Vol.18, No.
2 February 1996
Continuing Education Article
FOCAL POINT
★ Secondary causes of immune-
mediated disease must be ruled
out because they affect treatment
decisions and prognosis.
KEY FACTS
■ Immunologic tests cannot
differentiate primary
(autoimmune) from secondary
immune-mediated hematologic
disease.
■ Samples for immunologic
testing should be collected
before immunosuppressive
therapy is begun.
■ The direct immunoglobulin
(Coomb’s) test cannot detect
which antigen the antibodies are
directed against, nor does a
negative Coomb’s test rule out
immune-mediated hemolytic
anemia.
■ The direct immunofluorescence
test is more sensitive than the
platelet factor 3 test, but a
negative result does not rule
out immune-mediated
thrombocytopenia.
Diagnosis of Canine
Immune-Mediated
Hematologic Disease
Purdue University
Adam L. Honeckman, DVM
Deborah W. Knapp, DVM, MS
William J. Reagan, DVM, PhD
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
decreased production of platelets is mediated by antibodies and/or comple-
ment.1 Immune-mediated anemia or thrombocytopenia can occur alone or
concurrently or in conjunction with systemic lupus erythematosus (SLE).2,3
This article describes the pathophysiology, clinical signs, and differential diag-
nosis of canine immune-mediated hematologic disease and presents a classifica-
tion scheme and diagnostic plan.
PATHOPHYSIOLOGY
The immune-mediated disease process against erythrocytes or platelets may
be primary (autoimmune) or secondary. The terms autoimmune and immune-
mediated are not synonymous. In an autoimmune response, the immune sys-
tem recognizes and attacks self antigen. A secondary immune response is di-
rected at a foreign antigen but leads to inadvertent damage to normal host
tissue and cells. The frequency of primary and secondary immune-mediated
hemolytic anemia was similar in a recent study (42.9% and 57.1%, respective-
ly).4 Causes of secondary immune-mediated anemia or thrombocytopenia in-
clude neoplasia, infectious disease (parasitic, viral, bacterial, rickettsial, or fun-
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-
tient.9–11

SPHEROCYTES ■ WARM AGGLUTININS ■ COLD AGGLUTININS

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
Urinalysis

no yes
Reticulocyte count > 60,000 cells/µl?
or
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
Hemolysis
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

Neoplasia

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
and neutrophil chemotaxis.5
Differential Diagnosis Total protein should be eval-
uated carefully. In cases of
of Anemia in Dogs
anemia secondary to de-
creased production or in-
Decreased creased destruction of eryth-
erythrocyte production rocytes, total protein is usually
(nonregenerative anemia) normal. When anemia is sec-
■ Rickettsial disease ondary to blood loss, the
■ Lymphoma or other packed cell volume and total
neoplasia protein may be decreased be-
■ Chemotherapy, cause of loss of erythrocytes
and plasma proteins, with a
estrogens, or other
compensatory shift of fluid
drugs from the interstitial space to
■ Immune-mediated the intravascular compart-
disease ment.10
■ Chronic renal disease An absolute reticulocyte
■ Chronic inflammatory count or corrected reticulo-
cyte percentage should be
disease
used to determine whether
the anemia is regenerative or
Blood loss nonregenerative. An absolute
(regenerative anemia) number of reticulocytes
■ Trauma greater than 60,000 per mi-
■ Disseminated croliter or a corrected reticu-
intravascular locyte percentage (reticulo-
coagulopathy cyte percentage × observed
packed cell volume ÷ normal
■ Gastrointestinal ulcers
packed cell volume) greater
■ Anticoagulant than one represents a regen-
rodenticide erative response.9,10
■ Neoplasia Typical cases of immune-
■ Parasitic (fleas, mediated hemolytic anemia
hookworms) are regenerative because ma-
ture erythrocytes are being
Hemolysis hemolyzed but erythrocyte
(regenerative anemia) precursors are unaffected.5,8,12
Although immune-mediated
■ Immune-mediated
hemolytic anemia is usually
disease regenerative, immune-medi-
■ Disseminated ated anemia may be nonre-
intravascular generative if antibodies and/
coagulopathy or complement are directed
■ Microangiopathy (e.g., against erythrocyte precur-
sors in the bone marrow or if
hemangiosarcoma)
the onset is acute.1,5,8,12 There-
■ Heinz body anemia fore, lack of reticulocytosis
■ Pyruvate kinase does not rule out immune-
deficiency mediated hemolytic anemia.
■ Babesiosis A blood smear should be
examined microscopically to
evaluate erythrocyte mor-
RETICULOCYTE COUNT ■ REGENERATIVE ANEMIA ■ ROULEAUX
The Compendium February 1996
phology and to estimate platelet numbers. Spherocyto-
sis or agglutination strongly suggests immune-mediated
hemolytic anemia. Agglutination appears as a grapelike
clustering of erythrocytes in the blood smear. It must
be distinguished from rouleaux (erythrocyte clusters re-
sembling stacks of coins), which can occur in cases of
inflammatory disease.
This distinction can sometimes be made by examin-
ing a wet-mount preparation of the blood sample.
One small drop of blood should be mixed with one
large drop of isotonic saline, put on a clean micro-
scope slide, covered with a coverslip, and examined
under a microscope. Rouleaux, unlike agglutination,
should be dispersed by the addition of saline.1,9 This
test, however, is not infallible; false-negative results
may result from weak agglutinins. Other morphologic
features that may be present in the blood smear in-
clude polychromasia, anisocytosis, and metarubricyto-
sis.1,5,8,9
Bone marrow aspiration or biopsy is indicated in cas-
es of nonregenerative anemia (or thrombocytopenia) to
evaluate the cause. If nonregenerative anemia is im-
mune mediated, erythroid hyperplasia is often present.
A serum biochemistry profile and urinalysis should
be performed if immune-mediated hemolytic anemia is
possible. Dogs with immune-mediated hemolytic ane-
mia often have hyperbilirubinemia or bilirubinuria.12
The serum biochemistry profile and urinalysis are also
useful in ruling out other systemic diseases.
Immunologic Tests
The direct antiglobulin test (also known as the direct
Coomb’s test) is a useful additional test to support the
diagnosis of immune-mediated hemolytic anemia. It
detects antibodies or complement on erythrocytes. The
Coomb’s reagent, which contains species-specific anti-
bodies against various classes of antibodies and comple-
ment, is added to washed erythrocytes from the patient.
Agglutination occurs if the ratio between the antibodies
or complement on the erythrocytes and the antiglobu-
lins in the reagent is proper.5,9
The direct antiglobulin (Coomb’s) test is positive in
60% to 70% of cases of immune-mediated hemolytic
anemia.2,8,9 As the following list indicates, however,
there are many possible causes of false-negative results
and a negative Coomb’s test does not rule out immune-
mediated hemolytic anemia5,8,9,13:
■ Insufficient quantity of antibody or complement on
erythrocytes
■ Improper antiglobulin-to-antibody ratio
■ 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
neoplasia
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
deficiency
■ 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).

ANTIGLOBULIN TESTS ■ IgG ■ IgM

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

Megakaryocytes

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
thrombocytopenia

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

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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
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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
Compendium.
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.

H
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.

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

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
VOLUME 2 • NUMBER 10
OCTOBER 2000
INSIDE THIS ISSUE:

Peer-Reviewed Articles on
HEPATIC DISORDERS
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
1
■ 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

PLATELET COUNT ■ PLATELET SIZE ■ MARROW BIOPSY ■ PLATELET FACTOR 3

Small Animal
topenia (if the drug were not incorporated into the
test), and corticosteroid therapy.1,3,7 Corticosteroid ther-
apy has been reported to revert a patient’s platelet factor
3 test results from positive to negative in as little as 5
days.3 However, positive platelet factor 3 results have
also been reported after 21 days of treatment with pred-
nisone and cyclophosphamide.3
Direct immunofluorescence (DIF) has been used to
detect antimegakaryocyte antibodies. A bone marrow
aspirate is smeared on a slide, fixed in 95% ethyl alco-
hol for 10 minutes, washed with distilled water for 5
minutes, and then allowed to air dry. Fixed smears can
be stored up to 1 month at 4˚C. The slide is then ex-
posed to fluorescein-labeled rabbit anticanine im-
munoglobulin. Fluorescence of megakaryocytes indi-
cates a positive reaction.26
Direct immunofluorescence is reportedly more sensi-
tive than the platelet factor 3 test.3,6 False-negative find-
ings may result from corticosteroid therapy or from an
inadequate number of megakaryocytes to evaluate.1,3
Therefore, a negative test does not rule out immune-
mediated thrombocytopenia. The results of direct im-
munofluorescence have been reported to revert to nega-
tive as early as 9 days25 (but have reportedly remained
positive after 1 to 2 weeks) of corticosteroid therapy.3
Direct immunofluorescence is considered specific for
immune-mediated thrombocytopenia, and no causes of
false-positive results have been reported.
In addition, an enzyme-linked immunosorbent assay
(ELISA),27 a radioimmunoassay,28 and immunofluores-
cent assays 29,30,31 have been developed to detect an-
tiplatelet antibodies. The ELISA was reportedly more
sensitive than the platelet factor 3 test (88% versus
53%) in identifying immune-mediated thrombocy-
topenia. A recent report described an indirect platelet
immunofluorescent assay, which was found to be more
sensitive than the megakaryocyte immunofluorescent
assay in the detection of immune-mediated thrombocy-
topenia (70% versus 41%).31 These tests will likely gain
more widespread use.
CONCLUSION
Laboratory tests (e.g., the direct antiglobulin
[Coomb’s], platelet factor 3, and direct immunofluores-
cence tests) can aid in the diagnosis of canine immune-
mediated hematologic disease. These test results should
be taken into consideration, along with results of the
entire diagnostic workup. These tests cannot distinguish
primary (autoimmune) from secondary immune-medi-
ated disease. Therefore, a positive result is not an end
point in the diagnosis. The clinician must strive to rule
out underlying causes of immune-mediated hematolog-
ic disease before beginning therapy.
DIF ■ ELISA ■ ANTIPLATELET ANTIBODY TESTS
The Compendium February 1996
About the Authors
When this article was submitted, Dr. Honeckman was af-
filiated with the Department of Veterinary Clinical Sci-
ences of the School of Veterinary Medicine, Purdue Uni-
versity, West Lafayette, Indiana. He is currently affiliated
with Norwood Park Animal Hospital, Norridge, Illinois.
Dr. Knapp is affiliated with the Department of Veterinary
Clinical Sciences, School of Veterinary Medicine, Pur-
due University, and is a Diplomate of the American Col-
lege of Veterinary Internal Medicine (Oncology). Dr.
Reagan is affiliated with the Department of Veterinary
Pathobiology, School of Veterinary Medicine, Purdue
University, and is a Diplomate of the American College
of Veterinary Pathologists.
REFERENCES
1. Thompson JP: Immunologic diseases, in Ettinger SJ (ed):
Textbook of Veterinary Internal Medicine, ed 3. Philadelphia,
WB Saunders Co, 1989, pp 2297–2328.
2. Switzer WJ, Jain NC: Autoimmune hemolytic anemia in
dogs and cats. Vet Clin North Am Small Anim Pract
11:405–420, 1981.
3. Jain NC, Switzer JW: Autoimmune thrombocytopenia in
dogs and cats. Vet Clin North Am Small Anim Pract 11:421–
434, 1981.
4. Jones DR, Gruffydd-Jones TJ, Stokes CR, Bourne FJ: Use
of a direct enzyme-linked antiglobulin test for laboratory di-
agnosis of immune-mediated hemolytic anemia in dogs. Am
J Vet Res 53:457–465, 1992.
5. Halliwell RE, Gorman NT: Autoimmune blood diseases, in
Pedersen D (ed): Veterinary Clinical Immunology. Philadel-
phia, WB Saunders Co, 1989, pp 308–336.
6. Helfand SC, Couto CG, Madewell BR: Immune-mediated
thrombocytopenia associated with solid tumors in dogs.
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