Eosinophilic leukemia in three African pygmy hedgehogs (Atelerix albiventris) and

validation of Luna stain

David Martínez-Jiménez, Bridget Garner,1 Sheryl Coutermarsh-Ott, Caitlin Burrell, Sabrina

Clark, Mary Nabity, Josué Díaz-Delgado, Aline Rodrigues-Hoffmann, Karen Zaks, Laila

Proença, Stephen Divers, Corey Saba, Paola Cazzini

Loving Hands Animal Clinic (Martínez-Jiménez), Alpharetta, GA; Departments of Pathology

(Cazzini, Coutermarsh-Ott, Garner) and Small Animal Medicine and Surgery (Proença, Divers,

Saba), College of Veterinary Medicine, University of Georgia, Athens, GA; Departments of

Small Animal Clinical Sciences (Burrell) and Veterinary Pathobiology (Clark, Nabity, Díaz-

Delgado, Rodrigues-Hoffmann), College of Veterinary Medicine and Biomedical Sciences,

Texas A&M University, College Station, TX; and Department of Microbiology, Immunology

and Pathology (Zaks), College of Veterinary Medicine and Biomedical Sciences, Colorado State

University, Fort Collins, CO. Current affiliations: Cumming Veterinary Clinic, Cumming, GA

(Martínez-Jiménez); Easter Bush Pathology, Royal (Dick) School of Veterinary Studies, The

University of Edinburgh, Edinburgh, UK (Cazzini); Smithsonian Conservation Biology Institute,

Front Royal, VA (Burrell).

1Corresponding author: Bridget Garner, College of Veterinary Medicine, University of Georgia,

501 DW Brooks Drive, Athens, GA 30602. Email: garnerb@uga.edu

Running head: Eosinophilic leukemia and Luna stain in hedgehogs

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Abstract. Neoplasia is usually encountered in the African pygmy hedgehog at a mean age of 3.5

y, and malignancy is common. Myelogenous leukemias are rarely reported in hedgehogs. We

describe 3 cases of eosinophilic leukemia in adult, middle-aged (mean age: 2.3 y) hedgehogs, for

which prognosis appears grave. In 1 case, attempted treatment was unsuccessful, and in all 3

cases, the disease course was rapid and all died soon after diagnosis. Blood smear evaluation,

along with complete blood count, was critical in making the diagnosis in all cases. Luna stain

was validated and used to better visualize eosinophils in cytologic and histologic sections.

Electron microscopy confirmed the presence of specific granules in hedgehog eosinophils.

Key words: African pygmy hedgehog; Atelerix albiventris; eosinophilic leukemia; Luna stain;

myeloproliferative disease; neoplasia.

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Myeloproliferative diseases (MPDs) are neoplastic diseases affecting the bone marrow with

unregulated proliferation of clonal hematopoietic stem cells. This uncontrolled cell proliferation

leads to reduction of normal hematopoiesis and invasion of other tissues.21

Eosinophilic leukemia is an infrequent type of MPD and a variant of granulocytic leukemia

that has been described in animals and humans.2,4,14,15,20 Eosinophilic leukemia must be

differentiated from other conditions such as hypereosinophilic syndrome, a chronic idiopathic

condition in which high numbers of mature eosinophils are seen in the peripheral blood and

infiltrating various organs.4 In hypereosinophilic syndrome, the vast majority of the eosinophils

are mature and no atypical cells are seen, whereas in eosinophilic leukemia, immature cells can

be present in increased numbers in circulation and in tissues, and they predominate in the bone

marrow.20

Usually, eosinophils are easily recognized in cytologic and hematologic specimens given

the presence of their characteristic granules. Luna stain was originally developed to detect the

cytoplasmic granules within eosinophils.13 Although it is the most common special stain used to

improve visibility of eosinophils in histologic sections in domestic animals,5,7 this stain has not

been validated in the African pygmy hedgehog, to our knowledge.

Neoplasia is commonly reported in the African pygmy hedgehog (Atelerix albiventris).6

Hedgehogs have an average life span in captivity of 4–6 y, although some may live up to 8 y.

Neoplasia has been reported to occur in animals ranging from 1 mo to >5 y of age, with a mean

age of 3.5 y (Done L, et al. Necropsy lesions by body system in African hedgehogs (Atelerix

albiventris): clues to clinical diagnosis. Proc Joint Conf Am Assoc Zoo Vet and Am Assoc Wildl

Vet; Nov 1992; Oakland, CA).16 The most commonly reported neoplasms in African pygmy

hedgehogs are mammary gland adenocarcinoma, intestinal lymphoma, and oral squamous cell

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carcinoma.16 Myelogenous leukemia has been reported rarely in hedgehogs.8,10,17 In the current

report, we describe the clinical presentation and clinicopathologic features of 3 cases of

eosinophilic leukemia in African pygmy hedgehogs in the United States and the validation of

Luna staining to recognize eosinophils in this species.

Case 1. A 3-y-old male hedgehog was presented to the referring hospital because of a 2-wk

history of ataxia and weakness. The animal weighed 432 g, and had lost >25% of its body weight

from previous visits. While under general anesthesia, physical examination revealed a mass in

the mid abdomen and 3 additional cervical subcutaneous masses. Subsequently referred to the

College of Veterinary Medicine, University of Georgia (Athens, GA), the hedgehog was found to

have lost additional weight (404 g), and had left forelimb lameness. Mites were also present over

the axillary regions. Under isoflurane general anesthesia,a blood was collected from the cranial

vena cava and submitted for a complete blood cell count (CBC). The CBC was performed with

an automated analyzerb and revealed marked leukocytosis (Table 1). Most of the cells were

atypical and were classified as “other.” The atypical cells were large (15–20 µm diameter), round

cells with moderate nuclear-to-cytoplasmic (N:C) ratio and abundant, lightly to moderately

basophilic cytoplasm containing variable quantities of small, round, eosinophilic granules (Fig.

1A). The nuclei were round to reniform and had an open, ropy, chromatin pattern. These cells

were interpreted to be immature eosinophils. Rare non-granulated cells containing a single

nucleolus (presumed myeloblasts) were also present. Fine-needle aspirates of the abdominal and

skin masses were also taken and submitted for cytologic examination. The skin and abdominal

masses were cytologically similar, and were mainly composed of large, individualized, round

cells (88% of nucleated cells) compatible with the immature cells seen in circulation. Occasional

mitotic figures and binucleated cells were noted. Low numbers of mature eosinophils and

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neutrophils (6% of nucleated cells) were scattered throughout. Occasional lymphocytes and

plasma cells (6% of nucleated cells) were also seen. The presence of high numbers of

eosinophilic precursors (39.2 × 109/L and 88% of nucleated cells in the blood and the mass

aspirates, respectively) and the lack of orderly maturation or well-organized left shift in the

granular cells suggested that these were neoplastic eosinophils and not part of an inflammatory

response. Eosinophilic leukemia was considered to be the most likely diagnosis.

Following diagnosis, chemotherapy was begun (cytarabinec 100 mg/m2 subcutaneously

[SC q12h] for 2 d, and prednisoned 2 mg/kg orally [PO] q12h). Weight loss continued, and the

patient was then given 1 oral dose of ivermectine at 0.5 mg/kg, and started on tramadolf 15 mg/kg

PO q6h and mirtazapineg 1.35 mg/kg PO q12h. Because of severe bone marrow suppression by

day 4 post-initiation of the chemotherapy treatment, the patient was started on orbifloxacinh 20

mg/kg PO q12h, metoclopramidei 0.2 mg/kg PO q8h, and diazepamj 1 mg/kg PO q12h.

The clinical status of the animal continued to deteriorate with worsening of lethargy,

anorexia, diarrhea, and vomiting. Famotidinek 1 mg/kg SC q12h, maropitant citratel 2 mg/kg SC

q12h, and sucralfatem 10 mg/kg PO q8h were started. Despite supportive care, the hedgehog died

2 d after the last CBC (8 d after cytarabine treatment; Table 1) and was subjected to cosmetic

autopsy. At the time of autopsy, the animal weighed 342 g. Petechiae and ecchymoses were

present in the skin and subcutaneous tissues. Dark brown, hemorrhagic feces stained the

perineum, and moderate amounts of hemorrhagic feces were in the colon. Multiple, 3–6 mm,

pale tan, friable nodular masses were in the right and left cervical subcutaneous tissues, the

thoracic cavity, ribs, mesentery, and in the spleen. The liver was swollen and friable with a

prominent reticular pattern on the capsular and cut surfaces.

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A sample of the bone marrow was taken soon after euthanasia and routinely stained for

cytologic examination (Fig. 1B). The bone marrow was hypercellular, with no visible iron

deposits and normal megakaryocytes; the myeloid-to-erythroid (M:E) ratio was markedly

increased, indicating a large expansion of the myeloid line, and relative erythroid hypoplasia. In

a 500 cell count, the majority of the cells (68%) were immature myeloid cells ranging from

promyelocytes to metamyelocytes, many of which contained variable numbers of round

eosinophilic granules. Rare dysplastic features such as N:C maturation asynchrony, or

megalocytic cells, were noted in this population. Myeloblasts comprised 7% of the nucleated

cells, and band and segmented granulocytes comprised 15% of the nucleated cells. Erythroid

cells in different stages of maturation represented the remaining 10% of the nucleated cells.

Samples of bone marrow, liver, spleen, heart, lung, stomach, and intestine, as well as the

mediastinal, cervical, and abdominal masses, were submitted and processed for routine histologic

evaluation and Luna stain. Luna stain is performed on standard paraffin sections and employs a

combined hematoxylin–Biebrich scarlet solution to demonstrate eosinophil granules, which stain

red against a blue background. This combined solution was obtained by using Weigert iron

hematoxylin and 1% Biebrich scarlet solution as described elsewhere.13 Histologically, all organs

examined were diffusely infiltrated by cells similar to those seen in the marrow and peripheral

blood. Although the granules were not readily visible histologically, ~60% of the neoplastic cells

were positive for Luna stain, helping to identify them as immature eosinophils. A final diagnosis

of myeloid leukemia with eosinophilic differentiation and metastasis to multiple organs was

confirmed. The immediate cause of death was presumed to be cardiac dysfunction resulting from

myocardial necrosis associated with hypoxia.

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Case 2. A 2-y-old male hedgehog was presented to Texas A&M University College of

Veterinary Medicine Zoological Service (College Station, TX) for evaluation of an ulcerated, 7 ×

5 × 5 mm mass on the dorsal surface of the right hind foot. The animal was overweight (410 g).

Under isoflurane general anesthesia,a the mass was surgically removed and diagnosed as a

histiocytic sarcoma. The animal was subsequently discharged. Seven weeks later, the animal

returned with weight loss (355 g), ataxia, hind limb paresis, and progressive hyporexia; the

previously removed mass had not recurred. Blood samples were obtained under general

anesthesiaa from the cranial vena cava and submitted for CBC. The CBC was analyzedn and

revealed marked leukocytosis characterized by neutrophilia with a left shift including bands and

rarely metamyelocytes, monocytosis, and intermediate-to-large (12–30 µm) cells, classified as

“other” (Table 1), representing ~52% of the reported cells. These large cells had a high N:C ratio

and numerous, small, red cytoplasmic granules that often obscured the nucleus (Fig. 1C). Rare

large, immature cells with a round nucleus, but lacking cytoplasmic granules, and rare mature,

segmented eosinophils were also present. Neutrophils often displayed cytoplasmic basophilia

and anisocytosis, suggesting abnormal maturation. Mild anemia was also present, along with

polychromasia, which could indicate regeneration. Platelets were decreased. Based on the high

number and morphologic features of the large unclassified cells, eosinophilic leukemia was

considered the most likely diagnosis. The animal died in its cage shortly after anesthetic

recovery.

An autopsy was performed and revealed marked hepatomegaly and splenomegaly. The

bone marrow from the right femur was mottled red to tan and turgid. Bone marrow impression

smears were obtained, and tissues collected at autopsy were fixed and processed routinely for

light and electron microscopy. In a 500 cell count, 88% of the cells present in the bone marrow

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impression smears were immature eosinophils, similar to those observed in the blood smear, 8%

of the cells were neutrophils and neutrophil precursors, and the remaining 4% were erythroid

cells, lymphocytes, and plasma cells (Fig. 1D). On histologic examination, blood vessels and

organs were diffusely infiltrated by a population of 15–35 μm neoplastic round cells. The cells

had distinct cell borders and a mild-to-moderate amount of a granular bright eosinophilic

cytoplasm. Nuclei were variably round to indented to irregular, with coarsely stippled chromatin

and inconspicuous nucleoli. Anisocytosis and anisokaryosis were mild with a moderate mitotic

index, averaging 6 per ten 40× fields. Single cell necrosis and degranulation were common

features, the latter of which was often associated with intravascular aggregates of karyorrhectic

cellular debris, vascular endothelial necrosis, thrombosis, and hemorrhages, resembling typical

features of acute tumor lysis syndrome.9 The marrow cytoarchitecture was severely effaced by

neoplastic eosinophils (myelophthisis). Neoplastic cells identical to those seen in circulation

composed up to 80% of the myeloid population. This was supportive of a myeloproliferative

neoplasm. Luna staining was performed on liver sections, revealing strong positive cytoplasmic

staining. Control tissue from a different healthy hedgehog confirmed positive Luna staining of

hedgehog eosinophils compared with nonstaining neutrophils. Additionally, histologic

examination of the central nervous system revealed marked, multifocal white matter spongiosis

with mild, multifocal axonal degeneration involving the cerebrum, cerebellum, brainstem, and

spinal cord. This finding was strongly supportive of wobbly hedgehog syndrome.3

For electron microscopy, formalin-fixed tissues were immersed in 2% glutaraldehyde,

stained with 1% uranyl acetate in water, dehydrated, infiltrated, and polymerized in epoxy.o

Neoplastic cells observed within hepatic sinusoids ranged from 10 to 20 μm, and had a high N:C

ratio (Fig. 1E). The euchromatic nuclei were pleomorphic, large, and central to paracentral, with

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peripheral mildly aggregated chromatin and occasionally a single prominent nucleolus.

Approximately two-thirds of the neoplastic cells had irregularly indented nuclei, and one-third of

the cells had regularly round nuclei. The cytoplasm of these cells contained numerous electron-

dense granules (~0.5 µm diameter). Approximately 40–90% of the granules were round and

moderately and homogeneously electron dense (primary granules). Approximately 10–60% of

the granules were smaller, round to oval, and more electron-dense, often demonstrating a

crystalloid structure (specific granules; inset of Fig. 1E). These microgranules were larger than

those of other hedgehog species such as the European hedgehog (Erinaceus europaeus) and the

long-eared hedgehog (Hemiechinus auritus).18

Case 3. A 2-y-old male hedgehog was presented to the Colorado State University College

of Veterinary Medicine (Fort Collins, CO) for removal of a 1-cm diameter dermal nasal mass.

The mass was consistent with severe lymphocytic granulomatous dermatitis on initial

histopathology. The patient initially did well but was reevaluated 3 mo later for weight loss and

mass regrowth. The mass was removed again, and histopathology was reported as granulomatous

eosinophilic inflammation. The patient became lethargic and anorexic following surgery. Blood

was collected under isoflurane general anesthesiaa for CBC and revealed marked leukocytosis

(Table 1). Collected blood was analyzed,b as in case 1. The vast majority (97%) of the leukocytes

were large round cells that contained granules resembling those of eosinophils. The nuclei of

these cells ranged from rounded or reniform to appropriately segmented. Given the high

eosinophil count, with a high percentage of immature forms, eosinophilic leukemia was

considered as the most likely diagnosis. The immature cells were large, with rounded nuclei and

an indistinct-to-prominent nucleolus. Luna staining of the blood smear stained the cytoplasmic

granules (Fig. 1F). The hedgehog died 1 h after venipuncture, and an autopsy was declined.

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Our report describes 3 cases of eosinophilic leukemia in the African pygmy hedgehog. In

each case, the disease progressed rapidly, as all hedgehogs died soon after diagnosis. All animals

were adult to middle aged (mean age: 2.3 y), which is consistent with reports of other neoplastic

diseases in hedgehogs.16 In one of the cases presented (case 2), a histiocytic sarcoma had also

been diagnosed. This finding is consistent with previous reports in which 10% of hedgehogs with

cancer had more than 1 neoplasm.6 In all cases, clinical signs at presentation were nonspecific

and included weight loss. Ataxia was observed in 2 of the cases (cases 1 and 2). In case 2, a final

diagnosis of wobbly hedgehog syndrome was reached after histologic examination of the central

nervous system; in case 1, a cosmetic autopsy was elected and examination of the nervous

system was not performed, preventing a final evaluation.

In all 3 cases, moderate-to-marked leukocytosis characterized primarily by immature

eosinophils was present and ranged from 1.2 to 8.5 times the upper reference interval (Table 1).1

Eosinophilic leukemia should be differentiated from hypereosinophilic syndrome. In both

conditions, myeloid hyperplasia with eosinophilic predominance is seen in the bone marrow and

peripheral blood. However, the presence of high numbers of immature eosinophils in our cases,

the marked increase in the M:E ratio, the dysplastic changes observed, and the infiltration of

immature cells in other organs were characteristic of eosinophilic leukemia. Immature

eosinophils (hematopoietic cells with eosinophilic granules but no nuclear segmentation)

predominated within the marrow and multiple organs (cases 1 and 2). Crystalloid structures

typical of eosinophil-specific granules were also seen ultrastructurally in neoplastic cells (inset of

Fig. 1E).

Eosinophils are usually recognized easily in cytologic and hematologic specimens given

the presence of characteristic pink granules; however, these may not be as evident in histologic

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specimens. Luna staining is the most common special stain used to improve visibility of

eosinophils in histologic sections.5,7 Using healthy hedgehog tissue as a control, we demonstrated

positive staining in both the control and all eosinophilic leukemic cases. Eosinophils were

markedly positive, and Luna staining was widespread for cases 2 and 3. Interestingly, only 60%

of cells were positive for Luna in case 1. The lack of widespread, marked positivity could be the

result of either the immaturity of many of the cells or altered granule content in the atypical

neoplastic population. Antibodies for a specific eosinophil peroxidase and CD453 and CD348

have been used to confirm eosinophil lineage in dogs and cats, respectively14,19; however, the

validity of these antibodies has not been verified in other species and they were not attempted in

any of our cases.

Eosinophilic leukemia is commonly classified as a chronic leukemia.4,20 This classification

is likely because of the degree of differentiation, such as the presence of the characteristic

eosinophilic granules, which permits the recognition of the cells as eosinophil precursors. In our

cases, however, the immaturity of the cells and the rapid disease progression was more consistent

with an acute leukemia.

Although there is no literature-based evidence about the efficacy of chemotherapy for the

treatment of eosinophilic leukemia in hedgehogs, cytarabine was chosen because of its ease of

administration and its reported use in people with hypereosinophilic syndrome.12 In our report,

the chosen dosage was based on dosages used in dogs and cats. At this dose, it appeared to result

in a marked reduction in circulating neoplastic cells (Table 1). The other drugs were used in an

attempt to control the clinical course of the disease; dosages were all determined based on

scaling.11 However, despite treatment, the hedgehog died shortly after diagnosis, possibly, in

part, because of toxicities of chemotherapy. With that being said, conclusions about the efficacy

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of cytarabine or any chemotherapy regimen cannot be inferred from a single case. Future cases

and treatment attempts may provide more information about how to best manage this type of

cancer.

Acknowledgments

We thank Ms. Abigail M. Butler from the University of Georgia Department of Veterinary

Pathology for her help with the special stains in case 1; Dr. Ross Payne and Kelly Pruit from the

Texas A&M College of Veterinary Medicine and Biomedical Sciences for performing the

electron microscopy and Luna stain in case 2, respectively; Mr. Todd Bass from the Colorado

State University Diagnostic Laboratory for running the Luna stain; Mr. J. Brad Charles from the

CSU Animal Cancer Center for use of the camera for pictures of case 3; and Dr. Jorge Del Pozo

from the University of Edinburgh Easter Bush Pathology Department for his help with the

images.

Authors’ contributions

D Martínez-Jiménez contributed to conception and design of the study and to acquisition of data.

B Garner, S Divers, and C Saba contributed to conception and design of the study and to analysis

and interpretation of data. S Coutermarsh-Ott and C Burrell contributed to conception of the

study and to analysis and interpretation of data. S Clark, M Nabity, J Díaz-Delgado, A

Rodrigues-Hoffmann, K Zaks, and P Cazzini contributed to conception and design of the study

and to acquisition, analysis, and interpretation of data. L Proença contributed to conception and

design of the study and to analysis of data. All authors drafted the manuscript; critically revised

the manuscript; gave final approval; and agreed to be accountable for all aspects of the work in

ensuring that questions relating to the accuracy or integrity of any part of the work are

appropriately investigated and resolved.

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Sources and manufacturers

a. Isoflurane, Vedco, St. Joseph, MO.

b. Advia 120 automated analyzer, Siemens Healthcare, Malvern, PA.

c. Cytarabine, Zydus Hospira Oncology Private, Gujarat, India.

d. Prednisonlone, Hi-Tech Pharmacal, Amityville, NY.

e. Ivermectin, Merial Limited, Duluth, GA.

f. Tramadol, Amneal Pharmaceuticals, Hauppauge, NY.

g. Mirtazapine, Teva Pharmaceuticals, Sellersville, PA.

h. Orbifloxacin, Intervet, Roseland, NJ.

i. Metoclopramide, Teva Pharmaceuticals, Sellersville, PA.

j. Diazepam, Teva Pharmaceuticals, Sellersville, PA.

k. Famotidine, Carlsbad Technology, Carlsbad, CA.

l. Cerenia, Pfizer Animal Health, New York, NY.

m. Sucralfate, Teva Pharmaceuticals, Sellersville, PA.

n. Cell-Dyn 3700, Abbott Laboratories, Abbott Park, IL.

o. Mollenhauer’s Epon-Araldite formula, Electron Microscopy Sciences, Hatfield, PA.

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Table 1. Eosinophilic leukemia in 3 African pygmy hedgehogs (Atelerix albiventris):

comparison of complete blood counts performed.*

CBCCase 1

Case 2 Case 3 Reference interval1Pre-tx Post-txHematocrit 0.37 0.32 0.26 0.18 0.36 ± 0.07 (0.22–0.64) L/LHemoglobin 137 111 83 82 120 ± 28 (70–211) g/LRBC 4.3 3.67 4.07 1.8 6 ± 2 (3–16) × 1012/LMCV 85.3 87 62.6 101 67 ± 9 (41–94) fLMCH 31.9 30.3 20.3 46 11–31 pgMCHC 373 349 324 450 340 ± 50 (170–480) g/LPlatelets 137 142 Decreased 178 226 ± 108 (60–347) × 109/LMPV 11.6 10 ND 15.6 NAWBC 85.4 2.0 51.9 365 11 ± 6 (3–43) × 109/LSegmented 25.6 (30%) 0.220 (11%) 13.5 (26%) 11 (3%) 5.1 ± 5.2 (0.6–37.4) × 109/LBands 7.7 (9%) 0 (0%) 2.1 (4%) 0 NALymphocytes 2.6 (3%) 0.6 (32%) 5.2 (10%) 3.6 (1%) 4 ± 2.2 (0.9–13.1) × 109/LMonocytes 0 (0%) 0.1 (6%) 4.2 (8%) 0 0.3 ± 0.3 (0–1.6) × 109/LEosinophils 7.7 (9%) 0.3 (17%) 0 350.4 (96%) 1.2 ± 0.9 (0–5.1) × 109/LBasophils 2.562 (3%) 0.04 (2%) 0 0 0.4 ± 0.3 (0–1.5) × 109/LOther 39.28 (46%) 0.6 (32%) 26.99 (52%) 0 0 × 109/LNucleated RBC 2 3 1 50.6 0/100 WBC

* CBC = complete blood count; MCH = mean corpuscular hemoglobin; MCHC = mean

corpuscular hemoglobin concentration; MCV = mean corpuscular volume; MPV = mean platelet

volume; NA = not available; ND= not done; Pre-tx = before chemotherapy treatment; Post-tx =

after chemotherapy treatment; RBC = red blood cell count; WBC = white blood cell count

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Figure

Figure 1. Eosinophilic leukemia in African pygmy hedgehogs (Atelerix albiventris). A.

Peripheral blood smear from case 1. Note the large, immature cells, with high nuclear-to-

cytoplasmic (N:C) ratio and numerous, small, pink, cytoplasmic granules (arrow). A segmented

eosinophil is also present (arrowhead). Inset: In some areas, these immature cells predominated

(arrows). Modified Romanowsky stain. Bar = 20 µm. B. Bone marrow smear from case 1. The

hypercellular marrow consists predominantly of immature myeloid cells with pink granules

(arrows). Modified Romanowsky stain. Bar = 20 µm. C. Peripheral blood smear from case 2.

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Image of circulating neoplastic eosinophils represented by large cells with high N:C ratio and

numerous, small, red cytoplasmic granules (arrow). Notice the similarity to the cells in panel A.

Modified Romanowsky stain. Bar = 20 µm. D. Bone marrow smear from case 2. Immature

myeloid cells with pink granules represent the predominant cell type. Modified Romanowsky

stain. Bar = 20 µm. E. Transmission electron micrograph of liver from case 2. Note

representative ultrastructural features of neoplastic eosinophils within a hepatic sinusoid. The

eosinophils have many electron-dense granules. Bar = 2 μm. Inset. Crystalloid structures (arrow)

typical of eosinophil-specific granules. F. Peripheral blood smear from case 3. The cytoplasmic

granules stain strongly positive (bright red) with Luna stain. Note that many of these cells have

large, round nuclei, typical of more immature cells. Luna stain. Bar = 20 µm.

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