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CYTOLOGICAL ASSESSMENT OF LEUKOCYTES IN


REPTILES

Article · January 2013

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Albert Martinez Silvestre


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CYTOLOGICAL ASSESSMENT OF LEUKOCYTES IN REPTILES

A. Martínez Silvestre
DVM, MSc, PhD
Dipl ECZM (Herpetology)
Acred AVEPA (Animales Exoticos)
CRARC (Centro de Recuperacion de Anfibios y Reptiles de Catalunya) 08783 Masquefa,
Barcelona.

In reptiles practice, the information the blood smears provide on the cell appearance is
sometimes more useful than that of the cell types distribution. Used to counts, we rely on the total
or percentage number of each leukocyte category to issue a diagnostic (Martínez-Silvestre et al.
2011). However, the appearance of such leucocytes will provide probable diagnostics of various
diseases and even prognosis value. Counting 45% of heterophils does not result in the same
diagnostic than counting 45% of heterophils among which 80% have toxic granulation.

The sample analyzed must always be well extracted, without anticoagulants and stained; should
these conditions were not fulfilled, false positive results could occur due to diffusion artefacts, of
false negative results due to contact with inhibitor agents (various anticoagulant agents) or due to
aged preparations.

In hematologic cytology, it is better to interpret results of positive staining rather than the lack of
staining. When staining data are interpreted, the staining pattern (focal o discretely localized or
diffuse) and the staining extension in the sample must be considered, rather than just the staining
intensity. The species factor is also very important, since reactions observable in a species
should not be automatically extrapolated to other (Javanbkht et al. 2013).

Therefore, the main cytological variations and their diagnostic involvement in reptiles’ white blood
cells are next shown. All of them have been described using Diff Quick or May Grunwald Giemsa
staining. The special staining serve to outline some specific facts, but since they are not used
daily in the practice, we will mention them only if necessary.

The staining used in cytochemistry allows identifying the normal cell types and identifying what
line do the blast cells belong to, scarcely differentiated. This helps classifying the hematopoietic
neoplasm, so that the diagnostic can be improved and, sometimes, the initial morphologic
interpretation changed. The neoplastic cells contain the same enzymes or cell products than
those found in the cells of a health animal. However, because of the change in the development
of the neoplastic and dysplastic cells, and due to the low incidence of neoplasms in reptiles,
diagnostic irregularities may occasionally occur.
No matter the staining used, the blood smear must be performed immediately after the extraction
in order to avoid the presence of artefacts such as changes in leukocytes or thrombocytes
morphology, changes in staining properties of leukocytes, vacuolization of monocytes or
increased size of lymphocytes (Strik et al. 2007).

General variations of leukocytes:

Such variations use to be due to the use of certain anticoagulant agents. Thus, heparin causes
leukocytes aggregates that impact on their morphology. EDTA causes erythrocytes lyses and
changes in some labile leukocytes, such as heterophils. Citrate causes crystallization of
haemoglobin, which in turn may result in changes of both the erythrocyte (erythrolysis) and the
leukocytes close to the affected one.

The idiosyncrasy of reptiles regarding the blood smear also makes us to be cautious in the
diagnostics. Thus, the atavistic immune system of such species makes them to respond with high
mitotic activity even in absence of neoplastic diseases. The observation of mitosis in peripheral
blood is a physiologic finding. Only when they are present in high number we can consider a
pathologic condition, but in this case we must consider a chronic infectious condition, and even
parasitic, apart from neoplastic.

White blood cells degranulation is always associated to artefacts or the smear processing;
therefore, it should not be used as diagnostic sign.

Immature leukocytes show in general basophilic cytoplasm and variable segmentation of the
nucleus, depending on the cell type. Usually, they are bigger than the mature leukocyte.

In general, left-deviation should be seen as a sign of severe unbalance between cell production
and demand, so that circulating blood shows immature leukocyte forms (Pendl 2013).

Cell specific variations:

Heterophils

Functionally, this is the cell equivalent to mammals’ neutrophils and shows high morphologic
variation according to the reptile species. These cells are round and large, and although the
number and shape of granules varies with the species, in general they have spindle shaped
refractive cytoplasm granules, which stain deep orange with Romanowsky type stainings.
Opaque and refractive granules may appear in one single cell. The nucleus is eccentric, round to
oval in shape, with densely aggregated chromatin. Some species, such as lizards and iguanas,
have a lobed nucleus in their heterophils. The size varies with the species (10 – 23 μm).
Interpretation of variations:

Heterophils are mainly phagocytic cells and, therefore, significant increases in their appearance and
morphology are associated to inflammatory, especially infectious diseases or conditions involving tissue
damage. Non-inflammatory causes of heterophilia are stress (Davis et al. 2008) (because of excess
glucocorticoids) and the presence of neoplasies.
Circulating blood may show abnormal heterophils, among other immature heterophils and toxic cells. The
former (usually myelocytes and metamyelocytes) appear in blood of animals suffering from certain
diseases that cause excessive peripheral use of the mature heterophils. Immature heterophils show higher
degree of cytoplasmic basophilia, non-lobed nucleus, lower number of specific granules than mature cells
and, sometimes, immature granules (primary granules). Their presence in blood with heterophilia suggests
inflammatory diseases. The presence of left-deviation together with heterogeneous profile suggests over
demand because of an inflammatory response, probably associated to infectious aetiology.
The toxic heterophil is the one that fulfils the following conditions (Campbell 2012):
Increased cytoplasmic basophilia, appearance of abnormal granulation (deep blue or purple granules which
shape and response to staining are abnormal), cytoplasmic vacuolization.
The presence of toxic heterophils suggests inflammatory disease associated to the presence of infectious
agents. Depending on the degree of toxicity, they are classified into a +1 al + 4 score: +1 means the
presence of a cell with increased cytoplasmic basophilia; +2 means +1 plus slight abnormal granulation
(partial degranulation, granules which trend to fuse or abnormal granules) or vacuolization; +3 means
changes in the cell worse than the previous ones, and the nucleus may show slight karyorrhexis or
karyolysis; finally, +4 means very outlined changes both in the nucleus and in the cytoplasm. The nuclear
lobulation in reptile species that usually do not show it means severe inflammation.

In squamata and crocodiles, degranulated heterophils may be seen. It may be due to artefacts
during the sample handling, prolonged storage in anticoagulant, inadequate fixing or normal “in
vivo” aging of the cells.

Finding nuclear lobulation in species that do not show them physiologically (e.g. tortoises) may
suggest cell reaction against inflammation or parasitism (Wilkinson 2004).

Eosinophils
Reptiles’ eosinophils are large, round cells with spherical eosinophilic cytoplasm granules. Some
species, such as the green iguana show blue and even amber cytoplasmatic granules. The
nucleus shows variable shape, ranging from round to slightly elongated; it can be lobed in some
reptiles species. The size varies depending on the species; thus, snipes have the largest
eosinophils and lizards, the smallest ones (Strik et al. 2007).

Interpretation of variations:

Eosinophils may be associated to parasitic infections and stimulation of the immune system,
although cytology changes are rarely observed. In cases of chronic infections, trend to
degranulation can be observed in some species of tortoises.

Nuclear lobulations in species that have not them physiologically (e.g. tortoises) may suggest cell
reaction against inflammation of parasitism.
Basophils
Basophils are usually round, small cells containing a variable number of basophilic metachromatic
cytoplasmatic granules, which often mask the nucleus. When the nucleus is visible, it can be observed that
has not lobules. Lizards have small basophils and chelonian and crocodiles have large basophils.

Interpretation of variations:

Some reptiles show very high numbers of basophils, such as the iguana Cyclura or the tortoise Chelydra,
which allows seeing variations associated to the disease.
Their function is probably similar to the one of basophils in mammals. The appearance and staining
properties of basophils in reptiles no do seem to vary according to the seasonal changes, unlike other
granulocytes. Although and increased number is associated to the presence of parasitic infections and viral
infections, degranulations are rarely seen in diseased animals. In fact, basophils with unstained, clear
granules are often seen in some health tortoises, which diagnostic significance is unknown.

Monocytes
Monocytes are the largest leukocytes in peripheral blood. The cell morphology ranges from round to
amoeboid, and their nucleus is polymorphic (round, oval, lobed, kidney-shaped). The nucleus chromatin is
less condensed and stains paler than nuclear chromatin of lymphocytes. The cytoplasm of this cell turns
greyish-blue and may contain eosinophilic vacuoles or granules similar to powder particles, or azurophil.

Interpretation of variations:

The presence of monocytosis suggests inflammatory disease, particularly granulomatose


inflammation.

Lizards parasited with Karyolysus and snipes parasited with Hepatozoon have shown significant increased
monocytes (azurophil), probably associated to inflammatory response against parasites.

Often, monocytes in peripheral blood show phagocytic activity. Erythro and leukophagocytosis by
these cells may be associated to anaemia and presence of infectious diseases (Campbell 2012).

Diseases of advanced septic origin and/or terminal diseases cause high cell reaction, and
abundant mitoses may be observed in peripheral blood. In such cases, the cell appearance is not
easy to distinguish from the reactive lymphocyte.

Lymphocytes

Lymphocytes are round cells with scarce, moderately to weakly basophil cytoplasm, and their
nucleus shows also circular morphology, places centrally and has densely aggregated chromatin.
The cytoplasm is homogeneous and in general lacks vacuoles and granules, although in some
azurophil cytoplasmic granules may be found in some lymphocytes. They trend to “adjust” around
adjacent cells in the blood smear. They may show pseudopodia in the cell periphery.

Many health reptile species show higher count of lymphocytes than heterophils.

Interpretation of variations:
Reptiles have two main types of lymphocytes (B and T) involved in the immune system, but unlike
mammals and birds, which are homoeothermic, the immune responses of poikilothermic reptiles
are very influenced by the environment; thus, low temperatures may suppress or inhibit the
immune response.

Reactive lymphocytes are more basophilic, show irregular cell edges, increased size and even
some phagocytic activity. Reactive lymphocytosis can be associated to wounds heal,
inflammatory and parasitic and viral infectious diseases. In general, their presence suggests
stimulation of the immune system due to the presence of systemic antigens (kidney disease or
gout may stimulate the presence of such lymphocytes. Abundant reactive lymphocytes are
observed in the Inclusion Bodies Diseases (IBD) in snipes.

Sometimes, inclusion bodies are seen even intralymphocitic. These represent viral diseases,
such as herpesvirus of tortoises (Martínez-Silvestre et al. 2001). Some viral diseases also show
intraerythrocytic inclusion bodies (Davis & Holcomb 2008).

In advanced infectious septic diseases, lymphocytes use to appear polymorphic and with
abundant mitoses. Sometimes, nucleoli are seen and are poorly distinguished from monocytes. In
such cases, the appearance is not pathognomonic of neoplasia but of terminal severe reaction (of
infectious (e.g. terminal salmonella) or neoplastic origin).

The presence of plasmatic cells or lymphocytes precursors suggests septic disease usually of
infectious origin and chronic. Sometimes, the patient does not show symptoms prior to detection
of such cell types.

Sometimes it is not easy to morphologically distinguish between reactive lymphocytes and


activated thrombocytes (which could even perform phagocytosis) in reptiles; therefore, PAS,
Peroxidase or acid phosphatase stainings can be used to distinguish between thrombocytes
(usually PAS positive) and lymphocytes (PAS negative). However, some studies conducted with
lizards reported negative thrombocytes to this staining, and therefore idiosyncrasy of each
species must always be considered (Martínez-Silvestre et al. 2004).

Azurophils
This is an irregular shaped cell, slightly smaller than monocytes. The nucleus is not segmented and ranges
from round to irregular oval. The cytoplasm is basophil and darker than that of the monocytes, lavender
blue in colour. This cytoplasm contains low number of azurophil matt granules, of several sizes. This cell is
commoner in the suborder of snipes than in lizards of crocodilians and, therefore, it can be seen just
occasionally in chelonians.

Interpretation of variations:

Vacuolization and phagocyted material can be found in the cytoplasm of these cells. These are
phagocytic cells similar in shape to the monocytes, and able to unchain important oxidative
damage, similar to that of mammals’ neutrophils. Thus, variations in the appearance of these
cells are not yet well known. It has been suggested that they would be similar to those described
for heterophils and monocytes, being basically involved in unspecific inflammations, parasitism
and necrotic processes.
References

Campbell, T. 2012. Hematology of Reptiles. 277-297. In: Thrall, M.A., Weiser, G., Allison, A. &
Campbell, T. (eds.), Veterinary Hematology and Clinical Chemistry. John Wiley & Sons, Inc.

Davis, A.K., Maney, D.L.& Maerz, J.C. 2008. The use of leukocyte profiles to measure stress in
vertebrates: a review for ecologists. Functional Ecology, 22: 760-772.

Davis, A.K. & Holcomb, K.L. 2008. Intraerythrocytic inclusion bodies in painted turtles
(Chrysemys picta picta) with measurements of affected cells. Comparative Clinical Pathology,
17: 51-54.

Javanbkht, H., Vaissi, S.& Parto, P. 2013. The Morphological Characterization of the Blood Cells
in the Three Species of Turtle and Tortoise in Iran. Research in Zoology, 3: 38-44.

Martínez-Silvestre, A., Ramis, A., Majó, N., Soler Massana, J., Marschang, R.E.& Origgi, F.C.
2001. Analyse virale dans un cas de rhinite chronique chez une tortue mauresque (Testudo
graeca) en captivité. International Congress on Testudo Genus, 3: 29.

Martínez-Silvestre, A., Rodriguez-Dominguez, M.A., Mateo, J.A., Pastor, J., Marco, I., Lavín, S.&
Cuenca, R. 2004. Comparative haematology and blood chemistry of endangered lizards
(Gallotia species) in the Canary Islands. The Veterinary Record, 155: 266-269.

Martínez-Silvestre, A., Lavín, S.& Cuenca, R. 2011. Hematología y citología sanguínea en


reptiles. Clinica Veterinaria de Pequeños Animales, 31: 131-141.

Pendl, H. 2013. HEMATOLOGY IN BIRDS AND REPTILES FOR BEGINNERS. International


Conference on Avian, Herpetological & Exotic Mammal Medicine, 1: 59-63.

Strik, N.I., Alleman, A.R.& Harr, K.E. 2007. Circulating inflammatory cells. 167-218. In: Jacobson,
E.R. (eds.), Infectious Diseases and Pathology of Reptiles: Color Atlas and Text. Cabo Raton,
Florida. CRC Press.

Wilkinson, R. 2004. Clinical Pathology. 141-187. In: McArthur, S., Wilkinson, R. & Meyer, J.
(eds.), Medicine and Surgery of Tortoises and Turtles. Iowa. Blackwell Publishing Ltd.

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