of Care: Emergency
Because the cellularity of bone marrow aspirate Call 800-426-9119. and Critical Care Medicine.
varies, the number of megakaryocytes is difficult to de- Only $69 for 11 Concise. Authoritative. Cur-
information-packed issues.* rent. No general practice should
termine. In addition, the number of megakaryocytes *November/December is a combined issue.
be without it.
can be decreased, normal, or increased in cases of im-
mune-mediated thrombocytopenia.3,5,6,25


Small Animal The Compendium February 1996

topenia (if the drug were not incorporated into the

test), and corticosteroid therapy.1,3,7 Corticosteroid ther- About the Authors
When this article was submitted, Dr. Honeckman was af-
apy has been reported to revert a patient’s platelet factor
filiated with the Department of Veterinary Clinical Sci-
3 test results from positive to negative in as little as 5
ences of the School of Veterinary Medicine, Purdue Uni-
days.3 However, positive platelet factor 3 results have
versity, West Lafayette, Indiana. He is currently affiliated
also been reported after 21 days of treatment with pred-
with Norwood Park Animal Hospital, Norridge, Illinois.
nisone and cyclophosphamide.3
Dr. Knapp is affiliated with the Department of Veterinary
Direct immunofluorescence (DIF) has been used to
Clinical Sciences, School of Veterinary Medicine, Pur-
detect antimegakaryocyte antibodies. A bone marrow
due University, and is a Diplomate of the American Col-
aspirate is smeared on a slide, fixed in 95% ethyl alco-
lege of Veterinary Internal Medicine (Oncology). Dr.
hol for 10 minutes, washed with distilled water for 5
Reagan is affiliated with the Department of Veterinary
minutes, and then allowed to air dry. Fixed smears can
Pathobiology, School of Veterinary Medicine, Purdue
be stored up to 1 month at 4˚C. The slide is then ex-
University, and is a Diplomate of the American College
posed to fluorescein-labeled rabbit anticanine im-
of Veterinary Pathologists.
munoglobulin. Fluorescence of megakaryocytes indi-
cates a positive reaction.26
Direct immunofluorescence is reportedly more sensi- REFERENCES
tive than the platelet factor 3 test.3,6 False-negative find- 1. Thompson JP: Immunologic diseases, in Ettinger SJ (ed):
Textbook of Veterinary Internal Medicine, ed 3. Philadelphia,
ings may result from corticosteroid therapy or from an WB Saunders Co, 1989, pp 2297–2328.
inadequate number of megakaryocytes to evaluate.1,3 2. Switzer WJ, Jain NC: Autoimmune hemolytic anemia in
Therefore, a negative test does not rule out immune- dogs and cats. Vet Clin North Am Small Anim Pract
mediated thrombocytopenia. The results of direct im- 11:405–420, 1981.
munofluorescence have been reported to revert to nega- 3. Jain NC, Switzer JW: Autoimmune thrombocytopenia in
tive as early as 9 days25 (but have reportedly remained dogs and cats. Vet Clin North Am Small Anim Pract 11:421–
434, 1981.
positive after 1 to 2 weeks) of corticosteroid therapy.3 4. Jones DR, Gruffydd-Jones TJ, Stokes CR, Bourne FJ: Use
Direct immunofluorescence is considered specific for of a direct enzyme-linked antiglobulin test for laboratory di-
immune-mediated thrombocytopenia, and no causes of agnosis of immune-mediated hemolytic anemia in dogs. Am
false-positive results have been reported. J Vet Res 53:457–465, 1992.
In addition, an enzyme-linked immunosorbent assay 5. Halliwell RE, Gorman NT: Autoimmune blood diseases, in
(ELISA),27 a radioimmunoassay,28 and immunofluores- Pedersen D (ed): Veterinary Clinical Immunology. Philadel-
phia, WB Saunders Co, 1989, pp 308–336.
cent assays 29,30,31 have been developed to detect an- 6. Helfand SC, Couto CG, Madewell BR: Immune-mediated
tiplatelet antibodies. The ELISA was reportedly more thrombocytopenia associated with solid tumors in dogs.
sensitive than the platelet factor 3 test (88% versus JAAHA 21:787–794, 1985.
53%) in identifying immune-mediated thrombocy- 7. Thomason KJ, Feldman BF: Immune-mediated thrombocy-
topenia. A recent report described an indirect platelet topenia: Diagnosis and treatment. Compend Contin Educ
immunofluorescent assay, which was found to be more Pract Vet 7(7):569–576, 1985.
8. Cotter SM: Autoimmune hemolytic anemia in dogs. Com-
sensitive than the megakaryocyte immunofluorescent pend Contin Educ Pract Vet 14(1):53–59, 1992.
assay in the detection of immune-mediated thrombocy- 9. Tvedten H: Erythrocyte disorders, in Willard MD, Tvedten
topenia (70% versus 41%).31 These tests will likely gain H, Turnwald GH (eds): Small Animal Clinical Diagnosis by
more widespread use. Laboratory Methods. Philadelphia, WB Saunders Co, 1989,
pp 36–56.
CONCLUSION 10. Duncan JR, Prasse KW: Erythrocytes, in Veterinary Labora-
Laboratory tests (e.g., the direct antiglobulin tory Medicine Clinical Pathology. Ames, Iowa State University
Press, 1986, pp 3–30.
[Coomb’s], platelet factor 3, and direct immunofluores- 11. Cotter SM: Anemia, in Ettinger SJ (ed): Textbook of Veteri-
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mediated hematologic disease. These test results should 1989, pp 91–94.
be taken into consideration, along with results of the 12. Klag AR, Giger U, Shofer FS: Idiopathic immune-mediated
entire diagnostic workup. These tests cannot distinguish hemolytic anemia in dogs: 42 cases (1986–1990). JAVMA
primary (autoimmune) from secondary immune-medi- 202:783–788, 1993.
13. Jones DRE, Gruffydd-Jones TJ, Stokes CR, Bourne FJ: In-
ated disease. Therefore, a positive result is not an end vestigation into factors influencing performance of the ca-
point in the diagnosis. The clinician must strive to rule nine antiglobulin test. Res Vet Sci 48:53–58, 1990.
out underlying causes of immune-mediated hematolog- 14. Barker RN, Gruffydd-Jones TJ, Stokes CR, Elson CJ: Au-
ic disease before beginning therapy. toimmune hemolysis in the dog: Relationship between ane-


The Compendium February 1996 Small Animal

mia and the levels of red blood cell count, immunoglobulins, 23. Duncan JR, Prasse KW: Hemostasis, in Duncan JR, Prasse
and complement measured by the enzyme-linked antiglobu- KW (eds): Veterinary Laboratory Medicine Clinical Pathology.
lin test. Vet Immunol Immunopathol 34:1–20, 1992. Ames, Iowa State University Press, 1986, pp 73–86.
15. Jones DR, Darke PG: Use of papain for the detection of in- 24. Northern JR, Tvedten HW: Diagnosis of microthrombocy-
complete erythrocyte autoantibodies in autoimmune tosis and immune-mediated thrombocytopenia in dogs with
hemolytic anemia of the dog and cat. J Small Anim Pract 16: thrombocytopenia: 68 cases (1987–1989). JAVMA 200:
273–279, 1975. 368–372, 1992.
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