University of Edinburgh · Web viewPeripheral blood smear from case 1. Note the large, immature...
Transcript of University of Edinburgh · Web viewPeripheral blood smear from case 1. Note the large, immature...
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: [email protected]
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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immunophenotypical features. Vet Clin Pathol 2006;35:454–459.
3. Gibson CJ, et al. Anaplastic astrocytoma in the spinal cord of an African pygmy hedgehog
(Atelerix albiventris). Vet Pathol 2008;45:934–938.
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18. Schaefer HE, et al. Spezifische mikrogranula in eosinophilen. Eine vergleichende
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20. Valli VE. Chronic myeloproliferative diseases. In: Valli VE, ed. Veterinary Comparative
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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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