Pathology in Practice

Liza Bau-Gaudreault Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC J2S 2M2, Canada.

Search for other papers by Liza Bau-Gaudreault in
Current site
Google Scholar
PubMed
Close
 DMV, MSc
and
Carolyn Grimes Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Université de Montréal, St-Hyacinthe, QC J2S 2M2, Canada.

Search for other papers by Carolyn Grimes in
Current site
Google Scholar
PubMed
Close
 DVM

Click on author name to view affiliation information

History and Initial Clinicopathologic Findings

A 7-year-old 30.6-kg (67.3-lb) castrated male Old English Sheepdog was presented to the emergency service at a veterinary teaching hospital for evaluation of severe nonregenerative anemia. Two months earlier, the owner had noted that the dog had pale mucous membranes, but no other clinical signs were observed. A week before presentation, the dog was evaluated by the referring veterinarian. At that time, relevant clinicopathologic abnormalities included severe normocytic, hypochromic, nonregenerative anemia (Hct, 0.14 L/L; reference interval, 0.38 to 0.57 L/L), mild hyperkalemia (5.9 mmol/L; reference interval, 4.0 to 5.4 mmol/L), and slightly high symmetric dimethylarginine concentration (15 μg/dL; reference interval, 0 to 14 μg/dL). Radiography revealed mild splenomegaly and hepatomegaly. In the preceding 2 years, the dog had had 2 episodes of marked thrombocytopenia that resolved with immunosuppressive treatment (prednisone and azathioprine [dosages unknown]).

At the time of the referral evaluation (day 1), physical examination of the dog revealed pale mucous membranes and a 2/6 holosystolic heart murmur. No other abnormalities were noted. A CBCa revealed severe normocytic, hypochromic, nonregenerative anemia (Hct, 0.17 L/L [reference interval, 0.40 to 0.56 L/L]; reticulocyte count, 10,680 × 106 reticulocytes/L [reference interval, 0 to 91,000 × 106 reticulocytes/L]) with high RBC distribution width ([RDW] 26.3%; reference interval, 11.0% to 13.0%) and mild leukopenia (4.61 × 109 WBCs/L; reference interval, 5.10 × 109 to 14.20 × 109 WBCs/L). The platelet count was within the reference interval (253 × 109 platelets/L; reference interval, 153 × 109 to 400 × 109 platelets/L), and platelet morphology was unremarkable. Morphology flagging data, provided by the hematology analyzer,a indicated 3+ anisocytosis characterized by 3+ microcytic RBCs and 2+ macrocytic RBCs on the basis of percentages of RBCs (13.1% and 6.5%, respectively) that exceeded predefined thresholds for low and high RBC volume. These macrocytic and microcytic RBC subpopulations were visualized on an analyzer-generated RBC volume-to-hemoglobin concentration ratio scattergram (Figure 1). These changes prompted the evaluation of a fresh blood smear, which revealed 3+ ovalocytosis (10 to 15 ovalocytes/hpf [100X]) and anisocytosis characterized by macrocytosis and microcytosis.

Figure 1
Figure 1

Hematologic findings for a 7-year-old Old English Sheepdog that was presented for evaluation of severe nonregenerative anemia of 2 months’ duration. A—An RBC volume-to-hemoglobin concentration ratio scattergram obtained from the hematology analyzer used to perform a CBC. A microcytic subpopulation of RBCs is indicated by the arrowhead. A macrocytic population of RBCs is indicated by the arrow. The grid lines represent the reference interval for the volume (horizontal lines) and hemoglobin concentration (vertical lines). The normal RBC population should appear within the central square. B—Photomicrograph of a blood smear from the dog, in which normocytic, hypochromic, nonregenerative anemia had been identified. Ovalocytes (arrows), macrocytes, and microcytes are present. Modified Wright-Giemsa stain; bar = approx 20 μm.

Citation: Journal of the American Veterinary Medical Association 258, 7; 10.2460/javma.258.7.721

Additional Clinicopathologic Findings

Myelofibrosis associated with increased collagen deposits was suspected because of the presence of severe persistent nonregenerative anemia with macrocytosis and ovalocytosis without other noteworthy poikilocytes. A bone marrow core biopsy was performed. The bone marrow biopsy specimen was highly cellular with little adipose tissue (Figure 2) and a low myeloid cell-to-erythroid cell ratio (1:1) indicative of erythroid hyperplasia. Approximately 10% to 20% of the hematopoietic space was occupied by nonneoplastic mesenchymal cells (fibroblasts) admixed with fibers that stained with picrosirius red stain (collagen); the fibers did not stain with reticulin stain. On the basis of these findings, a diagnosis of moderate multifocal collagen myelofibrosis was made for the dog.

Figure 2
Figure 2

Photomicrographs of sections of a bone marrow core biopsy specimen obtained from the dog in Figure 1. A—In this section, notice the moderate fibrosis (pink fibrillar material). A moderate number of megakaryocytes and moderately abundant golden hemosiderin pigment are also observed. Hematoxylin phloxine saffron stain; bar = approx 40 μm. B—In this section, collagen fibrosis is evident on the basis of the picrosirius red staining of collagen fibers. Picrosirius red stain; bar = approx 100 μm.

Citation: Journal of the American Veterinary Medical Association 258, 7; 10.2460/javma.258.7.721

A direct Coombs test (gel microcolumn) was also performed, and the result was negative. Despite this negative test result, an immune-mediated component was suspected because of the dog's history and the clinicopathologic findings, and treatment with prednisone (1 mg/kg [0.45 mg/lb], PO, q 12 h) was initiated. Administration of doxycycline (10 mg/kg [4.5 mg/lb], PO, q 24 h for 3 weeks) was also initiated to treat possible underlying tick-borne disease because the dog had not been receiving tick-prevention medication.

Fifteen days after the initial referral evaluation, a repeated CBC was performed. Anemia had slightly improved (Hct, 0.23 L/L) but remained normocytic and nonregenerative (reticulocyte count, 65,550 × 106 reticulocytes/L) with a high RDW (24%). Blood smear findings were similar to those identified at the initial referral evaluation with a high number of ovalocytes (5 to 10 ovalocytes/hpf) and moderate to high anisocytosis with macrocytes and microcytes (Figure 3). However, mild polychromasia was present and, combined with the increase in reticulocyte count, compared with the count on day 1, was considered possibly reflective of an early erythroid regenerative response in the bone marrow. Additional examinations during the 8-month follow-up period revealed gradual improvement with continued immunosuppressive treatment. Ovalocytosis resolved after 7 months and, after 8 months, the dog's Hct had improved to 0.42 L/L.

Figure 3
Figure 3

Photomicrograph of a blood smear obtained from the same dog 15 days following the initial examination. Ovalocytes (types I and II) and macrocytes (Ma) are present. Modified Wright-Giemsa stain; bar = approx 20 μm. Inset—Type III ovalocytes and microcytes (Mi) are also present. Modified Wright-Giemsa stain; bar in main image applies to inset.

Citation: Journal of the American Veterinary Medical Association 258, 7; 10.2460/javma.258.7.721

Hematologic Diagnosis and Case Summary

Hematologic diagnosis: persistent nonregenerative anemia with ovalocytosis, macrocytosis, and microcytosis, suggestive of collagen myelofibrosis.

Case summary: persistent nonregenerative anemia associated with collagen myelofibrosis in a dog with presumed nonregenerative immune-mediated anemia (NRIMA).

Comments

For the dog of the present report, NRIMA was the most likely explanation for the changes observed. Nonregenerative immune-mediated anemia can be caused by antibody-mediated inhibition of erythropoiesis or destruction of late-stage erythroid precursors (precursor-directed immune-mediated anemia).1 Because the dog had erythroid hyperplasia, precursor-directed immune-mediated anemia was considered more likely2 despite the absence of evident rubriphagocytosis on evaluation of a bone marrow biopsy specimen. Destruction of late-stage erythroid precursors translates into nonregenerative anemia, erythroid hyperplasia (ineffective erythropoiesis), and, eventually, collagen myelofibrosis. The dog's positive response to immunosuppressive treatment further supported an immune-mediated component of the disease process.

Persistent nonregenerative anemia with ovalocytosis and anisocytosis characterized by macrocytosis is suggestive of collagen myelofibrosis. An interesting aspect of the case described in the present report was that the dog's mean corpuscular volume was within the reference interval. Consideration of additional information to characterize the RBC population was useful in this case. The RDW is a calculated value that indicates the amount of variation of RBC volume, and high RDW reflects anisocytosis. For the dog of the present report, the RDW was moderately high (24%). Moreover, the RBC volume-to-hemoglobin concentration ratio scattergram provided more complete information about the RBC subpopulations in terms of volume and hemoglobin content. In this dog, 13.1% of the RBCs were identified as microcytic and 6.4% were identified as macrocytic. The combination of macrocytic and microcytic RBC subpopulations yielded an elevated RDW and a mean corpuscular volume that was within the reference interval. The presence of a severe, persistent, nonregenerative anemia with anisocytosis characterized by macrocytosis and the presence of ovalocytosis has been strongly associated with collagen myelofibrosis, often secondary to NRIMA.3,b Histopathologic evaluation of the bone marrow biopsy specimen confirmed collagen myelofibrosis and revealed other erythroid changes (ineffective erythropoiesis) that supported a diagnosis of NRIMA.

Although ovalocytes are associated with the presence of myelofibrosis in dogs, they are also described in association with myelodysplastic syndrome, glomerulonephritis, and rare hereditary membrane abnormalities (β-spectrin or protein 4.1 mutation).4 Ovalocytes are subdivided into 4 categories according to their length and width; type I are almost circular, type II are oval, type III are elongated, and type IV are bacilliform.5 In the dog of the present report, type I, II, and III ovalocytes were identified. Macrocytosis may be an artifactual change (in vitro cell swelling or the presence of agglutinated RBCs) or may develop in association with regenerative anemias and hyperosmolar states or certain hereditary disorders (hereditary stomatocytosis and Poodle macrocytosis).6 Interestingly, dogs with myelofibrosis and macrocytosis have a better prognosis than do dogs with myelofibrosis but without macrocytosis.7 The exact pathogenesis of macrocytes and ovalocytes in cases of myelofibrosis remains unclear.

Myelofibrosis is characterized by an accumulation of fibrous tissue in the hematopoietic space.8 This fibrous tissue includes actively proliferating fibroblasts as well as accumulated connective tissue fibers. These fibers are either reticulin fibers (composed of primarily type III collagen), dense type I collagen fibers, or a combination of both. The type or types of fibers present can be determined with the application of special histologic stains (eg, Gomori, Masson trichome, picrosirius red, or reticulin stains) on bone marrow sections. It is important to distinguish the type of fibers present because different clinical syndromes are associated with different types of myelofibrosis; in early stages of fibrosis, reticulin fibers are more abundant and clinical disease is usually mild but progression to collagen fibrosis may occur. The presence of hematologic abnormalities and severe clinical disease is most strongly associated with collagen myelofibrosis.9

Primary myelofibrosis, sometimes called idiopathic myelofibrosis, has been described in human medicine10; it is a myeloproliferative neoplasm that arises from the clonal proliferation of hematopoietic stem cells. Primary myelofibrosis is rare in veterinary species but has been reported for 3 dogs and a cat.11,12 In animals, myelofibrosis is more commonly secondary to bone marrow damage. The most common causes of secondary myelofibrosis in dogs are NRIMA and intra- and extra-medullary neoplasms. These 3 causes combined are responsible for approximately 50% of cases (as determined in a study8 of 19 cases of canine myelofibrosis). Other causes of secondary myelofibrosis have been reported and include pyruvate kinase deficiency, myelonecrosis, congenital nonspherocytic anemia, and long-term administration of certain drugs.8,13

The pathophysiologic features of myelofibrosis are not completely understood. However, fibrogenic cytokines (eg, transforming growth factor-β, platelet-derived growth factor, and epidermal growth factor) produced by marrow platelets, megakaryocytes, and macrophages are thought to stimulate collagen secretion by fibroblasts.1,11

Collagen myelofibrosis is not a rare disorder. In 1 retrospective study,3 collagen myelofibrosis was identified in 47 of 717 (6.3%) bone marrow samples from dogs (1 sample/dog). Thus, it is important to identify meaningful hematologic evidence that may help signal its presence. Ovalocytosis, macrocytosis, and nonregenerative anemia are not individually suggestive of collagen myelofibrosis but when these changes are present concurrently, as in the dog of the present report, collagen myelofibrosis should be suspected and the animal's bone marrow should be evaluated.

Acknowledgments

This case was presented in part at a case session at the 2016 European Society of Veterinary Clinical Pathology annual meeting in Nantes, France.

The authors thank Drs. Magali Decôme and Lisa Carioto for submission of the case and Drs. Geneviève Langevin-Carpentier and Daniel Martineau for interpretation of bone marrow sections.

Footnotes

a.

Advia 120 hematology system, Siemens Healthcare, Tarrytown, NY.

b.

Hollinger C, Sirivelu MP, Scott MA. Hematologic findings predictive of myelofibrosis in dogs (abstr), in Proceedings. 46th Annu Meet Am Soc Vet Clin Pathol 2011;603–604.

References

  • 1.

    Weiss DJ, Wardrop KJ. Immune-mediated anemias in the dog. In: Schalm's veterinary hematology. 6th ed. Ames, Iowa: Wiley-Blackwell, 2010;219221.

    • Search Google Scholar
    • Export Citation
  • 2.

    Lucidi CA, de Rezende CLE, Jutkowitz LA, et al. Histologic and cytologic bone marrow findings in dogs with suspected precursor-targeted immune-mediated anemia and associated phagocytosis of erythroid precursors. Vet Clin Pathol 2017;46:401415.

    • Search Google Scholar
    • Export Citation
  • 3.

    Weiss DJ. A retrospective study of the incidence and the classification of bone marrow disorders in the dog at a veterinary teaching hospital (1996–2004). J Vet Intern Med 2006;20:955961.

    • Search Google Scholar
    • Export Citation
  • 4.

    Harvey JW. Evaluation of erythrocytes. In: Veterinary hematology: a diagnostic guide and color atlas. St Louis: Elsevier Saunders, 2012;51, 73.

    • Search Google Scholar
    • Export Citation
  • 5.

    Di Terlizzi R, Gallagher PG, Mohandas N, et al. Canine elliptocytosis due to a mutant β-spectrin. Vet Clin Pathol 2009;38:5258.

  • 6.

    Stockham SL, Scott MA. Erythrocytes. In: Fundamentals of veterinary clinical pathology. 2nd ed. Ames, Iowa: Wiley-Blackwell, 2008;142.

  • 7.

    Villiers EJ, Dunn JK. Clinicopathological features of seven cases of canine myelofibrosis and the possible relationship between the histological findings and prognosis. Vet Rec 1999;145:222228.

    • Search Google Scholar
    • Export Citation
  • 8.

    Weiss DJ, Smith SA. A retrospective study of 19 cases of canine myelofibrosis. J Vet Intern Med 2002;16:174178.

  • 9.

    Thiele J, Kvasnicka HM, Facchetti F, et al. European consensus on grading bone marrow fibrosis and assessment of cellularity. Haematologica 2005;90:11281132.

    • Search Google Scholar
    • Export Citation
  • 10.

    Quigley M, Linfesty RL, Bethel K, et al. Stubby elliptocytes are an invariable feature of leukoerythroblastosis. Blood 2007;109:2666.

  • 11.

    Kuter DJ, Bain B, Mufti G, et al. Bone marrow fibrosis: pathophysiology and clinical significance of increased bone marrow stromal fibres. Br J Haematol 2007;139:351362.

    • Search Google Scholar
    • Export Citation
  • 12.

    Breuer W, Darbès J, Hermanns W, et al. Idiopathic myelofibrosis in a cat and in three dogs. Comp Haematol Int 1999;9:1724.

  • 13.

    Stokol T, Blue JT, French TW. Idiopathic pure red cell aplasia and nonregenerative immune-mediated anemia in dogs: 43 cases (1988–1999). J Am Vet Med Assoc 2000;216:14291436.

    • Search Google Scholar
    • Export Citation
All Time Past Year Past 30 Days
Abstract Views 446 0 0
Full Text Views 1191 951 221
PDF Downloads 428 185 13
Advertisement