Microscopic studies on characterization of blood … · Microscopic studies on characterization of blood cells of endangered sea turtles J. Orós, A. B. Casal, and A. Arencibia Department

Microscopic studies on characterization of blood cells of endangered sea

turtles

J. Orós, A. B. Casal, and A. Arencibia

Department of Morphology, Veterinary Faculty, University of Las Palmas de Gran Canaria, Trasmontaña s/n, 35416

Arucas (Las Palmas), Spain

Because all species of sea turtles are included on the Red List of the World Conservation Union, the efforts to conserve sea

turtles, the advances in their medical management, and the studies on physiological parameters have increased in recent

years. The classification of blood cells in reptiles is inconsistent because variable criteria have been used to categorize

cells or because cellular lineages are uncertain. However, the classification of blood cells from sea turtles is very important

because these species are intensively scrutinized in captivity in clinical settings and in the wild during research projects.

Characterization of blood cells in reptiles is mainly based on microscopic studies including cytochemical stains,

morphometric studies, and ultrastructural characterization. Using as model a morphologic study of blood cells of

loggerhead sea turtles (Caretta caretta) we review the three aspects included in the study of characterization.

Cytochemical stains including benzidine peroxidase, chloroacetate esterase, alpha-naphthyl butyrate esterase (with and

without sodium fluoride), acid phosphatase (with and without tartaric acid), Sudan black B, periodic acid-Schiff, and

toluidine blue were used to identify six types of white blood cells: heterophils, eosinophils, basophils, lymphocytes,

monocytes and thrombocytes. Morphometric characterization using an image analysis program was also performed to

obtain the maximum length, minimum length, area, and perimeter for the cells and the nuclei. Ultrastructural

characteristics of blood cells were obtained using transmission electron microscopy. This triple approach permits an

adequate characterization of blood cells in reptiles and emphasises the usefulness of the microscopic studies in sea turtle

conservation.

Keywords blood cells; cytochemistry; sea turtle; transmission electron microscopy

1. Introduction

Two families and seven species of sea turtles are currently recognised [1]. The family Cheloniidae includes the green

turtle (Chelonia mydas), loggerhead (Caretta caretta), hawksbill (Eretmochelys imbricata), Kemp’s ridley

(Lepidochelys kempi), olive ridley (Lepidochelys olivacea), and flatback turtle (Natator depressus). The family

Dermochelyidae includes only the leatherback (Dermochelys coriacea). Because all species of sea turtles are included

on the Red List of the World Conservation Union [2] the research on anatomical, histological and physiological aspects

of sea turtles, the efforts to conserve sea turtles, and the advances in their medical management have increased in recent

years.

The classification of blood cells in reptiles is controversial because variable criteria have been used to categorize

cells or because cellular lineages are uncertain [3]. Descriptions of the morphologic characteristics of blood cells of sea

turtles are limited [3-6]. In addition, the classification of blood cells from loggerhead turtles (Caretta caretta) is

particularly important because this species is intensively scrutinized in captivity in clinical settings and in the wildness

during research projects.

Using as model a morphologic study of blood cells of loggerhead sea turtles (Caretta caretta) we review in this

chapter the three aspects included in the study: cytochemical characterization, morphometric characterization, and

ultrastructural characterization.

2. Reptilian blood cells

The blood of reptiles contains nucleated erythrocytes, heterophils, eosinophils, basophils, lymphocytes, monocytes, and

nucleated thrombocytes [7]. Mature erythrocytes of reptiles are permanently nucleated and blunt-ended ellipsoids [7].

The erythrocyte size for most reptiles range from a length x width of 14 x 8 µm to 23 x 14 µm [8]. The reptilian

erythrocyte has a centrally positioned oval to round nucleus. The nucleus has a dense coarse chromatin and the

cytoplasm is homogeneously eosinophilic. The cytoplasm stains orange-pink with Romanowsky stains.

Reptilian heterophils are generally round cells with eosinophilic fusiform cytoplasmic granules and clear cytoplasm.

Heterophils range between 10 and 23 µm in size [8]. The nucleus is typically round to oval and eccentric, with densely

clumped nuclear chromatin [9-13]. Some species of lizards have heterophils with lobed nuclei [8, 14]. The cytoplasmic

granules of reptilian heterophils are usually peroxidase negative, except for several species of snakes and lizards [9, 11,

15-17]. The peroxidase positive heterophils of green iguanas (Iguana iguana) suggest that these cells may have

bactericidal and oxidative properties similar to mammalian neutrophils [14].

Microscopy: Science, Technology, Applications and Education A. Méndez-Vilas and J. Díaz (Eds.)

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Reptilian eosinophils are large round cells with spherical eosinophilic cytoplasmic granules. The size varies with the

species, snakes having the largest eosinophils, and lizards having the smallest [8]. The nucleus is central and is variable

in shape. The cytoplasmic granules of eosinophils stain positive for benzidine peroxidase in some reptiles [11].

Reptilian basophils are small round cells that contain basophilic metachromatic cytoplasmic granules [7]. Basophils

vary in size but generally range between 7 and 20 µm [8]. Lizards tend to have small basophils, whereas turtles and

crocodiles have large basophils [8]. The cell nucleus is slightly eccentric in position and non-lobed.

Reptilian lymphocytes vary in size from small (5 to 10 µm) to large (15 µm) [8, 16]. They are round cells with a

round nucleus that is centrally positioned in the cell. Lymphocytes typically have a large nucleus to cytoplasm ratio, and

the nuclear chromatin is heavily clumped [7]. The typical small mature lymphocyte has scant slightly basophilic

cytoplasm. The cytoplasm of a normal lymphocyte is homogenous and lacks vacuoles and granules [7].

Reptilian monocytes are round or ameboid being generally the largest leukocytes of reptiles [7]. The nucleus is

variable in shape. The nuclear chromatin of monocytes is less condensed and stains relatively pale compared with the

nuclei of lymphocytes. The abundant cytoplasm of monocytes stains blue-gray, and may contain vacuoles or fine

eosinophilic or azurophilic granules. Although monocytes that have an azurophilic appearance to the cytoplasm are

often referred to as azurophils in the literature, their cytochemical and ultrastructural characteristics are often similar to

monocytes and therefore should be reported as monocytes rather than as a separate cell type [10, 14, 16, 17].

Reptilian thrombocytes are elliptical to fusiform nucleated cells. The centrally positioned nucleus has dense nuclear

chromatin that stains purple, and the cytoplasm is typically clear and may contain a few azurophilic granules. Activated

thrombocytes appear as clusters of cells with irregular cytoplasmic margins and vacuoles, and appear devoid of

cytoplasm when aggregated [7].

3. Cytochemical characterization

3.1 Sea turtles and samples preparation

Thirty-five juvenile loggerhead sea turtles were used in this study. The turtles had been rehabilitated in the Tafira

Wildlife Rehabilitation Centre (TWRC) (Las Palmas de Gran Canaria, Spain). Blood samples were obtained just before

the turtles were set free, when they were clinically normal and in good physical condition.

Two millilitres of blood were collected from the cervical sinus [18] in tubes without anticoagulant, using sterile

syringes and needles. Twenty blood smears for each turtle were prepared immediately and air-dried [19]. Two blood

smears of each turtle were stained with a quick Romanowsky-type stain, Diff Quick (DQ) (Everest, Barcelona, Spain).

The remaining 18 blood smears from each turtle were stained as follows: two blood smears from each turtle were

stained with each stain, except for the alpha-naphthyl butyrate esterase and acid phosphatase stains in which four blood

smears were stained. Stains included benzidine peroxidase (PER), chloroacetate esterase (CAE), alpha-naphthyl

butyrate esterase (NBE) (with and without sodium fluoride), acid phosphatase (ACP) (with and without tartaric acid),

Sudan black B (SBB), periodic acid-Schiff (PAS), and toluidine blue (TB) [20] using commercial kits (Sigma

Diagnostics, Sigma Aldrich Química S. A., Spain). Normal human blood smears were used as controls.

3.2 Benzidine peroxidase test

The commercial test uses the diaminobenzidine (DAB) as a benzidine substitute for peroxidase (myeloperoxidase)

cytochemistry. Historically, DAB has been much less attractive to histochemists because DAB has less tinctorial power

than benzidine [21]. However, DAB methodology was improved, making it more suitable for differentiating human

granulocytes, their precursors and monocytes from cells of lymphoid origin [22, 23]. According to their modification,

the brown reaction product is first intensified with copper salts followed by application of Gill’s modified Papanicolaou

stain, resulting in intense grey-black granules at sites of human neutrophil and monocyte myeloperoxidase [24].

The procedure used involves the following reactions:

DAB + H2O2 Oxidized DAB (light brown pigment)

Oxidized DAB + Cu(NO3)2 grey-black pigment

3.3 Naphthol AS-D chloroacetate esterase test

Cellular esterases are ubiquitous and appear to represent a series of different enzymes acting upon select substrates.

Under defined reaction conditions, it may be possible to determine hemopoietic cell types, using specific esterase

substrates. The described methods provide means to distinguish granulocytes from monocytes [25, 26]. To perform the

test, blood films are incubated with naphthol AS-D chloroacetate in the presence of a stable diazonium salt. Enzymatic


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hydrolysis of ester linkages liberates free naphthol compounds. These couple with the diazonium salt, forming highly

coloured deposits at the sites of enzyme activity.

3.4 Alpha-naphthyl butyrate esterase test

Nonspecific esterases hydrolyze synthetic ester compounds, liberating free naphthol compounds that couple with the

diazonium salt, forming insoluble coloured deposits at the sites of enzyme activity [27]. Alpha-naphthyl butyrate

esterase is found primarily in human blood cells of monocytic lineage, but lymphocytes and some mature granulocytes

also can show occasional positivity. To differentiate these cells conclusively from monocytes, sodium fluoride is

incorporated with the incubation system. The monocyte enzyme is inactivated in the presence of this compound.

3.5 Acid phosphatase test

Use of substituted naphthol AS phosphates in conjunction with diazonium salts for detection of acid phosphatase in

human leukocytes was reported in 1962 [28]. We use naphthol AS-BI phosphate and freshly diazotized fast garnet GBC

salt because this compound couples rapidly at acid pH, forming highly insoluble dye deposits. Most procedures employ

stable diazonium salts formed by reacting an arylamine with sodium nitrite in an acid medium [29]. The resulting

diazonium, usually unstable, can be treated with compounds such as zinc chloride, zinc sulphate or naphthalene-1,6-

disulfonate, forming stable salts. These stabilizers may exert marked inhibition upon some enzymatic systems, whereas

the diazonium chlorides are less inhibitory [29]. In the procedure, blood films are incubated in a solution containing

naphthol AS-BI phosphoric acid and freshly diazotized fast garnet GBC. Duplicate films may be treated with a solution

containing L(+)-tartrate. Naphthol AS-BI, released by enzymatic hydrolysis, couples with fast garnet GBC forming

insoluble brown dye deposits at sites of activity. Cells containing tartaric acid-sensitive acid phosphatase are devoid of

activity. Those mononuclear cells containing tartaric acid-resistant phosphatase are not affected by such treatment.

3.6 Sudan black B

The most sensitive and versatile of all these stains is Sudan black B [30]. Several lipids including phospholipids, neutral

fats and sterols are stained intensely by Sudan black B. Two distinct fractions were demonstrated in this dye using

chromatography and infrared spectroscopy: the first fraction stains neutral fats blue-black, whilst the second fraction

colours phospholipids grey [30].

The reaction of human neutrophil granules with the dye was described in 1947 [31]. The leukocyte Sudan black B

staining pattern usually parallels that of myeloperoxidase [32]. Cells committed along lymphoid pathways display

negative stain, whereas myeloid and monocytoid forms display characteristic positive reactions.

Previous methods utilized formaldehyde vapour fixation of blood films [32]. That technique may result in cell loss

and staining artifacts. The procedure used in our study utilizes a buffered glutaraldehyde fixative and shortened

incubation time that results in excellent staining without cellular loss or distortion [33].

3.7 Periodic acid Schiff

The PAS reaction is a useful indicator of the presence of tissue carbohydrates, and particularly so for glycogen when the

technique incorporates a diastase digestion stage [34]. The principle of the reaction is that periodic acid will bring about

oxidative cleavage of the carbon-to-carbon bond in 1.2-glycols or their amino or alkylamino derivatives, to form

dialdehydes [34]. These aldehydes will react with fuchsin-sulphurous acid, which combines with the basic

pararosaniline to form a magenta-coloured compound being alkyl sulphonate in type.

When this reaction is performed on blood films, glycols treated with periodic acid are oxidized to aldehydes. After

reaction with Schiff’s reagent (a mixture of pararosaniline and sodium metabisulfite), a pararosaniline adduct is released

that stains the glycol-containing cellular components [34].

3.8 Toluidine blue

Toluidine blue stain permits identify the metachromatic cytoplasmic granules of the basophils. The metachromasia can

be explained because tissue structures known as “chromotropes” carry acidic groups with a minimum surface density of

not more than 0.5 nm between adjacent negatively-charged groups. These chromatropes react with the metachromatic

dyes to produce a colour which is different to that normally exhibited by the dye. The dyes exist in a normal monomeric

(orthochromatic) form, and a potential polymeric (metachromatic) form when the negative charges of the chromotrope

attract sufficient positively-charged polar groups on the dye for aggregates to form and polymerise [34].

3.9 Cytochemical results

Erythrocytes stained with DQ were oval with a pale pink cytoplasm and a violet-blue oval or round nucleus. Some

erythrocytes had small basophilic intracytoplasmic inclusions. Erythrocytes did not take up any cytochemical stain

(Table 1).


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Heterophils were big and round cells with a dense, oval, strongly purple, often eccentric nucleus, which contained

clumped chromatin. When the nucleus was centrally located, it acquired a round form. The abundant cytoplasm showed

weak eosinophilia, containing numerous fusiform granules with the same coloration as that of the cytoplasm, as a result

of which, in most cases, heterophil granules were not seen easily. Heterophils were stained with ACP (with and without

tartrate), PER, CAE and SBB; and they were moderately stained with PAS (Table 1).

Eosinophils were round, with a generally eccentric, oval or round, strongly purple nucleus, which contained clumped

chromatin. The abundant cytoplasm was weakly basophilic, and contained scarce or a moderate number of round,

approximately 1 µm in diameter, well-defined eosinophilic granules. These granules were stained with ACP (with and

without tartrate), NBE (with sodium fluoride), CAE and PAS. They were also moderately stained with SBB (Table 1).

Table 1 Cytochemical staining characterization of blood cells of juvenile loggerhead turtles.

BLOOD CELL CYTOCHEMICAL STAINS

ACP* PER NBE** CAE SBB PAS TB

T NT F NF

Erythrocyte - - - - - - - - -

Heterophil + + + - - + + ± -

Eosinophil + + - + - + ± + -

Basophil - - - - - - - - +

Lymphocyte - - - - ± - - - -

Monocyte - - - - ± - - - -

Thrombocyte - - - - - - - + -

ACP: acid phosphatase; PER: benzidine peroxidase; NBE: alpha-naphthyl butyrate esterase; CAE: chloroacetate esterase; SBB: Sudan black B; PAS: periodic acid-Schiff; TB: toluidine blue

*Two ACP stains were made: with tartaric acid (T) and without tartaric acid (NT)

**Two NBE stains were made: with sodium fluoride (F) and without sodium fluoride (NF) +: positive; -: negative; ±: moderately positive

Basophils were difficult to find in the blood smears of loggerhead turtles. These cells were round, and contained a

dense, violet-blue, generally eccentric nucleus, which presented a clumped chromatin pattern. The cytoplasm contained

numerous big basophilic granules that often masked the nucleus. Basophil granules stained only with TB (Table 1).

Lymphocytes were small and round with a well-defined round purple-blue nucleus that contained prominently

clumped chromatin. The nucleus was surrounded by a rim of moderately granular basophilic cytoplasm. Lymphocytes

from loggerhead turtles stained only moderately with NBE (without sodium fluoride) (Table 1).

Monocytes were round or amoeboid with a purple-blue, generally oval, kidney-like form or fusiform, eccentric

location nucleus with a chromatin pattern slightly less clumped than that of the lymphocyte. The cytoplasm was weak to

moderately basophilic, sometimes containing variably sized intracytoplasmic vacuoles. Azurophilic granules were not

seen. Some monocytes had pseudopods. Monocytes were only moderately stained with NBE without fluoride (Table 1).

Thrombocytes were typically oval-shaped cells, although sometimes we observed round thrombocytes. The nucleus

was generally oval, strong violet-blue and with a clumped chromatin. The scant cytoplasm was seen accumulated in the

two poles of the cell when the thrombocyte presented an oval morphology; or a slim cytoplasmic halo around the

nucleus was seen when the cell acquired a round form. In all the cases, the cytoplasm presented a very pale coloration,

almost transparent, a characteristic that helped to distinguish this cell from the lymphocytes. Other important

characteristics of thrombocytes were their tendency to aggregate in the blood smears. Thrombocytes stained only with

PAS (Table 1).


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Fig. 1 (A) Heterophil of a loggerhead turtle showing positive acid phosphatase (without tartaric acid) stain. Scale bar = 7.3 µm. (B)

Eosinophil of a loggerhead turtle showing positive chloroacetate esterase stain. Scale bar = 4.7 µm. (C) Heterophil (arrow) and

eosinophil (arrowhead) of a loggerhead turtle stained with benzidine peroxidase stain. Scale bar = 7 µm. (D) A thrombocyte of a

loggerhead turtle showing intracytoplasmic positive cytochemical periodic acid-Schiff stain. Scale bar = 4.5 µm.

4. Morphometric characterization

4.1 Methodology

Two blood smears of each turtle (thirty-five juvenile loggerhead sea turtles) were stained with a quick Romanowsky-

type stain, Diff Quick (DQ) (Everest, Barcelona, Spain), according to the manufacturer’s instructions for differential

leukocyte count. Twenty erythrocytes and fifteen leukocytes from each turtle were measured using an image analysis

program, Image-Pro Plus, Version 4.1 (Media Cybernetics) obtaining the maximum length, minimum length, area, and

perimeter for the cells and the nuclei.

A statistical analysis of the cell dimensions based on the Student’s t test was made using the program SPSS 11.0 for

Windows, obtaining the following data: mean, standard deviation and range.

4.2 Image analysis procedures

The stages in obtaining measurements from an image analysis system are: image input, digitisation and display, image

processing, and image analysis [35]. Images for digital image analysis are most usually taken from a camera mounted

on a microscope. The video image is first digitised, so that the image is converted to a matrix of square elements called

pixels, which are then stored in the computer memory. Each pixel in the digital image is the brightness or intensity of

the image at that point, and is represented by a numerical intensity value called the grey level.

Once an image is in digital format (image capture), the numerical values allocated to each pixel in the computer

memory may be manipulated to alter the original image in order to enhance the information present in the image to

facilitate later image analysis [35].

The most commonly used morphological measurements are area, length, perimeter and diameter. The calculation of

the area of a cell is based on the surface of the cell in an image. The estimation of areas is used in morphometry in the

calculation of the relationship between surfaces which, stereologically, are equivalent to the relationship between

volumes according to the Delesse principle [36]. The calculation of the perimeter of a cell is based on the calculation of

the length of the outline of the cell. It must be borne in mind that this parameter depends significantly on the resolution


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of the cell being measured. As the quality of the resolution of the image decreases, the perimeter of the circle becomes

irregular and its calculated value initially increases, moving further away from the theoretical value extracted from the

formula p = 2πr, where r is the radius of the circle. It therefore follows that more precise results will be obtained in

higher resolution images [36]. The estimate of diameters is useful in cases where the objects of interest (cells or nuclei)

are regular circles. If they are oval shaped with various diameters, Feret’s diameter is used. Feret’s refers to the infinite

diameters of a shape. In the case of a circle, all the possible Feret’s have the same value. However, a regular oval has an

undefined number of Feret’s whose values vary between the major and minor axes [36].

4.3 Morphometric results

Dimensions of the blood cells identified in the loggerhead turtles are shown in Table 2. Due to the scarcity of basophils

in the blood of loggerhead turtles, it was not possible to determine their dimensions. The nucleus:cytoplasm area ratio

of the erythrocyte, heterophil, eosinophil, lymphocyte, monocyte, and thrombocyte was respectively 0.12, 0.14, 0.15,

0.63, 0.5, and 0.62.

Table 2 Dimensions (mean, standard deviations, and ranges) of the studied blood cells and their nuclei.

Maximum

length

diameter

Minimum

length

diameter

Area Perimeter

Erythrocyte

19.04 ± 1.25

18.89-19.21

12.86 ± 1.19

12.70-12.99

197.50 ± 26.45

191.06-197.50

51.23 ± 3.61

50.78-51.61 Nucleus 6.33 ± 0.72

6.26- 6.43

4.92 ± 0.54

4.86-4.99

24.58 ± 4.56

24.22-25.29

17.82 ± 1.70

17.67-18.07

Heterophil 17.85 ± 1.9

17.50-18.16

16.31 ± 1.78

15.97-16.62

228.58 ± 16.59

220.55-237.33

54.23 ± 4.34

53.16-55.16

Nucleus 8.54 ± 1.07

8.40-8.78

4.98 ± 1.22

4.80-5.23

32.24 ± 8.30

30.87-33.82

21.81 ± 2.48

21.39-22.27

Eosinophil 20.01 ± 2.19

19.54-20.49

16.84 ± 1.95

16.46-17.28

258.82 ± 31.10

248.92-270.29

58.70 ± 6.05

57.45-59.89

Nucleus 9.20 ± 1.42

8.92-9.53

5.94 ± 1.19

5.68-6.18

39.93 ± 10.22

37.95-42.30

24.65 ± 2.91

24.04-25.28

Lymphocyte 12.24 ± 1.33

12.05-12.56

11.06 ± 1.34

10.88-11.33

105.33 ± 7.65

102.64-111.00

36.89 ± 3.98

36.36-37.82

Nucleus 10.48 ± 1.18

10.29-10.68

8.09 ± 1.05

7.91-8.27

66.47 ± 2.84

64.29-68.66

29.98 ± 3.00

29.47-30.49

Monocyte 16.37 ± 1.90

16.04-16.78

14.75 ± 1.72

14.45-15.09

186.35 ± 11.22

179.92-195.69

49.54 ± 3.21

48.61-50.76

Nucleus 13.14 ± 1.36

12.91-13.44

9.10 ± 1.20

8.91-9.38

91.51 ± 7.52

89.11-95.23

36.46 ± 3.82

35.98-37.46

Thrombocyte 13.48 ± 2.35

13.08-13.92

6.18 ± 0.78

6.02-6.36

64.07 ± 5.35

61.72-67.32

32.82 ± 4.76

32.01-33.74

Nucleus 9.01 ± 1.05

8.81-9.17

5.48 ± 0.74

5.36-5.61

39.95 ± 7.61

38.84-41.44

23.56 ± 2.23

23.18-23.94 All dimensions are shown in µm, except area, in µm2

5. Ultrastructural characterization

5.1 Methodology

After collecting two millilitres of blood from the cervical sinus of each sea turtle in tubes with lithium heparin, blood

samples were immediately centrifuged, plasma was discarded and two cell layers were identified.

The layer of white blood cells and thrombocytes plus an small portion of the red blood cells layer were removed and

fixed primarily in phosphated buffered glutaraldehide 2% (0.1M-pH 7.4) , during 12 hours at 6 ºC. Samples were gently

centrifuged and washed three times for 15 minutes in the same solution. Buffered 2% osmium tetroxide was used as


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secondary fixative during 4 hours, and subsequently cells were washed three times in distilled water for 15 minutes and

left 4 hours in 1% uranyl acetate solution. Samples were dehydrated in ethanol (20º, 40º, 60º, 70º, 100º series) and

passed through ethanol-propylene oxide, propylene oxide and propylene oxide-Embed812 resin. Samples were

polimerized in fresh Embed812 resin, and ultrasectioned at 80nm in an Ultracut S (Leica, Austria). Finally, the sections

were stained with uranyl acetate (1% methanol) and Stato´s lead solution. The observation and microphotograph were

carried out using an EM910 Zeiss electron microscope at 80Kv.

5.2 Ultrastructural results

Ultrastructurally erythrocytes were oval, with an oval nucleus with dense heterochromatin. Some erythrocytes had small

pleomorphic and electron-dense intracytoplasmic inclusions without recognizable organelles. These inclusions were too

big to be viral particles, and they were not considered to be bacteria because there was no cell membrane or pili. Thus,

these inclusions were identified as degenerating organelles.

Five types of white blood cells were ultrastructurally identified in our study: heterophils, eosinophils, lymphocytes,

monocytes and thrombocytes. Ultrastructural characteristics of basophils were not determined due to the very low

number of basophils in the peripheral blood of the loggerhead turtle. Heterophils had a round shape with a round

eccentric nucleus containing moderate amounts of heterochromatin. The abundant cytoplasm contained numerous

electron-dense, round or elongated granules, and a scant number of pleomorphic granules of variable density. Some

mitochondria and endoplasmic reticulum were also observed.

Fig. 2 (A) Transmission electron micrograph of a heterophil from a loggerhead turtle. Scale bar = 1.6 µm. (B) Transmission

electron micrograph of an eosinophil from a loggerhead turtle. Scale bar = 1.3 µm. (C) Transmission electron micrograph of a

monocyte from a loggerhead turtle. Scale bar = 1.7 µm. (D) Transmission electron micrograph of a lymphocyte (arrow) and a

thrombocyte (arrowhead) from a loggerhead turtle. Scale bar = 1.8 µm.

Eosinophils presented a homogeneous round shape with a round or oval nucleus, containing variable amounts of

heterochromatin. Well-defined round electron-dense homogeneous granules were observed in the cytoplasm. Granules

did not contain crystalline structures. Some eosinophils contained numerous clear vacuoles or a big cytoplasmic

vacuole. Mitochondria, endoplasmic reticulum and golgi complexes were easily identified.

Lymphocytes were irregularly round, with a round nucleus, often indented, and with abundant amounts of

heterochromatin. The scant cytoplasm contained few mitochondria, polyribosomes, endoplasmic reticulum and small

electron-dense granules.


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Monocytes were round or fusiform, with a round nucleus containing scant heterochromatin. The cytoplasm had

mitochondria, a large golgi complex, endoplasmic reticulum and some small dense granules.

Thrombocytes were typically oval-shaped cells, although sometimes we observed round thrombocytes. The cell

edges presented some finger-like projections. The nucleus was generally oval containing peripherally located moderate

heterochromatin. The scant cytoplasm often contained clear canalicular structures and small variably dense granules.

6. Conclusions

The triple approach used in this study (cytochemical, morphometric, and ultrastructural characterization) permitted an

adequate characterization of the blood cells in loggerhead sea turtles.

The morphologic characteristics of erythrocytes from our juvenile loggerhead sea turtles were similar to those

reported in green turtles [3, 4] and in other terrestrial chelonians [11, 16]. The negative cytochemical stain of

erythrocytes from loggerhead turtles was similar to the results obtained from studies on erythrocytes of green turtles

[3].The erythrocytes identified in our study were 19 µm long. The erythrocytes of immature green turtles were 17-20

µm long [3]. However, there are no descriptions of the size of the erythrocytes in the haematological studies on black

turtles [37], Kemp’s ridley sea turtles [5], or young loggerhead turtles [19]. Erythrocytes from chelonians have a

nucleus:cytoplasm area ratio ranging 0.08-0.15 [38]. In our study this ratio was 0.12. There are no descriptions of the

nucleus:cytoplasm area ratio of the erythrocytes of other sea turtles. The intracytoplasmic inclusions observed in some

erythrocytes were similar to those described in the erythrocytes of several species of reptiles [39], including the green

iguana (Iguana iguana) [14], and the rhinoceros iguana (Cyclura cornuta) [40].

Neutrophils were not identified in our study. Neutrophils are rare in reptiles, but have been described in the tuatara

(Sphenodon punctatus) [41]. There are several descriptions of neutrophils in sea turtles [4] although probably are large

degranulated eosinophils [3].

In our study, heterophils were stained with ACP (with and without tartrate), PER, CAE and SBB; and they were

moderately stained with PAS. There are no previous references on cytochemical characterization of heterophils from

loggerhead turtles. In a similar study, heterophils from green turtles stained only with NBE and PAS [3]. Heterophils

from other terrestrial reptiles stained with ACP and alkaline phosphatase (ALP) [11, 16]. The differences in the

cytochemical characteristics of heterophils from different species of sea turtles show that the heterophils of these

species have different enzymes. The ultrastructural characteristics were similar to those described for heterophils from

green sea turtles [3]. However, other reptiles, particularly some snakes, had two morphologic variations of heterophils;

one variant contained a homogeneous population of electron-dense intracytoplasmic granules, and the other variant

contained some granules that were electron dense, whereas other granules similar in size and shape had a lighter matrix,

suggesting different developmental stages of heterophils in circulation [13, 42, 43].

Eosinophils from loggerhead turtles were homogeneous in size, 20 µm in diameter, unlike eosinophils from green

turtles, which are large as well as small [3]. Large and small eosinophils have also been described in Kemp’s ridley

turtle [5]. Large eosinophils in green turtles may represent activated cells that contain degranulated or coalescent

granular material in response to a parasitic infection or other inflammatory stimulus [3]. In our study, granules from the

eosinophils were stained with ACP (with and without tartrate), NBE (with sodium fluoride), CAE and PAS. They were

also moderately stained with SBB. Large and small eosinophils from green turtles stained strongly only with CAE, and

marginally with NBE and PAS [3]. Small eosinophils from Kemp’s ridley turtles stained strongly with ACP whereas

large eosinophils stained moderately with ALP, PAS, and SBB [5]. Except for the absence of crystalline structures,

eosinophils from loggerhead turtles had similar ultrastructural characteristics to those described for small eosinophils

from green sea turtles [3].

Basophils were scarce in loggerhead turtles, similar to those described in green turtles[3]. Basophils were not

identified in Kemp’s ridley turtles [5]. In our study, basophils were slightly smaller than heterophils, and basophil

granules stained only with TB. This cytochemical pattern is similar to that described in green turtles [3]. However,

basophils of some snakes stain with PAS, but not with TB [13].

Lymphocytes from loggerhead turtles were morphologically similar to lymphocytes reported for green turtles [3] and

Kemp’s ridley turtles [5]. Lymphocytes from loggerhead turtles were not difficult to distinguish from thrombocytes

using DQ stain because freshly prepared smears were used. However, lymphocytes from other reptilian species can be

difficult to distinguish from thrombocytes [13, 42], specially if blood smears are prepared from blood that has been

chilled [3]. Lymphocytes from loggerhead turtles stained only lightly with NBE (without sodium fluoride) whereas

lymphocytes from green turtles did not stain with any cytochemical stain [3]. However, lymphocytes from Kemp’s

ridley turtles stained with nonspecific esterase [5]. Lymphocytes from loggerhead turtles were homogeneous in size, 12

µm in diameter, unlike lymphocytes from green turtles, which were large as well as small [4]. Lymphocytes from

loggerhead turtles had an area that was 77 % lesser than that of monocytes, and were thus easy to distinguish from

monocytes.

Monocytes from loggerhead turtles were similar to the monocytes from green turtles [3]. Other authors did not

identify monocytes from green turtles [4, 6] or Kemp’s ridley turtles [5]. Monocytes are more difficult to differentiate

from lymphocytes when smears are made from blood that has been chilled 8 h, partly because of cellular shrinkage [3].


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Monocytes from loggerhead turtles stained only moderately with NBE (without sodium fluoride). Monocytes from

green turtles stained with ACP and PAS, and only some of them with CAE and NBE [3].

In our study, azurophils were not identified in the blood of loggerhead turtles. References on azurophils in the blood

of sea turtles are rare [44]. Most authors did not identify azurophils in sea turtles [3-6, 19].

Thrombocytes from loggerhead turtles to be similar to those reported for green turtles [3]. Thrombocytes were not

reported from Kemp’s ridley turtles [5]. Thrombocytes from other reptilian species can be difficult to distinguish from

lymphocytes [11, 42]. Thrombocytes usually retain their morphologic characteristics on freshly prepared smears [3].

However, if blood smears are prepared from blood that has been chilled, cellular shrinkage makes differentiation more

difficult. Thrombocytes from loggerhead turtles had an area that was 39 % lesser than that of lymphocytes. In our study,

thrombocytes were differentiated from lymphocytes cytochemically by staining only with PAS. Thrombocytes from

green turtles stain with NBE and PAS [3]. Thrombocytes from other terrestrial reptiles stain also with ACP [16, 42].

Ultrastructurally thrombocytes from loggerhead turtles had open canalicular systems, which is characteristic for this

cell type in many reptiles, including green sea turtles [3]. Similar open canalicular systems have been described for the

platelets in mammals [20].

This study provides a morphologic classification of blood cells in this endangered species, useful for the scientists

involved in sea turtle conservation around the world, and emphasises the usefulness of the microscopic studies in sea

turtle conservation.

Acknowledgements The authors would like to thank P. Castro, Department of Morphology, University of Las Palmas de Gran

Canaria (ULPGC), and F. Freire, Electronic Microscopy Service, ULPGC, for technical assistance. They are grateful to Dr P.

Calabuig (TWRC) and members of Consejería de Medio Ambiente, Cabildo Insular de Gran Canaria, for providing us the turtles.

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Microscopic studies on characterization of blood … · Microscopic studies on characterization of blood cells of endangered sea turtles J. Orós, A. B. Casal, and A. Arencibia Department