December 2013, Vol. 7, No. 12, pp. 1228-1235 Journal of Life Sciences, ISSN 1934-7391, USA

Extramedullary Hematopoiesis Mimicking a Neoplasm in

a Goeldi’s Monkey (Callimico goeldii)

Rute Marina Noiva1, Hugo Pissarra1, Teresa Fernandes2, Rui Bernardino2, Luis Madeira de Carvalho1, Fernando

Alves Afonso1 and Maria Conceicao Peleteiro1

1. Interdisciplinary Research Centre on Animal Health, Faculty of Veterinary Medicine, Technical University of Lisbon

(CIISA/FMV/UTL), Avenida da Universidade Tecnica, Lisbon 1300-477, Portugal

2. Jardim Zoologico de Lisboa, Estrada de Benfica 158-160, Lisbon 1549-004, Portugal

Received: August 23, 2013 / Accepted: November 04, 2013 / Published: December 30, 2013. Abstract: Extramedullary hematopoiesis consists in the appearance and proliferation of hematopoietic cells outside the bone marrow. In

this article, the authors describe a case of hepatosplenic hematopoiesis in a 9-year-old, male Goeldi’s monkey concurrent with a

Calodium hepaticum infestation, belonging to the Lisbon’s Zoo primate collection (Portugal). Lesions were identified upon necropsy

after euthanasia due to the presence of an apparently non-excisable, metastatic aortic mass. Histopathological analysis of samples

taken was carried out and immunohistochemical staining was used to characterize the cellular population involved, confirming the

diagnosis of extramedullary hematopoiesis. To the best of the authors’ knowledge, this is the first report of hepatosplenic

extramedullary hematopoiesis in a Goeldi’s monkey.

Key words: Extramedullary hematopoiesis, Goeldi’s monkey, Callimico goeldii, Calodium hepaticum, liver, spleen.

1. Introduction

In the mature animals, homeostatic hematopoiesis is

confined to the marrow cavity of the flat bones and the

epiphysis of long bones [1]. EMH (extramedullary

hematopoiesis) is the typically asymptomatic

appearance of hematopoietic elements outside of the

bone marrow as a result of compensation for

hematopoietic malfunction, serious chronic anemia,

myelofibrosis, chronic myelocytic leukemia and

metastasis of malignant tumors to the bone marrow,

among other causes [2, 3].

Extramedullary hematopoiesis usually occurs in

tissues with an environment that supports the

proliferation of primitive hematopoietic bone marrow

elements [4]. The organs of the reticuloendothelial

system, like the liver, spleen and lymph nodes are the

sites most frequently involved, although, in humans,

Corresponding author: Rute Marina Noiva, Ph.D., research

field: veterinary pathology. E-mail: rute.noiva@gmail.com.

EMH has also been observed in the posterior

mediastinum, kidney, central nervous system and

peripheral nerves, meninges, middle ear, pancreas,

urethra, thyroid and adrenal glands, gastrointestinal

tract, pharynx, lung, pleura and pericardium, heart,

peritoneum and retroperitoneum, skin, kidney, breast,

ovary, prostate gland, endometrium, epididymis and

thymus [1, 3-6].

In humans, EMH often resembles more common

conditions such as benign or malignant

lymphadenopathy or a metastasis on radiographic

studies [3]. Ultrasound findings vary from solitary

heterogenous hypoechoic nodules to multiple

echogenic masses with central necrosis. Computerized

tomography findings are also variable, ranging from

multiple hypodense to solitary hyperdense masses [7].

In adult humans, hepatic EMH is seen in a variety

of conditions, including hematologic disorders, sepsis,

transplantation, massive hepatic necrosis, but is also

D DAVID PUBLISHING

Extramedullary Hematopoiesis Mimicking a Neoplasm in a Goeldi’s Monkey (Callimico goeldii)

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notably prominent within hepatoblastomas, hepatic

adenomas and hepatocellular carcinoma [8].

In new world monkeys, nodular foci of

extramedullary hematopoiesis and myelolipomas are

occasionally found in the spleen and liver. However,

the pathogenesis and significance of these foci is

unknown [9].

The current work aims at reporting a case

of extramedullary hematopoeisis mimicking a

metastatic neoplasm in a Goeldi’s monkey (Callimico

goeldii).

2. Materials and Methods

In November 2009, a 9-year-old male Goeldi’s

monkey (Callimico goeldii), belonging to the

zoological collection of the Lisbon’s Zoo (Portugal)

was taken for clinical examination, due to a 4-day

history of behavioural changes. The animal had been

increasingly unresponsive and spent progressively

longer periods of time on top of a heating unit. During

a routine clinical evaluation, the animal was found to

be thin in spite of a normal appetite and an otherwise

uneventful clinical history. A large abdominal mass

(about 4 cm in diameter) was detected in routine

abdominal palpation. Blood was collected for CBC

(complete blood count) and biochemical analysis,

which revealed anemia and mild leucocytosis with

monocytosis and lymphocytosis, along with elevated

billirrubin levels. Other complementary exams,

namely ultrasound, did not reveal the origin of the

abdominal mass but justified a strong suspicion of

aortic involvement.

Exploratory laparotomy revealed the aortic mass to

be impossible to remove and intra-surgical euthanasia

was decided upon.

The necropsy was carried out at the zoo’s medical

facilities and organ samples were sent to the

Laboratory of Pathological Anatomy and to the

Laboratory of Parasitology of the FMV/UTL, for

histopathological and parasitological analysis.

Immunostaining was performed for specific

myeloid and lymphoid markers, using the following

antibodies: CD3 (Dako, polyclonal), Pax5

(Novocastra, monoclonal), Macrophages/Monocytes

(AbDSerotec, monoclonal), Myeloperoxidase (Dako,

polyclonal), CD10 (Zymed, monoclonal), CD20cy

(Dako, monoclonal), light λ-chains (Dako, polyclonal)

and CD117 (Dako, polyclonal).

A commercial labelling system was used with the

NovolinkTM Polymer (Novocastra).

3. Results

At necropsy, the 4 cm abdominal mass was shown

to envelope a section of the abdominal aorta. All other

organs appeared unchanged, except for the liver which

exhibited a pale nodule and several scattered

yellowish spots, and the spleen which appeared

diffusely enlarged.

Histopathology revealed the aortic mass to be a

collection of fibrinous exudate arranged in layers and

enveloped by a congestive fibrous capsule (Fig. 1).

Histochemical staining with PAS (periodic acid-Schiff

stain) revealed several organisms consistent with

yeasts, sometimes arranged in pseudohyphae, within

the mass.

The pale liver nodule corresponded to infiltration of

the hepatic parenchyma by hematopoietic precursor

cells with disruption of the normal hepatic architecture,

while the yellowish spots were found to be

granulomas surrounding nematode eggs.

Parasitological analysis of liver samples identified the

eggs to be from Calodium hepaticum. Adult forms of

the nematode were also visible in histological liver

sections. PAS staining revealed microorganisms

similar to those present in the aortic mass in the serosa

lining the liver, spleen, kidney and intestine.

The splenomegaly was due to infiltration by

hematopoietic precursor cells similar (although

less extensive) to that observed in the liver, along

with moderate hyperplasia of the white splenic

pulp.

Immunostaining for specific myeloid and lymphoid

Extramedullary Hematopoiesis Mimicking a Neoplasm in a Goeldi’s Monkey (Callimico goeldii)

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Fig. 1 Photomicrographs of necropsy samples (Hematoxylin & Eosin). (A) and (B) Liver sections exhibiting marked infiltration by hematopoietic cells, among which megakaryocytes are the most flagrant example (*). Calodium hepaticum eggs

can also be identified (arrow) (40 and 100); (C) Spleen section with multifocal infiltration by hematopoietic cells (40); (D)

External surface of the thick fibrous capsule surrounding the aortic mass, with focal calcification and infiltration by

inflammatory cells (40), Inset: Several yeasts within the fibrinous exudate of the aortic mass (PAS, 400).

markers revealed that the cellular population in the

liver and spleen was heterogenous in nature and

compatible with a diagnosis of hepatosplenic

extramedullary hematopoiesis. Table 1 summarizes

the results obtained upon immunostaining for both the

lymphoid and myeloid markers mentioned, namely in

what concerns the intensity of marker expression

(reflecting percentage of cells marked and intensity of

staining) and the pattern of distribution of positive

cells within both hepatic and splenic parenchyma.

Figs. 2-6 illustrate areas of stained tissues

representative of the overall positive cell distribution

for each marker in the liver and spleen. As it can be

estimated from both images and table, the positively

marked cells are distributed through the affected aeas

of the hepatic parenchyma in a diffuse manner,

without any discernible pattern. Most of the cells

expressed lymphoid (CD3, CD20 and Pax5) and

macrophage/granulocyte (macrophage and

myeloperoxidase) surface markers, with no apparent

expression of neural crest/mastocyte markers (CD117).

Infiltrating positively marked cells exhibited a

different pattern of organization in the spleen, with the

lymphoid cells arranging themselves in a follicular

pattern similar to that usually seen in a healthy organ.

All other cells expressing the remaining markers

tested were found diffusely distributed through the

splenic parenchyma. Contrarily to the cellular

population infiltrating the liver, cells expressing

macrophage/granulocyte markers were less frequently

observed in the spleen. Again, no CD117-positive

cells were present.

Both kidneys exhibited multifocal interstitial

lymphocitic infiltrates and the presence of intratubular

hyaline cylinders, while the intestine showed MALT

hyperplasia.

Extramedullary Hematopoiesis Mimicking a Neoplasm in a Goeldi’s Monkey (Callimico goeldii)

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Fig. 2 Immunohistochemical staining of liver and spleen sections (NovolinkTM polymer, Mayer’s hematoxylin). (A) Liver,

CD3 marker (100); (B) Spleen, CD3 marker (100); (C) Liver, CD10 marker (100); (D) Spleen, CD10 marker (100).

Fig. 3 Immunohistochemical staining of liver and spleen sections (NovolinkTM polymer, Mayer’s hematoxylin). (A)

Liver, CD20 marker (100); (B) Spleen, CD20 marker (100); (C) Liver, CD117 marker (100); (D) Spleen, CD117

marker (100).

Extramedullary Hematopoiesis Mimicking a Neoplasm in a Goeldi’s Monkey (Callimico goeldii)

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Fig. 4 Immunohistochemical staining of liver and spleen sections (NovolinkTM polymer, Mayer’s hematoxylin). (A) Liver,

Lambda chains marker (100); (B) Spleen, Lambda chains marker (100); (C) Liver, Macrophage marker (100); (D) Spleen,

Macrophage marker (100).

Fig. 5 Immunohistochemical staining of liver and spleen sections (NovolinkTM polymer, Mayer’s hematoxylin). (A) Liver,

Myeloperoxidase marker (100); (B) Spleen, Myeloperoxidase marker (100); (C) Liver, Pax5 marker (100); (D) Spleen,

Pax5 marker (100).

Extramedullary Hematopoiesis Mimicking a Neoplasm in a Goeldi’s Monkey (Callimico goeldii)

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Fig. 6 Expression of the different immunohistochemical markers along the chain of hematopoietic differentiation [10, 11].

BFU-E: Burst Forming Unit-Erythroid Precursor; CFU: Colony Forming Unit; E: Erythroid; Eo: Eosinophil; G: Granulocyte; GEMM: Granulocyte, Erythrocyte, Monocyte, Megakaryocyte; GM: Granulocyte, Monocyte.

Table 1 Intensity and pattern of immunostaining of the infiltrating hematopoietic precursor cells in the liver and spleen.

Immunohistochemical marker Liver Spleen

Intensity Pattern Intensity Pattern

CD3 +++ Diffuse + Perivascular

CD10 + Diffuse +/- Diffuse

CD20 ++ Diffuse ++ Follicular

CD117 - - - -

Light-λ chains + Diffuse ++ Diffuse

Macrophage ++++ Diffuse + Diffuse

Myeloperoxidase +++ Diffuse + Diffuse

Pax5 ++ Diffuse ++ Follicular

4. Discussion

Extramedullary hematopoiesis results when there is

hormonal induction for increased cell production and

pluripotential hematopoietic stem cells available.

These cells normally circulate in very low numbers

and, in preparation for extramedullary hematopoiesis,

they return to embryonic sites of colonization, sparing

the germinal centers and periarteriolar lymphoid

sheaths [12].

In general, three situations underlie the abnormal,

extramedullary proliferation of normal hematopoietic

elements: filtration, where immature cells are trapped

by the spleen or other sites and proliferate; inadequate

marrow space to produce appropriate numbers of

marrow elements or damage to the bone marrow

microenvironment leading to increased numbers of

circulating hematopoietic stem cells; and abnormal

cytokine or other circulating hematopoietic growth

factors causing stem cell differentiation to

hematopoietic cells or local effects simulating the

marrow microenvironment. The third cause may occur

independently as a result of hematopoietic growth

factors production by a tumor or at sites of tissue

Extramedullary Hematopoiesis Mimicking a Neoplasm in a Goeldi’s Monkey (Callimico goeldii)

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damage and repair [1, 8]. In human adults, hepatic

EMH is seen in a variety of conditions, including

hematologic disorders, sepsis, transplantation, massive

hepatic necrosis, but is also notably prominent within

hepatoblastomas, hepatic adenomas and hepatocellular

carcinoma. The cause of these proliferations is not

well understood, and may be due to local hypoxic

conditions, local effects of tumors that simulate the

embryonic liver and are supportive of hematopoietic

development, or production of growth factors that

attract and/or support hematopoiesis [8]. When

anemia is excessively prolonged and severe, there may

be some degree of extramedullary hematopoiesis by

the liver and spleen [13].

The organs of the reticuloendothelial system, like

the liver, spleen and lymph nodes are the preferential

target organs for EMH, while the most common site

involved by non hepatosplenic EMH in humans seems

to be in or surrounding the vertebral column [4, 5].

Other preferential sites include the inguinal,

para-aortic, cervical and paratracheal lymph nodes;

the retroperitoneum; the lungs, pleura or both; the

genitourinary system; the skin; the right thalamus, the

right atrium of the heart; the oral mucosa and the

rectus femoris muscle, in that order [5].

Extramedullary hematopoiesis is characteristically

trilineage [12]. In the liver, it can appear as

focal aggregates of myeloid cells in the perisinusoidal

compartment as this area retains its embryonic

hospitality for EMH throughout the animal’s

life [12-14].

Megakaryocytes are the most obvious

hematopoietic precursor and characteristically lie

adjacent to the smooth muscle splenic trabeculae

when stimulation is mild, but may become diffusely

distributed in the sinus areas when stimuli are

pronounced and prolonged [12].

In the case here described, extramedullary

hematopoiesis was restricted to the liver and spleen. It

was not possible, in this case, to determine the

primary cause for the aortic mass.

EMH is a frequent finding in human patients

undergoing liver transplantation for massive hepatic

necrosis. This may also be a consequence of the

anaemia associated with this condition. Alternatively,

there may be a possibility that intrahepatic

hematopoiesis is linked with hepatopoiesis [15]. In

this monkey’s case, although impossible to prove, it is

justified to theorize that the chronic and diffuse

hepatic aggression caused by the presence of the

numerous Calodium hepaticum granulomas (along

with all secondary effects associated to the infection),

in conjunction with the exuberant response to the

aortic mycotic infection, may have been the primary

stimulus for the development of EMH, possibly as an

organic compensatory mechanism. The lesions of

chronic interstitial nephritis would most likely have

contributed to the overall metabolic state of

dysfunction. Although most reported cases of

Calodium hepaticum infection in primates do not

mention extramedullary hematopoiesis, it is not

unreasonable to assume that these two conditions

might coexist in sporadic cases, especially if adjuvant

lesions are also present.

Clinically, non hepatosplenic-EMH may present as

an incidental finding or with a symptomatic disease or

condition, including pleural effusion, ascites,

neurologic deficit, cardiac tamponade, chronic renal

failure, acute respiratory failure, orbital proptosis and

subglottic stenosis. Antemortem diagnosis can be

made by tissue biopsy, FNA (fine needle aspiration)

biopsy, or radionuclide scanning [5].

Similarly, in the case here described all of the

animal’s clinical signs (progressive unresponsiveness

and weight loss without loss of appetite) and

complementary exam results (anemia and mild

leucocytosis with monocytosis and lymphocytosis,

and elevated bilirubin levels) were not indicative of

extramedullary hematopoiesis, but could easily be

attributed to the on-going concurrent conditions later

detected during necropsy and confirmed by

histopathological analysis.

Extramedullary Hematopoiesis Mimicking a Neoplasm in a Goeldi’s Monkey (Callimico goeldii)

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5. Conclusion

In humans, EMH often resembles more common

conditions such as benign or malignant

lymphadenopathy or a metastasis on radiographic

studies [3]. This report describes a case of

hepatosplenic extramedullary hematopoiesis

mimicking a metastatic paraaortic neoplasm with

hepatic and splenic involvement in a Goeldi’s monkey,

with particular emphasis on the immunohistochemical

phenotype of the cells involved in this condition. No

other published reports similar cases in these animals

were found up to the completion of this study

suggesting this might be the first of that kind.

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