Cytological Bone Marrow Cell Differential Counts and Morphologic Findings in Healthy Cynomolgus Monkeys ( Macaca fascicularis) from Nonclinical Toxicology Studies.

Cytological bone marrow evaluation is utilized in nonclinical toxicology studies to characterize hematopoietic effects when the combined interpretation of histologic and complete blood count data does not yield sufficient information. Results from cytological bone marrow examination should be interpreted in the context of variability observed in concurrent control animals with consideration of cytologist experience and historical/published data. Cytological bone marrow differential counts and cellular morphologic findings from 130 (66 male, 64 female) healthy control cynomolgus monkeys from nonclinical toxicology studies were retrospectively analyzed. Myeloid to erythroid (M:E) ratios and the percentage of total cells for each cell type were determined from differential cell count data. M:E ratios ranged from 0.6:1 to 2.3:1. Percentages of total granulocytic cells, total erythroid cells, and lymphocytes ranged from 26.6% to 60.6%, 25.7% to 52.2%, and 5.5% to 40.4%, respectively. Monocytes, plasma cells, mast cells, and mitotic figures were typically <1% of total cells. Notable morphologic findings included occasional giant neutrophilic metamyelocytes and band neutrophils, ring-shaped band neutrophil nuclei, metarubricyte nuclear blebbing and binucleation, multiple or nonfused megakaryocyte nuclei, and emperipolesis. These results represent cytological bone marrow findings from healthy control cynomolgus monkeys utilized in nonclinical toxicology studies and provide insight into expected background variability.

Retrospective study of 613 canine cytologically normal bone marrows associated to altered hemograms: description, possible interpretation, and review of the principal physiological hematopoietic mechanisms.

Background: In clinical routine, it can happen that to an abnormal hemogram corresponds an unexpected cytological normal bone marrow examination that can be difficult to interpret and to manage. Aim: This cytologically retrospective study wants to evaluate a consistent number of qualitative and quantitative normal bone marrow exams according to the hematological and clinical-pathological data to judge if this normality is by itself a pathologic state. Methods: Six hundred and thirteen bone marrow samples were examined. The bone marrow cytological examinations were performed using morphological and numerical criteria together with a complete hemogram, after the identification of clinical or hematological alterations such as multiple lymph nodes enlarged, positive leishmania serological result, staging of neoplasia, cytopenia, increased number of cells, or suspicion of malignant blood disorders. Results: Of the 613 bone marrow samples evaluated, 85 (14%) were classified as normal or without cytological abnormalities; however, only 28 (33%) of those cases had a normal hemogram associated, whereas 55 (65%) had one or more cytopenia and 2 (2%) had increased blood cells count. Conclusion: From this study emerges that cytological bone marrow examinations without any morphological or numerical abnormalities are often associated with altered hematological exams and for this reason, they should not be considered normal and should lead to other deepened investigations.

A Primer for the Evaluation of Bone Marrow.

The traditional role of cytologic and histologic evaluation of bone marrow remains important in understanding diseases and conditions that affect this tissue. It is only through correlation of historical and clinical findings with hematologic, bone marrow, and other ancillary data that an accurate diagnosis can be made. Thus, the clinician is an essential link in helping establish a correct diagnosis. This article is a primer for understanding key features of bone marrow evaluation and provides practical tips for developing the best practices for optimal patient care.

Retrospective study of 152 feline cytological bone marrow examinations: preliminary classification and ranges.

OBJECTIVES: Cytological assessment of the bone marrow is an essential tool for understanding and investigating haematological abnormalities. Sometimes it represents the only way to reach a definitive diagnosis. The purpose of this study was to provide a general overview regarding the prevalence of feline bone marrow disorders encountered in a private European laboratory setting, to classify them and to assess the differential cell counts related to such disorders. METHODS: In total, 152 bone marrow samples were classified using cytological and numerical criteria. The bone marrow cytological examinations were performed after the identification of haematological alterations, such as cytopenias, increased number of cells or suspicion of malignant blood disorders. RESULTS: Of the 152 bone marrow samples evaluated, 71 (46.7%) were classified as hyperplastic, primarily granulocytic and erythroid hyperplasia (50.7% and 45.1%, respectively, of the total hyperplasia); 23 (15.1%) showed dysmyelopoiesis, mainly in the form of myelodysplastic syndrome (39.1% of the total dysmyelopoiesis); 21 (13.8%) had no cytological abnormalities; 17 (11.2%) were malignant blood disorders; 15 (9.9%) had hypoplastic conditions; and two (1.3%) were miscellaneous diseases. Metastatic disease was detected in only two cases (1.3%). Differential cell counts and myeloid-to-erythroid (M:E) ratios were reported for normal, erythroid hypoplastic, erythroid and granulocytic hyperplastic and dysplastic conditions. CONCLUSIONS AND RELEVANCE: This study provides a general overview of the prevalence and incidence of feline bone marrow disorders together with ranges for differential nucleated cell counts and M:E ratios for the various conditions reported.

Hemophagocytic syndrome in dogs: 24 cases (1996-2005).

OBJECTIVE: To determine the frequency, potential causes, and clinical and clinicopathologic features of hemophagocytic syndrome in dogs. DESIGN: Retrospective study. 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.

Diagnostic bone marrow sampling in cats.

Practical relevance: Hematologic disorders are relatively common in cats, as inflammatory, immune-mediated and infectious diseases have the potential to impact erythroid, myeloid and thrombopoietic lines within the bone marrow. Clinical challenges: Clinicians often benefit from information obtained from bone marrow cytology and histopathology in diagnosing hematologic disorders in feline patients. However, these sampling procedures are ones that many veterinarians are intimidated by and thus not comfortably performing in clinical practice. AUDIENCE: This review, aimed at all veterinarians who treat cats, highlights the reasons a clinician may elect to sample bone marrow, and details the collection of bone marrow for both cytology and histopathology. Potential complications and limitations of each procedure are also reviewed. Evidence base: The collection of bone marrow for cytology and histopathology is not new to feline medicine and several research studies have investigated various sampling modalities in cats. This article offers a comprehensive review of the currently accepted best practices.

Bone Marrow Aspirate Evaluation.

Important steps in bone marrow aspirate evaluation include determining if bone marrow evaluation is indicated; using appropriate aspirate collection, smear preparation, and staining techniques; and performing a systematic approach for the cytologic evaluation. The cytologic evaluation of bone marrow requires knowledge of the morphology of bone marrow cell types, the proportion of these cell types normally present, and the ability to evaluate overall cellularity of bone marrow. Accurate interpretation of bone marrow cytologic findings depends on evaluation of a current complete blood cell count. These components are the pillars of getting the most useful information in the diagnosis of hematologic disorders.

Evaluation of dysmyelopoiesis in cats: 34 cases (1996-2005).

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 myelodysplastic 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.

Suspected myelodysplastic/myeloproliferative neoplasm in a feline leukemia virus-negative cat.

A 10-year-old castrated Domestic Short-Haired cat was presented to a primary care veterinarian for a wellness examination and laboratory examination for monitoring of diabetes mellitus. The CBC revealed marked thrombocytosis, leukopenia and macrocytic, normochromic anemia. The cat tested negative for FeLV and feline immunodeficiency virus, but was positive for Mycoplasma haemominutum by PCR. Hematologic abnormalities were not responsive to therapy, so a repeat CBC and a bone marrow aspiration for cytology were performed. Additional blood smear findings included anisocytosis with megaloblastic erythroid precursors, large platelets, eosinophilic myelocytes and metamyelocytes, and rare unidentified blasts. The bone marrow smear was highly cellular, and the cytologic pattern was consistent with myelodysplastic syndrome with an erythroid predominance. At that time, 15% blasts were present. The cat was treated with a vitamin K2 analog, doxycycline, and prednisolone, but without a clinical response. Within 3 months, euthanasia was elected due to declining quality of life, and a necropsy was performed. Postmortem bone marrow smears were highly cellular and dominated by monomorphic blasts of unknown line of origin (52%), persistent marked erythroid and megakaryocytic dysplasia, and ineffective erythropoiesis and granulopoiesis. Immunohistochemical, immunocytochemical, and cytochemical stains resulted in a diagnosis of acute myeloid leukemia of unclassified type. Additional histologic findings included mixed hepatitis with trematode infestation and lymphoplasmacytic interstitial nephritis with fibrosis. The marked thrombocytosis with myelodysplastic syndrome and the FeLV-negative status of this cat were unusual. The difficulty in classifying the myelodysplasia and subsequent leukemia highlights a need for further reporting and characterization of these types of disease.

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.

Canine bone marrow cytological examination, classification and reference values: A retrospective study of 295 cases.

Cytologic assessment of bone marrow with knowledge of the hemogram represents an effective method to investigate hemic tissue and its function. To determine the spectrum and prevalence of canine bone marrow disorders over a 2 year period in a diagnostic laboratory setting achieved through a standard approach to cytologic bone marrow assessment. A retrospective study of bone marrow fine needle aspirates sample preparations, blood smears, hemogram data and case records. Of the 295 bone marrow samples evaluated, 90 (30.5%) were nondiagnostic samples. Of the remaining samples, 25.1% were classified as hyperplasia of which most were granulocytic hyperplasia (58.1% of the total hyperplasia), 19.3% had no cytological abnormalities, 12.9% had malignant hemopathy and 7.8% had hypo-aplastic conditions. Only a small proportion of cases involved dysplasia (1.7%) and metastatic disease was detected in only one case (0.3%). Reference values of nucleated cells and the M/E ratio were calculated for normal and erythroid and granulocytic hyperplastic bone marrow. This study provides the spectrum and the prevalence of canine bone marrow disorders as well as a differential bone marrow cell counting and determination of reference intervals for diseases.

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.

Anemia of inflammation in dogs infected with Ehrlichia platys.

Ten adult male dogs were inoculated with Ehrlichia platys, and blood samples were collected throughout the infection to evaluate the hematologic changes with respect to serum biochemical analytes. All dogs developed a mild, normocytic, normochromic anemia by postinoculation day 7, with significantly (P less than 0.05) decreased serum iron concentration and total iron-binding capacity. Stainable bone marrow iron appeared normal or increased throughout the infection. By postinoculation day 31, the PCV was not significantly different from the pretreatment value. All dogs became hypergammaglobulinemic, leukopenic, hypoalbuminemic, and hypocalcemic during the infection. These findings were compatible with the syndrome of anemia of inflammation.

Effect of canine parvovirus on erythroid progenitors in phenylhydrazine-induced regenerative hemolytic anemia in dogs.

The effects of canine parvovirus (CPV) infection in dogs with hemolytic anemia was compared with the clinical effects of human parvovirus-induced aplastic anemia in human beings with chronic regenerative anemias. Phenylhydrazine was used to induce a transient, severe, hemolytic anemia in dogs to evaluate the effects of CPV infection on rapidly dividing bone marrow precursors. Erythrocyte colony-forming unit bone marrow cultures and cytologic examination of bone marrow were used to determine the effects of CPV infection on erythroid bone marrow precursors. The induced hemolytic anemia regenerated rapidly and although the bone marrow was infected, it was determined that CPV infection did not induce a detectable decrease in erythroid progenitors in dogs with severe hemolytic anemia.

Bone marrow necrosis in the dog.

Bone marrow necrosis in 4 dogs was characterized by refractory anemia with or without leukopenia and thrombocytopenia. Romanovsky-stained bone marrow had a diffuse blue discoloration, and marrow particles were elongated. Degenerating cells, cellular debris, and vacuolated macrophages also were seen. Examination of bone marrow sections revealed extensive replacement of normal stroma by necrotic debris and hemorrhage. The cause of the necrosis was not determined. Concomitant disorders included chronic ehrlichiosis, estrogen toxicosis, malignancy, endometrial cystic hyperplasia, and glomerulonephritis.

Anemia associated with feline leukemia virus infection.

One hundred feline leukemia virus-positive cats with evidence of anemia were examined to determine characteristics of the anemia. The anemia was usually normochromic and normocytic, with low reticulocyte counts but with normal white blood cell and platelet counts. About one third of the cats had splenomegaly. The bone marrow was usually hypocellular or normally cellular, with an increased myeloid to erythroid ratio. A history of recent stress or infection in many cases indicated that the immunosuppressive effect of feline leukemia virus may have been involved. Supportive treatment with periodic blood transfusions was successful in prolonging survival. Corticosteroids and androgens may have been beneficial in some cases.

Primary pure red cell aplasia in dogs: 13 cases (1996-2000).

OBJECTIVES: To examine clinical features, laboratory test results, treatment, and outcome of dogs with pure red cell aplasia (PRCA). DESIGN: Retrospective study. 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.

Bone marrow biopsy and evaluation.

Bone marrow evaluation provides valuable diagnostic and prognostic information about neoplastic, metabolic, and inflammatory diseases. Bone marrow biopsies should be done only after examination of peripheral blood, to avoid performing unnecessary biopsies. A blood sample should be taken at the time of the bone marrow biopsy, for complete hematopoietic evaluation. It is preferable to take both an aspiration and core biopsy simultaneously. A good sample is mandatory for accurate evaluation and interpretation. The method of evaluation should be systematic, complete, and cover the following points: adequacy of specimens; estimation of cellularity; identification of number, maturation pattern, and morphology of megakaryocytes, myeloid cells, and erythroid cells; estimation of M:E ratio; and identification of abnormal cells, cellular reactions, infectious agents, or abnormal stromal reactions. Bone marrow findings should be interpreted in conjunction with signalment, history, physical findings, and laboratory results. Reference or institutional laboratories should be contacted for proper handling of bone marrow specimens for special procedures, such as histopathology, cytochemistry, immunopathology, and electron microscopy.

Pyruvate kinase deficiency anaemia in a Basenji dog.

Pyruvate kinase deficiency anaemia was suspected in an 18-month-old male Basenji dog after other known causes of canine haemolytic anaemia had been excluded. Anaemia of moderate severity (packed cell volumes 0.20 to 0.26 1/1) and reticulocytosis (uncorrected reticulocyte counts 8 to 43%) persisted during 5 months' observation, and biopsies showed development of bone marrow fibrosis and sclerosis. The diagnosis of pyruvate kinase deficiency anaemia was presumptive because erythrocyte pyruvate kinase concentrations in the affected dog were inconclusive and related animals were not available for enzyme assay. However, the gene for pyruvate kinase deficiency is known to occur among Basenji dogs in Australia.

Immunocytological confirmation of bone marrow metastases in a dog with cholangiocarcinoma.

The case history of a four-year-old, male Bernese mountain dog is presented. Carcinoma cells were detected in the liver by ultrasound-guided fine-needle aspiration. Bone marrow aspirated from the iliac crest and the left femur showed a distinct infiltration by carcinoma cells. Immunocytological examination of the liver and bone marrow metastases showed a negative staining result for large spectrum cytokeratin (CK) KL1, a strong positive result for CK7 and a focal weak positive result for CK20. The dog was euthanased due to the grave prognosis. Histopathological examination revealed metastatic cholangiocarcinoma. The authors conclude that cytological and immunocytological examination of bone marrow aspirates should be used more frequently for the detection of distant metastases of carcinomas in small animal medicine.