Type of smear may influence thrombopoietic cell counts in the bone marrow of clinically healthy dogs

BACKGROUND: The expected number of thrombopoietic cells in normal canine bone marrow is poorly defined and there is no consensus on the most appropriate way to prepare cytologic smears to evaluate these cells nor on the optimum method for their quantification. OBJECTIVE: The purpose of this study was to determine total and differential counts of thrombopoietic cells in the bone marrow of clinically healthy Beagle dogs by comparing 4 different smear types and bone marrow core biopsies. METHODS: Twenty-two clinically healthy, male Beagle dogs, 10 to 12 months old, were used in the study. Following bone marrow aspiration and core biopsy from the iliac crest, Giemsa-stained smears were prepared by 4 techniques: drop-squash, particle-squash, buffy coat, and fat-layer smears. Thrombopoietic cells were counted in up to 100 low-power fields (LPF, X10 objective) in the aspiration smears and in all possible high-power fields (HPF, X40 objective) in H&E-stained biopsy sections. RESULTS: Mean total thrombopoietic cell counts were 2.76 cells/LPF (drop-squash), 1.55 cells/LPF (particle-squash), 8.05 cells/LPF (buffy coat), and 3.08 cells/LPF (fat-layer). Core biopsies yielded 5.31 cells/HPF but frequently failed to provide interpretable specimens. There was a significant difference in cell counts among the 4 smear types (P <.001). Based on evaluation of buffy coat smears, thrombopoietic cells included 1.23% megakaryoblasts, 8.77% promegakaryocytes, and 90% megakaryocytes, with a mean maturation index of 0.11. CONCLUSION: Thrombopoietic cell counts in canine bone marrow are influenced by the smear technique. Buffy coat and fat-layer smears may be useful to obtain cellular smears in hemodiluted or small aspirate samples.     

Frequency and severity of mastocytemia in dogs with and without mast cell tumors: 120 cases (1995-1997).

OBJECTIVE: To elucidate frequency of detection on blood smears and severity on quantitative buffy coat evaluation of mastocytemia between dogs without mast cell tumors (MCT) and dogs that had MCT, and to expand the list of diseases associated with mastocytemia in dogs without MCT. DESIGN: Retrospective study. ANIMALS: 94 dogs without MCT and 26 dogs with MCT. PROCEDURE: Medical records of all dogs with mast cells detected on blood or buffy coat smears during a 2-year period were reviewed. Dogs with mastocytemia were grouped by disease into dogs with MCT and dogs without MCT. Twenty-five of the dogs without MCT that had mast cells detected on blood smears also had evaluations of buffy coat smears. Quantitative buffy coat results of the 25 dogs without MCT were compared with those of the 26 dogs with MCT. RESULTS: 95.5% of blood smears with mast cells detected during CBC determination were from dogs without MCT. For these dogs, diagnoses included inflammatory disease (28.2%), regenerative anemia (27%), neoplasia other than MCT (25.9%), and trauma (11.8%). Dogs with MCT had a mean of 71.4 mast cells/buffy coat smear, whereas dogs without MCT had a mean of 276.2 mast cells/buffy coat smear. The 2 highest counts of mast cells/buffy coat smear were for dogs without MCT. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of results of quantitative buffy coat evaluations, severity of mastocytemia in dogs without MCT often exceeds that detected during tumor staging in dogs with MCT. Random detection of mast cells in blood smears during CBC determination in dogs is usually not secondary to MCT.     

Systemic Mastocytosis in 16 Dogs

The clinical and pathologic features of systemic mastocytosis in 16 dogs are reported. There was no apparent breed or sex predilection, and the median age at presentation was 9.5 years. In 14 of 16 cases there was a primary cutaneous mast cell tumor (MCT). When cutaneous tumor location was compared with previous reports, there was no association between location and systemic dissemination. The most common presenting signs associated with the cutaneous tumor were regional dissemination, edema, ulceration, and abscessation. They were present in 12 dogs (69%). Signs of systemic illness, including anorexia, vomiting, and diarrhea, were seen in eight dogs (50%). Other than the cutaneous tumors, the most consistent physical and radiographic abnormalities included lymphadenopathy, splenomegaly, and hepatomegaly. Eosinophilia and basophilia were seen in two and five dogs, respectively. Six dogs had increased numbers of mast cells in peripheral blood or buffy coat smears. Five of the nine dogs evaluated had increased numbers of mast cells in bone marrow aspirates. Bone marrow aspiration was superior to both peripheral blood and buffy coat smears in predicting mastocytosis. Coagulation abnormalities were seen in three of five dogs tested. Using a conventional histomorphologic grading system, 10 of 13 (77%) tumors were classified as Grade III or undifferentiated and were overrepresented when compared with previous reports of cutaneous MCTs. Eighty-eight percent of the dogs either died or were euthanatized because of their tumors. Organs commonly involved at necropsy included lymph nodes, spleen, liver, and bone marrow; four dogs had gastroduodenal ulcers.     

Telomerase activity in clinically normal dogs and dogs with malignant lymphoma

OBJECTIVES: To determine whether telomerase activity was present in lymph nodes, buffy coat, and serum samples from dogs with malignant lymphoma (ML) and in liver, lymph node, buffy coat, and serum samples from clinically normal dogs SAMPLE POPULATION: Tissue specimens and blood samples were obtained from 11 clinically normal adult dogs (age range, 1 to 4 years) and 14 client-owned dogs with ML. PROCEDURE: The telomere repeat amplification protocol assay was used to quantify telomerase activity in the tissues from clinically normal dogs and dogs with ML. RESULTS: Of 11 clinically normal dogs, 8 had lymph node samples, 5 had liver samples, and 1 had buffy coat samples with detectable telomerase activity. None of the serum samples from the clinically normal dogs had detectable telomerase activity. Of 14 dogs with ML, 9 had lymph node samples, 3 had buffy coat samples, and 1 had serum samples with measurable telomerase activity. CONCLUSIONS AND CLINICAL RELEVANCE: Telomerase activity was not specific to tumor cells and overlapped with that found in cells from clinically normal dogs. Telomerase activity in neoplastic lymph nodes was not substantially different from that found in lymph nodes from clinically normal dogs. The determination of telomerase activity cannot be used as a sole diagnostic test for cancer. Therapeutic modalities directed toward the telomerase enzyme may not be feasible in dogs, because somatic tissues from clinically normal dogs possess variable amounts of telomerase activity.     

Mixed Ehrlichia canis, Hepatozoon canis, and presumptive Anaplasma phagocytophilum infection in a dog

A 5-month-old, female, mongrel dog was admitted to the Clinic of Companion Animal Medicine, Aristotle University of Thessaloniki, Greece, with depression, anorexia, fever, peripheral lymphadenopathy, splenomegaly, oculonasal discharge, nonregenerative anemia, and mild thrombocytopenia. Cytology of Giemsa-stained buffy coat, bone marrow, and lymph node aspiration smears revealed numerous morulae in mononuclear leukocytes and in neutrophils, and Hepatozoon canis gamonts in neutrophils. The dog was seropositive to Ehrlichia canis (immunofluorescence assay [IFA]) and Hepatozoon canis (ELISA) but not to Anaplasma phagocytophilum (IFA). A nested polymerase chain reaction performed on bone marrow aspirates was positive for E canis. This method was not applied for the detection of A phagocytophilum. Treatment with doxycycline and imidocarb dipropionate resulted in both clinical and parasitologic cure. This is the first reported case of a mixed infection with E canis, H canis, and presumptive A phagocytophilum. The findings emphasize the value of cytology in offering a quick and inexpensive diagnosis in mixed tick-borne infections of dogs.     

The utility of staging in canine mast cell tumours

Current staging of canine mast cell tumours (MCTs) practiced by many veterinarians involves a minimum of lymph node (LN) assessment, abdominal ultrasound and thoracic radiography. Historically, some have advocated buffy coat and bone marrow evaluation. Two hundred and twenty dogs with MCT seen at a referral clinic were staged using LN palpation/cytology, thoracic radiography and abdominal ultrasound. The utility of each method was evaluated by considering prevalence of spread and future behaviour. At presentation, 30.9% of dogs had metastases to the local LN; 6.8% of all the dogs also had distant metastases. No dog had or developed distant metastasis in the absence of LN metastasis. No dog had convincing evidence of pulmonary metastasis. In this series, the local LN was sentinel to metastasis and in the absence of local LN metastasis, the utility of further staging was low. Thoracic radiography was not useful in the staging of canine MCT.     

Bone marrow mastocytosis in dogs with myelosuppressive monocytic ehrlichiosis (Ehrlichia canis): a retrospective study

BACKGROUND: Bone marrow mastocytosis has been reported rarely in naturally occurring canine monocytic ehrlichiosis (CME). OBJECTIVES: The aims of the present study were to estimate the prevalence and magnitude of bone marrow mastocytosis in a case series of dogs with natural CME and to assess the association, if any, between mastocytosis and the clinical severity of the disease. METHODS: Seventy-six dogs with confirmed CME (Ehrlichia canis) were included in the study. Affected dogs were allocated into group A (n = 51) without bone marrow hypoplasia and group B (n = 25) with bone marrow hypoplasia. Twenty clinically healthy Beagles not previously exposed to E canis served as controls (group C). The main inclusion criteria for group A were documentation of normocellular to hypercellular bone marrow and complete clinical cure following a 4-week treatment with doxycycline, while those for group B were bone marrow hypoplasia and lack of response to doxycycline. Bone marrow aspirate smears from all 96 dogs were Giemsa-stained and examined for the presence of mast cells, which were calculated as a percentage of 1,000 nucleated cells (NCs). The prevalence of mastocytosis was compared among the 3 groups by the Pearson's chi-square test. RESULTS: Bone marrow mastocytosis (>0.1% of NCs) was found in 5 (20%) dogs in group B (range, 0.5-2.5% of NCs; median, 1% of NCs). One dog in each of groups A and C had 0.1% mast cells in the marrow. The prevalence of bone marrow mastocytosis in dogs in group B was significantly higher (P = .004) than in groups A and C. CONCLUSION: Bone marrow mastocytosis can be seen in a substantial number of dogs with E canis-induced myelosuppression.     

Evaluation of PCR to detect Theileria parva in field-collected tick and bovine samples in Tanzania

The ability of PCR to detect infections of Theileria parva, the cause of East Coast Fever, in field-collected tick and bovine samples from Tanzania was evaluated. PCR-detected infection prevalence was high (15/20, 75%) in unfed adult Rhipicephalus appendiculatus ticks that fed as nymphs on an acutely-infected calf, but low (22/836, 2.6%) in unfed adult R. appendiculatus collected from field sites in Tanzania. Tick infection prevalence was comparable to that in previous studies that used salivary gland staining to detect T. parva infection in field-collected host-seeking ticks. Of 282 naturally-exposed zebu calves, seven had PCR-positive buffy coat samples prior to detection of Theileria spp. parasites in stained buffy coat cells or lymph node biopsies. Evidence of Theileria spp. infections was detected in stained smears of lymph node biopsies from 109 calves (38.6%) and buffy coat samples from 81 (28.7%), while buffy coat samples from 66 (23.4%) were PCR-positive for T. parva. Implications of these findings for the sensitivity and specificity of the PCR are discussed.     

Mammary gland carcinoma in a dog with peripheral blood and bone marrow involvement associated with disseminated intravascular coagulation

A 7-year-old female Leonberger dog was referred to the National Veterinary School of Lyon Teaching Hospital with a 2-day history of anorexia and bleeding. A mammary mass had been removed 7 months earlier, but histologic examination was not performed. On physical examination, the dog was depressed and had pale mucous membranes and numerous petechiae and hematomas. Significant laboratory findings were moderate thrombocytopenia, prolonged prothrombin, activated partial thromboplastin, and thrombin times, hypofibrinogenemia, and increased concentration of fibrin(ogen) degradation products. A peripheral blood smear, buffy coat preparation, and bone marrow aspirate contained low numbers of large atypical cells that had moderate nuclear:cytoplasmic ratios, oval nuclei with multiple prominent nuclei, and basophilic cytoplasm with villous projections. A small nodule was found in the left inguinal mammary gland, and a fine-needle aspirate contained cells similar to those in blood and bone marrow. In samples of blood, bone marrow, and the mammary mass, the neoplastic cells were immunoreactive for cytokeratin. The diagnosis was mammary carcinoma with secondary disseminated intravascular coagulation (DIC) and disseminated tumor cells in bone marrow and circulating tumor cells in blood; this diagnosis was not confirmed by histopathologic examination. Owing to clinical deterioration and the poor prognosis, the dog was euthanized and a necropsy was not performed. This is the first report of a canine mammary carcinoma with circulating tumor cells and secondary DIC.     

Nested PCR for diagnosis of canine leishmaniosis in peripheral blood, lymph node and bone marrow aspirates

A nested polymerase chain reaction (PCR) assay was developed using primers selected from the genomic DNA of Leishmania infantum and applied to the diagnosis of leishmaniosis in peripheral blood in dogs. Blood of 39 dogs of different breeds, all sampled in Catalonia (Spain), were tested for leishmaniosis by enzyme linked immunosorbent assay (ELISA), western blotting (WB) and peripheral blood mononuclear cell (PBMC) culture and nested PCR. Twenty negative controls (healthy dogs less than 1-year-old that had not been exposed to a sandfly season) were also studied. Nineteen of the 39 dogs studied were positive by ELISA and/or WB, and 18 of these had a positive PBMC nested PCR. PBMC nested PCR was negative in all the remaining animals that were negative by serological examination, including the 20 negative controls. Parasitological examination and nested PCR of bone marrow and lymph node aspirate from the 19 dogs positive by serological examination, were also positive. These results indicate that PBMC nested PCR is a sensitive and specific tool to diagnose leishmaniosis in dogs. The use of PBMC has the advantage over bone marrow and lymph node aspirates in that it is a less invasive sample.     

Effects of platelet clumping on platelet concentrations measured by use of impedance or buffy coat analysis in dogs.

OBJECTIVE: To determine whether platelet clumps are homogeneously distributed in blood samples, and whether platelet concentrations (PC) obtained by use of impedance and buffy coat analysis can be considered minimum values when platelet clumps are present. DESIGN: Prospective study. SAMPLE POPULATION: 50 blood samples obtained from 30 dogs. PROCEDURE: 10 blood samples containing platelet clumps were used and 10 smears were made from each sample; amount of platelet clumping was graded for all 100 smears. Blood from each of 20 healthy dogs was divided between 2 EDTA tubes before and after platelet clumping was induced by adenosine diphosphate (ADP). The PC for each ADP-treated and untreated sample were measured, using impedance and quantitative buffy coat analyzers. RESULTS: Platelet clumps were evident in all 100 blood smears, but the amount of clumping varied considerably within some samples. Using the impedance analyzer, the PC of ADP-treated samples were significantly lower and never higher than the PC of untreated samples. Using the buffy coat analyzer, some ADP-treated samples had increased PC; however, significant differences were not detected between treated and untreated samples. CONCLUSIONS AND CLINICAL RELEVANCE: Platelet clumping was not homogeneous within blood samples. When platelet clumps were identified by direct examination of blood smears, the PC detected by use of the impedance analyzer could be considered minimum values. In contrast, the PC detected by use of the buffy coat analyzer were sometimes increased. Useful information can be obtained by measuring PC in blood with platelet clumps; values obtained by use of impedance can be considered minimums, and values obtained by use of buffy coat analysis may be either minimum values or reasonable estimates of PC.     

Evaluation of cytology in the diagnosis of acute canine monocytic ehrlichiosis (Ehrlichia canis): a comparison between five methods

The purpose of this study was the comparison of the diagnostic sensitivity between buffy coat (BC), peripheral blood (PB), lymph node (LN), bone marrow (BM) and short-term culture (P-D) cytology that has been based on the detection of Ehrlichia canis morulae, in the acute phase of canine monocytic ehrlichiosis (CME). Their cellular localization, total numbers and microscopic differentials were also investigated. The highest sensitivities were achieved after evaluating 1000 oil immersion fields (OIFs) in BC (66%) and an equal number in LN (60.9%) smears, separately or together (74%). The morulae were more often detected into lymphocytes than monocytes. The highest total number of morulae (n=143) were found in P-D smears. Finally, to avoid false positive diagnoses, platelets, lymphocytic azurophilic granules, lymphoglandular bodies and phagocytosed nuclear material should not be confused with the morulae.     

Immunophenotypic comparison of blood and lymph node from dogs with lymphoma

Peripheral blood and lymph node tissue from 12 dogs with lymphoma was immunophenotyped. Additionally, the bone marrow was immunophenotyped in 6 dogs. The lymphomas were characterized as B-cell in 11 dogs and T-cell in 1 dog. Immunophenotypic patterns in the peripheral blood and bone marrow were variable. The trend in dogs with B-cell lymphoma was normal to increased percentage of IgG-positive cells, decreased percentage of pan-T-positive cells, decreased percentage of CD4-positive cells, and decreased CD4/CD8 ratio. Simultaneous immunophenotyping of lymph node, blood and bone marrow cannot be recommended routinely without further studies to document its value as an independent prognostic indicator. However, it is potentially useful for tumor staging and monitoring remission, especially in lymphoma patients with a leukemic phase.     

Bone Marrow Cytological Findings in 4 Dogs and a Cat With Hemophagocytic Syndrome

Hemophagocytic syndrome or hemophagic histiocytosis was diagnosed in 4 dogs and 1 cat by evaluation of bone marrow aspirate smears. One of the dogs had a suspected infection with canine parvovirus and a confirmed infection with Salmonella spp, 2 dogs had presumptive diagnoses of myeloproliferative and lymphoproliferative disease, respectively, and 1 dog died without a diagnosis. The cat had hepatic lipidosis and lesions compatible with feline calicivirus infection. All animals had cytopenias involving 2 or more cell lines, and fragmented erythrocytes in the blood, along with mild to moderate increases in the number of macro-phages in the bone marrow. Numerous marrow macro-phages contained phagocytized hematopoietic cells. Other cytological features of the bone marrow were variable in each patient, but the degree of response in the blood was inadequate, even in those with bone marrow hyperplasia. The phagocytosis of hematopoietic elements did not appear to be caused by a primary immune disorder, but rather by the inappropriate activation of normal macrophages secondary to infectious, neoplastic, or metabolic diseases. These findings suggest that hemophagocytic syndrome may be an important factor in the development of cytopenias; the data also support the cytological evaluation of bone marrow aspirates as an aid in the diagnosis of hemophagocytic syndrome. J Vet Intern Med 1996;10:7–14. Copyright © 7996 by the American College of Veterinary Internal Medicine .     

Quantification of thrombopoietic activity in bone marrow aspirates of dogs

The aims of the study were to investigate possible influences of different numbers of counted fields on slides, and different slides on the counting of platelet precursors, and to establish guidelines for bone marrow examination in dogs. The study was based on bone marrow slides of 131 healthy dogs. The slides were prepared by a squash technique. On each slide the number of thrombopoietic cells at different levels of maturation were counted in all fragment-containing fields. The results of a variance component model revealed no influence of different slides, in contrast to a high variance related to different fields. Between 20 and 25 low power fields (100x magnification) had to be examined to achieve an imprecision of 20%. An imprecision of 10% required the counting of 100 fields. The lowest thrombopoietic activity was seen in the bone marrow of dogs aged one to six years. The total number of platelet percursors per field was significantly higher in adult female (10.4 +/- 2.67) than in male dogs (7.84 +/- 2.04, P = 0.0008) as was the number of megakaryocytes. We recommend assessment of thrombopoiesis on the basis of 20-25 fragment-containing low power fields and that age- and sex-related differences should be considered.     

Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood samples collected using ethylenediaminetetraacetic acid as an anticoagulant. Bovine viral diarrhea virus antigen was detected by immunofluorescence between 3 and 11 days postinfection in lymphocyte smears and 3 to 12 days postinfection in buffy coat smears. Isolation of virus from both lymphocytes and buffy coat preparations correlated with detection of immunofluorescence. Serum neutralizing antibody to bovine virus diarrhea virus was detected on day 10 postinfection. Buffy coat smears were as sensitive as lymphocyte smears for the detection of antigen by immunofluorescence. It appeared that immunofluorescent staining of white blood cells was an effective method of detecting bovine virus diarrhea viral antigen.     

Hemorrhagic disease in dogs infected with an unclassified intraendothelial piroplasm in southern Brazil

A hemorrhagic disease affecting dogs in Brazil, referred to popularly as "nambiuvú" (bloody ears) and believed to be transmitted by ticks, has been observed in animals infected with an organism described originally in 1910 as a piroplasm, and known locally as Rangelia vitalli. In this series of 10 cases, the disease was characterized by anaemia, jaundice, fever, spleno- and lymphadenomegaly, hemorrhage in the gastrointestinal tract, and persistent bleeding from the nose, oral cavity and tips, margins and outer surface of the pinnae. The ixodid ticks Rhipicephalus sanguineus and Amblyomma aureolatum infested affected dogs from suburban and rural areas, respectively. Laboratory findings included regenerative anaemia, spherocytosis, icteric plasma and bilirubinuria. Those intracellular organisms were found in bone marrow smears but not in blood smears. Microscopically, zoites were seen within the cytoplasm of blood capillary endothelial cells. Parasitized and non-parasitized endothelial cells were positive immunohistochemically for von Willebrand factor (vWF). Langhans-type multinucleate giant cells were observed in the lymph nodes and choroid plexus. There was prominent erythrophagocytosis by macrophages in the lymph node sinuses and infiltration of the medullary cords by numerous plasma cells. Ultrastructurally, this organism had an apical complex that included a polar ring and rhoptries but no conoid. This parasite was contained within a parasitophorous vacuole that had a trilaminar membrane with villar protrusions and was situated in the cytoplasm of capillary endothelial cells. This organism tested positive by immunohistochemistry for Babesia microti. This pathogen was also positive by in situ hybridization for B. microti. Tentative clinical diagnosis in these cases was based on the history, clinical picture, haemogram and favorable response to therapy, and confirmed through microscopic examination of smears from the bone marrow or histological sections of multiple tissues, especially lymph nodes where zoites were most frequently found. The disease was reproduced by intravenous inoculation of blood from a naturally infected dog into an experimental dog. The authors demonstrate in this study that this organism is a protozoa of the phylum Apicomplexa, order Piroplasmorida. This piroplasm seems to be different from Babesia since it has an intraendothelial stage. Molecular phylogenetic analysis is necessary to better characterize this parasite and clarify its taxonomic status.     

Experimental infection of dogs with a feline endogenous retrovirus RD-114

Background

The feline endogenous retrovirus RD114 is contained in the genome of cats. The virus may contaminate live canine vaccines based on cultured feline cells. The in vivo infectivity, acute and subacute pathogenicity, and viral proliferation of the RD114 virus were evaluated by experimental infection of dogs.

Methods

Nine specific pathogen free dogs were divided into three groups, with each group consisting of one female and two male dogs. Dogs were subcutaneously inoculated in the neck with either 1 ml RD114 stock virus (group A), inactivated RD114 virus suspension (group B), or cell culture medium (group C) as a negative control. To assess blood cell counts and biochemical properties, blood samples from each group were collected 5 days before inoculation, just prior to inoculation, and 1, 3, 7 and 10 days post-inoculation.

Result

During the experimental period of 51 days, none of the dogs inoculated with RD114 virus showed any clinical signs, significant increases in rectal temperature or abnormal blood biochemical characteristics including C-reactive protein when compared with the negative controls. We were not able to re-isolate the RD114 virus from buffy coat cells of group A dogs. Additionally, we could not detect RD114 provirus in the genomic DNA isolated from peripheral blood leukocytes, lymph node, spleen and sternal bone marrow cells.

Conclusions

Signs of RD114 virus proliferation were not found after subcutaneous infection of dogs. Although the potential risk caused by infection with RD114 virus in dogs could not be assessed in this study, we suspect that RD114 virus has little or no virulence in dogs.     

Acute megakaryoblastic leukemia with erythrophagocytosis and thrombosis in a dog

A 7-year-old, intact male Dachshund was presented to the Lyon veterinary school for lethargy and anorexia of several weeks duration. The main clinical signs were pale and icteric mucous membranes, hepatomegaly, splenomegaly, and lymphadenopathy. Results of a CBC and plasma biochemistry tests revealed severe nonregenerative anemia, thrombocytopenia, and increased alanine aminotransferase and alkaline phosphatase activities. Blood smear evaluation and cytologic examination of lymph node and bone marrow aspirate specimens revealed a large population of poorly differentiated blast cells with morphologic features suggesting megakaryocytic lineage. A low number of well-differentiated but dysplastic megakaryocytes also were observed in lymph node and bone marrow smears. A few blast cells were erythrophagocytic. Blast cells were positive for glycoprotein IIIa, factor VIII-related antigen, and factor XIII using immunocytochemistry. The dog was euthanized and necropsied. Histologic findings consisted of diffuse, massive infiltration of lymph nodes, liver, and spleen by megakaryoblasts and atypical megakaryocytes, with widespread thrombosis. This case confirms the usefulness of immunochemistry, including for factor XIII, in the diagnosis of megakaryoblastic leukemia, and demonstrates the unique features of tumor cell erythrophagocytosis and marked fibrinous thrombosis, which have not been reported previously in dogs.     

Flow cytometric evaluation of hemophagocytic disorders in canine

Background — Hemophagocytic macrophages in canine bone marrow are observed in malignant histiocytosis as well as benign hemophagocytic histiocytosis. Cytomorphologic evaluation alone may be inadequate to consistently differentiate between benign and malignant forms of hemophagocytic disorders. Objective — The purpose of this study was to evaluate the ability of flow cytometry and immunophenotyping to differentiate between benign and malignant types of hemophagocytic disorders in dogs. Methods — Blood smears and bone marrow differential cell counts were evaluated for 10 dogs with hemophagocytic disorders. Bone marrow samples were labeled with monoclonal antibodies to CD18, MCH class‐II, Thy‐1, CD14, CD3, and CD21. Using flow cytometry, forward‐angle versus side‐angle light scatter plots were analyzed and immunophenotypes were determined. Results — Scatter plots from 3 dogs with a necropsy diagnosis of malignant histiocytosis revealed 2 atypical cell clusters. One cluster contained cells of similar size or larger than immature myeloid cells and metamyelocytes. Cells in the other cluster were highly granular, with granularity similar to or greater than that of metamyelocytes. In bone marrow from dogs with malignant histiocytosis that was labeled with anti‐CD14 antibody, macrophages represented 29–48% of nucleated cells. Seven dogs had a clinical or histopathologic diagnosis of benign hemophagocytic syndrome. Three of the dogs had normal cell distribution in scatter plots. Two dogs had 2 abnormal cell clusters: 1 within the immature myeloid and metamyelocyte gates and the other with granularity similar to or greater than that of metamyelocytes. The remaining 2 dogs had an atypical cell population, mostly within the immature myeloid gate. For dogs with benign hemophagocytic syndromes, 6–17% of cells in the bone marrow were CD14 positive. Conclusions — The cellular distribution in scatter plots and the total number of macrophages in bone marrow may be useful in differentiating malignant histiocytosis from benign hemophagocytic syndromes in dogs.