History

An 11-year-old 4.3-kg spayed, female Longhaired Dachshund was referred because of severe anemia. The dog had been evaluated by the referring veterinarian because of lethargy and decreased appetite. At that point, the dog was bright, alert, and responsive, and had pale-pink mucous membranes with a capillary refill time < 2 seconds. A CBC performed by the referring veterinarian revealed severe, macrocytic (mean corpuscular volume, 89 fL; reference interval [RI], 58 to 79 fL), normochromic (mean corpuscular hemoglobin concentration, 34 g/dL; RI, 30 to 38 g/dL) anemia (PCV, 11%; RI, 36% to 60%) with fragmentation morphology (schistocytes) and a thrombocytosis (693,000 platelets/µL; RI, 170,000 to 400,000 platelets/µL). Radiography revealed no clinically meaningful abnormalities, and immune-mediated anemia was the working diagnosis. The referring veterinarian initiated treatment with dexamethasone sodium phosphate (4 mg, intravenously [IV]), doxycycline (25 mg, per os [PO], q 12 h), and prednisolone (5 mg, PO, q 12 h). The patient was returned for a follow-up examination 2 days later, at which time the doxycycline dosage was increased to 50 mg PO q 12 h. The patient was re-evaluated 2 days later, and another injection of dexamethasone sodium phosphate (4 mg, IV) was administered. At a recheck examination 2 days later, the patient was noted to have been very quiet and to have had pale mucous membranes. An in-house CBC revealed severe, normocytic, normochromic anemia, with a PCV of 9%. Another injection of dexamethasone (4 mg) was administered, and the patient was referred for further evaluation of severe anemia.

Clinical and Clinicopathologic Findings

On physical examination, the dog was quiet and alert, and had pale mucous membranes; bounding femoral pulses; and an undetectable capillary refill time. A CBC and serum biochemical analyses were performed.

The CBC confirmed the previously documented severe nonregenerative anemia, with a PCV of 10% and an absolute reticulocyte concentration of 17,200 cells/µL (RI, 8.0 X 10³ to 65.0 X 10³ cells/µL). Peripheral blood smear review revealed marked RBC anisocytosis with 2+ elliptocytes (3 to 8 cells/100X field), 1+ schistocytes (1 to 2 cells/100X field), and 1+ codocytes (3 to 5 cells/100X field) (Figure 1). The dog had mild leukocytosis (22.5 X 10³ cells/µL; RI, 5.0 X 10³ to 13.0 X 10³ cells/µL) characterized by mature neutrophilia (20.0 X 10³ cells/µL; RI, 2.7 X 10³ to 8.9 X 10³ cells/µL) with mild (1+) toxic change and monocytosis (1.8 X 10³ cells/µL; RI, 0.1 X 10³ to 0.8 X 10³ cells/µL), consistent with an inflammatory leukogram. Thrombocytosis (717 X 10³ thrombocytes/µL (RI, 134 X 10³ to 396 X 10³ thrombocytes/µL) was also present. Fibrinogen concentration was slightly high (0.5 g/dL; RI, 0.1 to 0.4 g/dL). Biochemical abnormalities noted were high activities of aspartate aminotransferase (144 IU/L; RI, 16 to 53 IU/L), alanine aminotransferase (556 IU/L; RI, 23 to 93 IU/L), and alkaline phosphatase (230 IU/L; RI, 7 to 116 IU/L), with mildly low concentrations of total calcium (8.5 mg/dL; RI, 8.7 to 10.4 mg/dL) and creatinine (0.56 mg/dL; RI, 0.6 to 1.5 mg/dL). Both albumin and total protein concentrations were within reference limits.

Photomicrograph of a peripheral blood smear prepared from a blood sample from an 11-year-old 4.3-kg spayed, female Longhaired Dachshund that was referred because of severe anemia. A—Elliptocytes (arrow), codocytes (arrowhead), and RBCs exhibiting fragmentation morphology (schistocytes [asterisk]) are shown. Wright-Giemsa stain; bar = 10 µm. B—Elliptocytes (arrow) and a schistocyte (asterisk) are evident, along with frequent platelets. Wright-Giemsa stain; bar = 5 µm.

Citation: Journal of the American Veterinary Medical Association 259, S2; 10.2460/javma.20.12.0656

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Photomicrograph of a peripheral blood smear prepared from a blood sample from an 11-year-old 4.3-kg spayed, female Longhaired Dachshund that was referred because of severe anemia. A—Elliptocytes (arrow), codocytes (arrowhead), and RBCs exhibiting fragmentation morphology (schistocytes [asterisk]) are shown. Wright-Giemsa stain; bar = 10 µm. B—Elliptocytes (arrow) and a schistocyte (asterisk) are evident, along with frequent platelets. Wright-Giemsa stain; bar = 5 µm.

Citation: Journal of the American Veterinary Medical Association 259, S2; 10.2460/javma.20.12.0656

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Photomicrograph of a peripheral blood smear prepared from a blood sample from an 11-year-old 4.3-kg spayed, female Longhaired Dachshund that was referred because of severe anemia. A—Elliptocytes (arrow), codocytes (arrowhead), and RBCs exhibiting fragmentation morphology (schistocytes [asterisk]) are shown. Wright-Giemsa stain; bar = 10 µm. B—Elliptocytes (arrow) and a schistocyte (asterisk) are evident, along with frequent platelets. Wright-Giemsa stain; bar = 5 µm.

Citation: Journal of the American Veterinary Medical Association 259, S2; 10.2460/javma.20.12.0656

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Additional Clinicopathologic Findings

In addition to the severe anemia, erythrocyte morphology was of particular clinical importance. The shape changes noted included elliptocytes, which in dogs have been associated with bone marrow pathology such as myelofibrosis,¹ and schistocytes, an indication of increased RBC shear force, fragility, or both. The dog’s anemia was judged nonregenerative because sufficient time (6 days) had elapsed for an appropriate regenerative response to become evident between the initial documentation and the CBC performed on referral at the University of Florida. Based on these findings, a bone marrow aspirate was attempted but yielded no identifiable hematopoietic tissue for cytologic evaluation (dry tap) and raised clinical concern for a myelophthisic condition such as myelofibrosis. A core bone marrow biopsy specimen was obtained and then submitted for histopathologic evaluation.

Histopathologic Findings

The bone marrow core biopsy specimen was submitted in neutral-buffered 10% formalin, decalcified, embedded in paraffin, sectioned, stained with H&E stain, and then examined with light microscopy. Ample intact trabecular marrow spaces were available for examination. Sixty percent to 70% of the available marrow spaces contained a markedly high amount of mature fibrovascular tissue deposition, with fibroblasts and intermittent small-caliber vessels frequently aligned in linear streams that canalized hematopoietic precursors (Figures 2 and 3). Hematopoietic precursors represented approximately 10% to 20% of overall nucleated cellularity in the marrow spaces. The majority of hematopoietic cellularity consisted of mature or band-form granulocytes. There were high numbers of large cells with high nuclear-to-cytoplasmic ratios, open vesicular chromatin patterns, and 1 to 3 distinct basophilic nuclei in the paratrabecular regions (areas of expected granulocytic proliferation) that were interpreted as early myeloid precursors and indicated relative, mildly left-shifted granulocytic hyperplasia, consistent with the peripheral neutrophilia observed. Erythroid precursors were observed in occasional small aggregates adjacent to bone spicules. The granulocytic-to-erythrocytic ratio was 10:1 (RI,¹ 0.75 to 2.53:1). Eosinophils and plasma cells were present in low numbers, < 3% of total nucleated cellularity. Iron stores were present, reflected as scant to low numbers of hemosiderin-laden macrophages and mild amounts of free hemosiderin, scattered in the marrow spaces. One to 3 megakaryocytes were present per trabecular space and were judged to be adequate in number. Overt evidence of erythroid precursor phagocytosis was not identified. Bone spicules were smooth, well formed, and composed of well-organized lamellar bone. A Masson trichrome stain of the bone marrow revealed the deposition of moderate amounts of thin to mildly thickened bright-blue collagen fibers in areas of fibrovascular proliferation. No intralesional microorganisms or evidence of neoplasia were observed in the examined bone marrow sections.

Photomicrograph of a bone marrow core biopsy specimen obtained from the dog described in Figure 1. Most of the marrow spaces are filled with fibrovascular tissue that replaces much of the hematopoietic tissue. H&E stain; bar = 200 µm.

Citation: Journal of the American Veterinary Medical Association 259, S2; 10.2460/javma.20.12.0656

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Photomicrograph of a bone marrow core biopsy specimen obtained from the dog described in Figure 1. Most of the marrow spaces are filled with fibrovascular tissue that replaces much of the hematopoietic tissue. H&E stain; bar = 200 µm.

Citation: Journal of the American Veterinary Medical Association 259, S2; 10.2460/javma.20.12.0656

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Photomicrograph of a bone marrow core biopsy specimen obtained from the dog described in Figure 1. Most of the marrow spaces are filled with fibrovascular tissue that replaces much of the hematopoietic tissue. H&E stain; bar = 200 µm.

Citation: Journal of the American Veterinary Medical Association 259, S2; 10.2460/javma.20.12.0656

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Photomicrograph of a bone marrow core biopsy specimen obtained from the dog described in Figure 1 showing granulocytic cells and myeloid precursors admixed with streaming fibrovascular tissue. H&E stain; bar = 50 µm.

Citation: Journal of the American Veterinary Medical Association 259, S2; 10.2460/javma.20.12.0656

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Photomicrograph of a bone marrow core biopsy specimen obtained from the dog described in Figure 1 showing granulocytic cells and myeloid precursors admixed with streaming fibrovascular tissue. H&E stain; bar = 50 µm.

Citation: Journal of the American Veterinary Medical Association 259, S2; 10.2460/javma.20.12.0656

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Photomicrograph of a bone marrow core biopsy specimen obtained from the dog described in Figure 1 showing granulocytic cells and myeloid precursors admixed with streaming fibrovascular tissue. H&E stain; bar = 50 µm.

Citation: Journal of the American Veterinary Medical Association 259, S2; 10.2460/javma.20.12.0656

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Morphologic Diagnosis and Case Summary

Morphologic diagnosis: myelofibrosis, coalescing, marked with marked erythroid hypoplasia, bone marrow, left humerus; resulting in severe, nonregenerative anemia.

Case summary: myelofibrosis in a dog.

Comments

Myelofibrosis is a bone marrow disorder characterized by inappropriate fibroblast proliferation of the medullary spaces with replacement of hematopoietic tissue. This disorder has been documented in patients with a variety of conditions (eg, myeloproliferative, lymphoproliferative, metastatic, or infiltrative neoplasms) or in response to conditions that cause bone marrow injury and necrosis (eg, idiosyncratic drug responses).² Nonregenerative immune-mediated anemia targeting bone marrow erythroid precursors has been linked with myelofibrosis^3–5 and, in turn, with the RBC morphology changes observed in this case.⁵ One proposed mechanism for myelofibrosis is that direct and or indirect cytokine production within the marrow microenvironment stimulates fibroblast proliferation and deposition of reticulin (initially) and collagen (later).¹ Special histochemical stains, such as reticulin stain and Masson trichrome stain, can be useful in detecting small amounts of reticulin or collagen fiber formation. When present in large amounts, as in this case, the myelofibrosis can be clearly identified on H&E sections. Bone marrow aspiration for cytologic evaluation can be unrewarding in cases of myelofibrosis as a result of increased fibrous tissue and a relatively decreased amount of hematopoietic cellularity (myelophthisis, or displacement of marrow hematopoietic cellularity). The resulting anemia leads to generalized systemic tissue hypoxia, which likely contributed to the hepatocellular injury (ie, centrilobular) and associated serum liver enzyme increases observed for the dog in this report. This dog’s neutrophilia, monocytosis, lymphopenia, and thrombocytosis were consistent with cytokine-driven systemic inflammation, although an inciting cause was not identified.

This case highlights the importance of manual blood smear review, and interpreting its findings in the context of the automated CBC analyzer results, specifically with regard to erythrocyte morphology. The erythrocyte shape changes identified at the time of referral were pivotal to informing considerations for advanced diagnostic procedures, such as bone marrow evaluation. Elliptocytes or ovalocytes suggest erythrocyte shape deformation as a consequence of advanced bone marrow disease and have been associated with myelofibrosis and myelodysplastic syndrome in dogs.^1,5

A bone marrow core biopsy was performed in this case because of repeated, unsuccessful bone marrow aspiration attempts. Cytologic evaluation of bone marrow aspirates, although of limited sensitivity in detecting focal disease or fibroproliferative conditions, is useful for distinguishing cell lineages and morphology or identifying increased rubriphagocytic activity that indicates precursor-directed immune-mediated anemia (PIMA), which was suspected—although not confirmed—this case. Bone marrow histopathology provides a view of overall tissue architecture, and the collected specimen may be used for additional histologic or immunohistochemical staining, as in this instance. This underscores the importance of obtaining bone marrow core biopsies in cases of severe, nonregenerative anemia when myelofibrosis is suspected and bone marrow aspiration attempts yield little or no diagnostic cellularity.

The dog in this report received a blood transfusion and was started on an immunosuppressive dosage of prednisone (1.15 mg/kg, PO, q 12 h) based on a presumptive diagnosis of PIMA, with the addition of cyclosporine (6 mg/kg, PO, q 12 h) 6 weeks later. After discharge from the veterinary teaching hospital, the dog received continued care at a regional specialty hospital. Approximately 1 year after the initial presentation, the dog was reported to have had a PCV of 38% and to have been doing well at home.