Objective—To develop a clinically applicable assay for detection of serum anti-neutrophil antibodies in dogs.
Sample Population—Serum samples of 20 healthy dogs and 20 sick dogs.
Procedures—An indirect immunofluorescence assay was developed in which canine serum was incubated with paraformaldehyde-fixed neutrophils and subsequently incubated with fluorescein-conjugated rabbit anti-dog IgG. Neutrophil median fluorescence intensity and the percentage of neutrophils with an increase in fluorescence intensity were determined by use of a flow cytometer.
Results—Neutrophils incubated with serum from healthy and sick dogs had a normally distributed curve when displayed as a histogram. Alloantibodies or immune complexes that significantly affected test results were not detected. Hyperglobulinemia did not appear to affect test results. The neutrophil donor did not significantly affect test results. With 1 exception, results for the sick dogs did not differ appreciably from those for healthy dogs. Serum from a dog with steroid-responsive neutropenia had a greater neutrophil fluorescence value and percentage of neutrophils with an increase in fluorescence intensity, compared with either healthy or sick dogs.
Conclusions and Clinical Relevance—The indirect immunofluorescence test gave consistent results for healthy and sick dogs and detected anti-neutrophil antibodies in a dog with steroid-responsive neutropenia. Definitive evaluation of the test will be dependent on evaluation of persistently neutropenic dogs and correlation of test results with a response to immunosuppressive therapy.
Objective—To evaluate monoclonal antibodies that
may be useful for immunophenotyping myeloid cells
in bone marrow of dogs.
Sample Population—Bone marrow specimens
obtained from 5 dogs.
Design—Specimens were labeled with monoclonal
antibodies that detected CD18, major histocompatability
antigen class-II (MHC class-II), CD14, and Thy-1.
Cells labeled with each of the antibodies were isolated
by use of a fluorescence-activated cell sorter.
Differential cell counts of sorted cells were used to
determine cells that were labeled by each of the various
Results—Myeloid cells labeled with anti-CD18 antibody
included granulocytes, lymphocytes, and monocytes-
macrophages. Immature and mature granulocytes
were labeled. Lymphocytes, monocytesmacrophages,
and eosinophils were labeled with anti-Thy-1 antibody. Cells labeled with anti-MHC-class II
antibody included approximately 9% of bone marrow
cells, which consisted almost exclusively of lymphocytes
and monocytes-macrophages. Approximately
4% of bone marrow cells were labeled with anti-CD14 antibody, with > 90% of sorted cells being
Conclusions and Clinical Relevance—Four monoclonal
antibodies for use in detecting subpopulations
of canine bone marrow cells were evaluated. These
antibodies should be useful in differentiating the origin
of leukemic cells in dogs. (Am J Vet Res 2001;62:1229–1233)
Objective—To evaluate the potential usefulness of 2
flow cytometric methods for determination of differential
cell counts in feline bone marrow.
Sample Population—10 bone marrow specimens
from client-owned cats.
Procedure—Bone marrow specimens were stained
with 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and
evaluated by use of flow cytometry. Differential
counts were also determined by analysis of scatterplots
of forward-angle versus side-angle light scatter
of unstained specimens, obtained by use of flow
cytometry (scatterplot method). Results of both flow
cytometric methods were compared with differential
cell counts determined by manually counting 1,000
cells on slides of Wright-stained smears.
Results—Staining with DiOC6 resulted in identification
of mature and immature erythroid and myeloid
cells and lymphocytes. Use of the scatterplot method
resulted in identification of mature and immature erythroid
and myeloid cells and metamyelocytes.
However, to identify lymphocytes by use of the scatterplot
method, bone marrow specimens were first
labeled with an anti-major histocompatability class-II
antibody. Comparison of results of the scatterplot
method with manual counts yielded higher correlation
coefficients and more similar mean values than did
comparison of results of the DiOC6 method.
Conclusion and Clinical Relevance—The scatterplot
method provided more accurate and precise
results than the DiOC6 method for determination of
bone marrow differential cell counts in cats by use of
flow cytometry. When combined with fluorescent
labeling of lymphocytes, the scatterplot method has
potential to provide rapid semiquantitative assessment
of bone marrow differential cell counts in cats.
(Am J Vet Res 2001;62:474–478)
Objectives—To evaluate use of monoclonal antibodies
to increase accuracy of flow cytometric differential
cell counting of canine bone marrow cells.
Sample Population—Bone marrow specimens from
Procedures—Specimens were labeled with monoclonal
antibodies that detected CD18, major histocompatability
antigen class-II (MHC class-II), CD14,
and Thy-1. Location of fluorescent and nonfluorescent
cells within gates of a template developed for canine
bone marrow differential cell counting was determined,
the template was revised, and 10 specimens
were analyzed by use of the old and revised templates
and by labeling cells with anti-MHC class-II and
Results—Data confirmed the presumptive location of
marrow subpopulations in scatter plots, permitted
detection of lymphocytes and monocytemacrophages,
and was used to revise the analysis
template used for differential cell counting. When differential
cells counts determined by the original and
revised templates were compared with results of
manual differential cell counts, the revised template
had higher correlation coefficients and more similar
mean values. Labeling cells with anti-MHC class-II
and anti-CD14 permitted identification of lymphoid
and monocyte-macrophages cells in bone marrow
Conclusions and Clinical Relevance—Use of the
revised flow cytometric analysis template combined
with anti-CD14 and anti-MHC class-II antibody labeling
provides reliable differential cell counts for clinical
bone marrow specimens in dogs. These techniques
have potential applications to clinical bone marrow
examination and preclinical toxicity studies. (Am J Vet
Objective—To determine the frequency, potential causes, and clinical and clinicopathologic features of hemophagocytic syndrome in dogs.
Animals—24 client-owned dogs.
Procedures—Records for dogs in which diagnostic bone marrow specimens (including an aspiration smear and core biopsy material) were obtained from 1996 to 2005 were reviewed. Inclusion criteria were presence of bicytopenia or pancytopenia in the blood and > 2% hemophagocytic macrophages in the bone marrow aspirate.
Results—Of 617 bone marrow specimens evaluated, evidence of hemophagocytic syndrome was detected in 24 (3.9%). The Tibetan Terrier breed was overrepresented among dogs with hemophagocytic syndrome. Clinical signs associated with hemophagocytic syndrome included fever, icterus, splenomegaly, hepatomegaly, and diarrhea. Hemophagocytic syndrome was associated with immune-mediated, infectious, and neoplastic-myelodysplastic conditions and also occurred as an idiopathic condition. Overall, dogs with infection-associated hemophagocytic syndrome had better 1-month survival rates than dogs with immune-associated and idiopathic hemophagocytic syndrome.
Conclusions and Clinical Relevance—Results indicated that hemophagocytic syndrome may occur more frequently in dogs than has previously been suspected on the basis of the paucity of reported cases. Although most dogs had definable underlying disease conditions, idiopathic hemophagocytic syndrome was also identified. Hemophagocytic syndrome of any cause is potentially life-threatening; however, the prognosis should be adjusted on the basis of the associated disease process and potential for successful treatment.
Objective—To identify the incidence, potential causes,
and clinical and clinicopathologic features of bone
marrow necrosis in dogs.
Animals—34 client-owned dogs.
Procedures—Reports of cytologic examinations of
bone marrow specimens performed between 1996
and 2004 were reviewed. All reports that indicated
the presence of necrosis, stromal disruption, phagocytic
macrophages, individual cell necrosis, or
myelofibrosis were evaluated further.
Results—Of 609 reports of bone marrow evaluations
performed during the study period, 34 (5.6%) had evidence
of bone marrow necrosis. Nine dogs had no
evidence of associated diseases or drug or toxin
exposure, and 25 dogs had associated disease conditions
or drug exposures. All 9 dogs with idiopathic
bone marrow necrosis were anemic (mean Hct,
14%), but only 3 had neutropenia, and 3 had thrombocytopenia.
All 9 had myelofibrosis. Of the 25 dogs
with associated disease conditions or drug exposures,
only 14 (56%) had anemia (mean Hct, 33%). In
addition, 14 (56%) had neutropenia and 18 (72%) had
thrombocytopenia. Only 10 (40%) had myelofibrosis.
Conclusions and Clinical Relevance—Results suggest
that bone marrow necrosis may be common in dogs
with hematologic disorders. In most dogs, bone marrow
necrosis was associated with an underlying disease condition
or drug exposure, but idiopathic bone marrow
necrosis was also identified. Disease conditions that
should increase suspicion of possible bone marrow
necrosis include sepsis, lymphosarcoma, and systemic
lupus erythematosus; drug exposures that should
increase suspicion of possible bone marrow necrosis
include chemotherapeutic agents, phenobarbital, carprofen,
metronidazole, and mitotane. (J Am Vet Med Assoc 2005;227:263–267)
Objective—To further classify dysmyelopoiesis as diagnosed by use of a general classification scheme and to determine clinical features and laboratory test results that could be used to differentiate between the various forms of dysmyelopoiesis in cats.
Design—Retrospective case series.
Sample Population—Bone marrow slides from 34 cats.
Procedures—Medical records of cats in which dysmyelopoiesis was diagnosed on the basis of blood and bone marrow analyses from 1996 to 2005 were reviewed. Criteria for inclusion in the study were findings of > 10% dysplastic cells in 1 or more hematologic cell lines in the bone marrow and concurrent cytopenias in the blood. Cats that met these criteria were classified into subcategories of myelodysplastic syndromes or secondary dysmyelopoiesis on the basis of reevaluation of slides.
Results—Of 189 bone marrow slides reviewed, 34 (14.9%) had > 10% dysplastic cells in 1 or more cell lines. Cats were subcategorized as having myelodys-plastic syndrome with excessive numbers of blast cells (n = 13), myelodysplastic syndrome with refractory cytopenias (8), a variant form of myelodysplastic syndrome (1), and secondary dysmyelopoiesis (12). Findings of dysmyelopoiesis and autoagglutination in cats with myelodysplastic syndrome and in those with immune-mediated anemia complicated differentiating between the 2 conditions.
Conclusions and Clinical Relevance—Differentiating cats with myelodysplastic syndromes from cats with immune-mediated hemolytic anemia was difficult because severe anemia and autoagglutination may be concurrent findings in both conditions. Differentiating between myelodysplastic syndrome with excessive numbers of blast cells and myelodysplastic syndrome with refractory cytopenias was useful in predicting clinical outcome.
Objectives—To examine clinical features, laboratory
test results, treatment, and outcome of dogs with
pure red cell aplasia (PRCA).
Animals—13 dogs with severe nonregenerative anemia
and bone marrow erythroid aplasia.
Procedures—Medical records of dogs determined to
have PRCA on the basis of results of blood and bone
marrow analysis between 1996 and 2000 were
reviewed. Criteria for inclusion in the study were
severe nonregenerative anemia (Hct < 20%; reticulocyte
count < 1.0%), selective erythroid aplasia in
bone marrow, and lack of underlying diseases that
may have caused the anemia.
Results—Median age of dogs was 6.5 years.
Females were significantly overrepresented. Median
Hct was 10%, and median reticulocyte count was
0.1%. Direct Coombs' test results were negative for
all dogs tested, and spherocytosis was evident in 2
dogs. All dogs were treated with prednisolone, and 2
dogs were treated with prednisolone and cyclophosphamide.
Responses to treatment were complete,
partial, and poor in 10, 1, and 2 dogs, respectively.
Median time required to achieve an increase of 5% or
more in Hct was 38 days, and median time to complete
remission was 118 days. Of 10 dogs for which
follow-up information was available, only 1 required
long-term immunosuppressive treatment.
Conclusions and Clinical Relevance—Dogs with
PRCA appear to respond readily to treatment with
immunosuppressive drugs; however, hematologic
responses may not be observed for weeks to months
after initiation of treatment. (J Am Vet Med Assoc
Objective—To evaluate the activation status of neutrophils
in blood samples obtained from horses with
naturally occurring colic associated with strangulating
obstruction, nonstrangulating obstruction, or inflammatory
Animals—30 horses with naturally occurring colic
and 30 healthy control horses.
Procedure—Activation status of neutrophils was
determined by assessing the number of neutrophils
that could pass through filters with 5-µm pores, cellsurface
CD11-CD18 expression, and alterations in size
and granularity of neutrophils.
Results—Horses with impaction or gas colic did not
have evidence of activated neutrophils. Horses with
inflammatory bowel disease consistently had evidence
of activated neutrophils, including decreased
leukocyte deformability, increased CD11-CD18 expression,
increased neutrophil size, and decreased neutrophil
granularity. Horses with strangulating colic had
variable results. Of horses with strangulating colic, 7
of 14 had marked changes in filtration pressures, 5 of
14 had increased CD11-CD18 expression, 6 of 14 had
changes in neutrophil size, and 5 of 14 had changes in
neutrophil granularity. Among horses with strangulating
colic, changes in deformability, size, and granularity
of neutrophils correlated with an adverse outcome.
Conclusions and Clinical Relevance—Activated
neutrophils were detected in all horses with inflammatory
bowel disease and a few horses with strangulating
colic. Correlation of activated neutrophils
with horses that had strangulating colic that died or
were euthanatized indicates that activated neutrophils
are a negative prognostic indicator. Additional
studies are needed to determine whether activated
neutrophils contribute directly to the adverse outcome
in horses with strangulating colic. (Am J Vet Res 2003;64:1364–1368)
Objective—To evaluate lipopolysaccharide (LPS)-
induced activation of equine neutrophils in blood.
Sample Population—Blood samples from 5 healthy
Procedure—Neutrophil integrin (CD11/CD18)
expression, size variation, degranulation, and
deformability were measured with and without incubation
with LPS. Time and concentration studies were
done. The mechanism of endotoxin-induced neutrophil
activation was investigated by inactivating
complement or preincubating neutrophils with
inhibitors of tumor necrosis factor-α (TNF-α) synthesis,
prostaglandin-leukotriene synthesis, or plateletactivating
Results—Incubation of equine neutrophils with LPS
increased cell surface expression of CD11/CD18,
decreased neutrophil deformability, increased and
decreased neutrophil size, and induced neutrophil
degranulation. The LPS-induced neutrophil activation
was attenuated by addition of inhibitors of TNF-α and
Conclusion and Clinical Relevance—Equine neutrophils
are readily activated in vitro by LPS, resulting
in increased expression of integrin adhesion molecules,
decreased deformability, variation in neutrophil
size, and degranulation. The tests used to detect activated
neutrophils in this study may be useful in
detecting in vivo neutrophil activation in horses with
sepsis and endotoxemia. (Am J Vet Res