Morfología plaquetas caninos

Acta Veterinaria Hungarica 53 (3), pp. 337–350 (2005)
MORPHOLOGICAL EVALUATION OF CANINE PLATELETS
ON GIEMSA- AND PAS-STAINED BLOOD SMEARS
Dóra HALMAY1*, P. SÓTONYI2, P. VAJDOVICH3 and T. GAÁL3
1Triovet Veterinary Surgery, Budapest, Hungary; 2Department of Anatomy and
Histology and 3Department of Internal Medicine, Faculty of Veterinary Science,
Szent István University, H-1078 Budapest, Hungary
(Received December 3, 2004; accepted December 16, 2004)
The morphology of canine platelets (changes in size, shape, staining charac-teristics,
degree of activation and clump formation, distribution of granules, appear-ance
of vacuoles on Giemsa-stained smears) was investigated in 20 healthy control
and 181 diseased dogs. In the group of the sick dogs 84 animals suffered from dis-orders
affecting directly the haematological parameters or the haematopoietic or-gans
such as bleeding, thymic haemorrhage, haemolytic disorders, lymphoma, im-mune-
mediated thrombocytopenia, and other 97 dogs were affected by other dis-eases
(hepatopathy, nephropathy, hepatic, splenic or intestinal neoplasm, skin dis-eases,
diabetes mellitus, Cushing’s syndrome, sepsis). The alterations found in
platelet morphology were not specific for any disorder. The most common platelet
abnormalities were polychromasia and the presence of giant platelets. These
changes occurred in a high number in disorders accompanied by bleeding or hae-molysis.
Anisocytosis was the most frequent finding in hepatic, splenic or intestinal
neoplasms and in certain endocrinopathies. Microcytosis was observed in immune-mediated
thrombocytopenia, hepatic neoplasms and endocrine disorders. Extreme
platelet activation was common in haemolysis, hepatopathies, neoplastic diseases
and sepsis. Vacuolisation was present in thymic haemorrhage, pancreatitis, diabetes
mellitus and Cushing’s syndrome. A new morphologic phenomenon, i.e. a ring-like
formation of granules, was described in the cytoplasm of the platelets both in
healthy and diseased animals. In addition, two forms of pathologic granulation were
also described for the first time in Giemsa-stained blood smears: the pseudonuclear
and the spot-like formation of granules, which were observed especially in disorders
affecting the blood cells. The granulation and morphological characteristics of plate-lets
on smears stained by periodic acid–Schiff reaction (PAS) were also studied.
Three localisations of granulation were observed, such as peripheral, eccentric and
diffuse. The ratio of PAS-positive and -negative platelets was evaluated in several
diseases. Our findings support the diagnostic value of platelet evaluation by light mi-croscopy
and help clinicians/clinical pathologists to understand why morphologic
changes of thrombocytes might be expected in several diseases.
Key words: Platelet, thrombocyte, light-microscopic morphology, dogs,
0236-6290/$ 5.00 © 2005 Akadémiai Kiadó, Budapest
Giemsa staining, PAS staining
*Corresponding author; E-mail: Halmay.Dora@aotk.szie.hu; Fax: +36 (1) 478 4137

338 HALMAY et al.
The examination of platelets, the smallest blood cells, is difficult by light
microscopy when the smear is stained with routine haematological dyes such as
May-Grünwald, Pappenheim, Giemsa and Diff Quick, as their structure is very
poor. Other cytochemical staining methods, which are well known in haematol-ogy,
do not provide too much additional information. The main energy source of
platelets is glycogen spread all over the cytoplasm. Fortunately, this compound
can be easily visualised by special dyes, such as periodic acid–Schiff reaction
(PAS). This staining makes glycogen granules and their distribution visible, pro-viding
an easy way for the evaluation of platelet morphology (Jain, 1993).
Giemsa staining is widely used in haematology as it is simple and the ap-propriately
stained smear shows the fine structure of platelets (Szász et al., 1981;
Powers, 1989; Reagan et al., 1998; Cowell et al., 1999). PAS-stained smears are
less commonly used for the morphological evaluation of platelets.
Morphology of platelets in healthy dogs in Giemsa-stained smears
The platelets in canine blood are light blue and anucleated (Bessis, 1972;
Jain, 1986; Bush, 1991; Hoffbrand and Pettit, 1997; Day et al., 2000). Most of
them are medium sized (Cowell et al., 1999). They are round or oval (Bessis,
1972; Erslev and Gabuzda, 1975; Handagama et al., 1986; Powers, 1989; Jain,
1993). If the smear was not fixed immediately after blood collection, bizarre-shaped
platelets may appear (Bessis, 1972). During blood collection thrombo-cytes
may become activated and then grow thin cytoplasmic processes or pseu-dopods
(Hoffbrand and Pettit, 1997; Cowell et al., 1999). Platelets have multiple,
fine purple granules in the cytoplasm, which are grouped in the centre or dis-persed
(Bessis, 1972; Jain, 1986; Bush, 1991; Hoffbrand and Pettit, 1997; Day et
al., 2000). The granulation of platelets is less visible in dogs than in cats (Bush,
1991). Aged platelets are smaller and have lower density than young ones (Bes-sis,
1972; Bush, 1991; Hoffbrand and Pettit, 1997). In normal condition they have
no vacuoles but if blood was collected into an EDTA-containing tube and was
stored for more than 24 h, platelets could swell and show vacuolisation (Bessis,
1972). Even on a well-prepared blood smear small platelet clumps may form (Bes-sis,
1972; Hoffbrand and Pettit, 1997). Large clumps of platelets on smears suggest
unsuitable blood collection and thromboplastin contamination (Jain, 1986).
Morphologic features of thrombocytes are altered by a variety of inherited
and acquired conditions (Stobbl, 1959; Nolte and Mischke, 1995). Usually, platelet
morphology studies comprise the following aspects when using common staining
procedures such as Giemsa staining (Jain, 1986; Jain, 1993; Cowell et al., 1999):
(1) Staining characteristics. Like in the evaluation of red blood cells (RBC), ‘poly-chromasia’
may be used for the different staining of platelets on the smear.
This means the simultaneous occurrence of hypochromic (poorly stained), hy-perchromic
(strongly stained or basophilic) and normochromic thrombocytes.
Acta Veterinaria Hungarica 53, 2005

MORPHOLOGICAL EVALUATION OF CANINE PLATELETS 339
(2) Size. The following terms can be used to characterise the size of platelets
when comparing them to the size of red blood cells (RBC):
– Microcytes: platelets which are smaller than one-fourth of an RBC (1–2 μm)
– Medium-sized platelets: their size is one-fourth to one-third of an RBC (2–
3 μm)
– Macrocytes: RBC-sized platelets (7 μm)
– Giant platelets: larger than an RBC (> 7 μm)
– Anisocytosis: simultaneous occurrence of platelets of different size on the
smear
(3) Polymorphism
(4) Presence of activated (with pseudopods) and non-activated (without pseudo-pods)
platelets
(5) Presence of pathologic granulation
(6) Vacuolisation of the cytoplasm
One to three percent of the abnormalities described above may be normal
in healthy dogs (Bessis, 1972). Generally, platelets are pathologic on smears if:
– there are extreme large and small ones simultaneously on the smear,
– staining characteristics of the hyalomere (pale homogeneous peripheral zone)
is abnormal (e.g. basophilia),
– the granulomere (chromomere) is very strong (granules are striking) – it is
typical of human platelets (Stobbl, 1959; Mende et al., 1975; Keller, 1986)
and has not been described yet in the platelets of dogs.
Morphology of platelets in healthy dogs on PAS-stained smears
The staining of blood cells with PAS is usual in haematological diagnostic
work (Szász, 1981), but the PAS-staining characteristics of thrombocytes have
not been intensively studied so far. Glycogen granules are scattered in the hya-lomere
and chromomere. These granules are irregularly shaped, often forming
clumps (Bessis, 1972). The staining features of blood cells on the smear of a
healthy dog are characteristic. The glycogen content of platelets is greater than
that of granulocytes (Jain, 1986). In the majority of studies quantitative changes
in the glycogen granules of thrombocytes were examined by electron microscopy
and there were just a few investigations that used light-microscopic evaluation of
PAS-stained smears (Bessis, 1972).
As there are only few detailed descriptions about the light-microscopic
morphology of canine platelets (Bessis, 1972; Yamashiro et al., 1983; Jain, 1986;
Keller, 1986; Fijnheer et al., 1989; Hopper et al., 1989; Tablin et al., 1989; Jain,
1993; Reagan et al., 1998; Cowell et al., 1999), our goal was to investigate ca-nine
thrombocytes on Giemsa- and PAS-stained smears in healthy animals and in
dogs with pathological conditions.

340 HALMAY et al.
Materials and methods
Animals
Blood samples of 201 dogs of several breeds and of different age and sex
presented to the Small Animal Clinic of the Department of Internal Medicine,
Faculty of Veterinary Science, Budapest were used in the study. Based on the fi-nal
clinical diagnosis, two main groups were formed retrospectively (Table 1):
(1) Group 1 (n = 20 healthy, control dogs)
(2) Group 2 (n=181 diseased dogs). Of them, 84 animals were suffering from
diseases affecting directly the haematological parameters or the haematopoi-etic
tissues. Group 2 comprised further 97 diseased dogs suffering from other
diseases.
Sampling procedure, staining and microscopic evaluation
For Giemsa staining venous blood was collected from all patients into a
vacutainer tube with 3.8% sodium citrate as anticoagulant, followed by the
preparation of two blood smears stained by Giemsa (Bessis, 1972) in 24 h to
prevent platelets from forming dendritic projections on the glass slide. Stained
blood smears were first scanned at 400-fold magnification to evaluate platelet
count and to notice platelet clumps. Detailed examination of intracellular morphol-ogy
required a 1000-fold magnification with oil-immersion objective. Depending
on the patient’s platelet count, 50 to 100 platelets were evaluated in each case.
Aspects of evaluation were staining characteristics of platelets, presence of
anisocytosis, polymorphism, activated and non-activated forms, normal and
pathologic granulation, vacuolisation of cytoplasm, and other findings.
For PAS staining venous blood samples were collected into K-EDTA-coated
vacutainer tubes. Thin blood smears were made immediately and stained
by the PAS method (Bessis, 1972; Szász, 1981). We did not use the method of
PAS reaction with diastase digestion for differentiation of glycogen from any
other PAS-positive material as in platelets only the glycogen is stained by PAS.
In each smear 50–100 platelets were evaluated according to the following
aspects:
– PAS-negative platelets: cytoplasm is pale with indefinite cell border
– PAS-positive platelets: cytoplasm is pale and acidophilic with fine or rough,
purple granules
Like in the case of the Giemsa-stained smears, 50–100 platelets were stud-ied.
The proportion of PAS-positive and -negative platelets was given and the
type of granulation was evaluated.
All reagents for Giemsa and PAS staining were purchased from Sigma
Ltd., Hungary.

Table 1
Groups of animals
Group 1 (control)
n = 20
Healthy dogs
Group 2 (diseased dogs) (some animals belong
to more than one disease subgroup)
n = 181
n = 84 with affected haematological parameters
n = 97 with non-affected haematological parameters
Bleeding, haemolysis (other than babesiosis) (n = 18)
Babesiosis (n = 48)
Immune-mediated haemolytic anaemia (n = 19)
Disorders of the haematopoietic tissues (n = 15)
(idiopathic thrombocytopenic purpura, feline leukaemia virus infection, lymphoma,
chronic lymphoid leukaemia, erythroid myelosis, chronic myeloid leukaemia, essential
thrombocythaemia, polycythaemia vera, panmyelophthisis)
Hepatopathy (n = 60)
(posthaemorrhagic or posthaemolytic hepatopathy, hepatic lipidosis, hepatitis, hepatopathy
in pancreatitis, hepatogenic encephalopathy, toxic hepatopathy, hepatic neoplasm, idio-pathic)
Nephropathy (n = 41)
(tubular nephrosis, acute and chronic renal insufficiency, glomerulonephritis, renal
dysplasia, oxalate nephrosis)
Neoplasm (n = 22)
(thoracic, abdominal, intestinal, urinary bladder, splenic, gastric, liver, mammary, skin,
uterus, vascular neoplasm)
Gastrointestinal disorders (n = 35)
(haemorrhagic enteritis, ascaridosis, protein-losing enteropathy, foreign body in the gut,
pancreatitis)
Heart diseases (n = 11)
(Hypertrophic cardiomyopathy, dilated cardiomyopathy, AV block, mitral/tricuspid in-sufficiency,
myocarditis, aortic stenosis, haemopericardium)
Endocrine disorders (n = 11)
(diabetes mellitus, Cushing’s syndrome, hypoadrenocorticism, hypothyroidism)
Sepsis (n = 20)

342 HALMAY et al.
Results
(1) Giemsa staining
Staining characteristics of platelets. In the control dogs (Group 1) plate-lets
uniformly stained pale blue, usually with a distinguished granulomere and
hyalomere zone (Fig. 1). Polychromasia, with the appearance of basophilic (hy-perchromic)
and mildly stained (hypochromic) platelets, occurred in the diseased
animals with different frequencies (Fig. 2). Most often it was found in haemor-rhagic
(especially thymic apoplexy) and haemolytic disorders (immune-mediated
haemolytic anaemia, IMHA), nephropathies, gastrointestinal disorders and endo-crinopathies
(first of all in diabetes mellitus and Cushing’s syndrome).
Anisocytosis. In healthy dogs the dominant platelets were medium sized
with some macrocytes. Anisocytosis was found in almost all diseases in Group 2,
with the exception of immune-mediated thrombocytopenia. The most obvious
anisocytosis was noticed in hyperadrenocorticism and diabetes mellitus and it
was less frequently found in association with neoplasms.
Predominance of microcytes or giant platelets was rarely observed. The
number of microcytes exceeded the number of any other-sized platelets in neph-ropathies,
hepatic neoplasm, Cushing’s syndrome and diabetes mellitus (Fig.
3A). Presence of giant platelets characterised especially the haemorrhagic and
haemolytic disorders including thymic haemorrhage and IMHA (Fig. 3B). In
hepatopathy, splenic haemangioma, pancreatitis and hypoadrenocorticism a
small number of giant platelets and macrocytes also occurred.
Polymorphism. In the control dogs only round- and oval-shaped platelets
were found. In diseased animals irregular forms, usually comma-, ribbon- or but-terfly-
shaped platelets were seen. In certain haematopoietic disorders such as
lymphoma and idiopathic thrombocytopenic purpura we did not find any altera-tions
in the shape of platelets. In thymic haemorrhage severe polymorphism in-volving
almost all the platelets was found (Fig. 4).
Presence of activated and non-activated platelets. A morphological sign of
activation is the appearance of fine cytoplasmic processes (spiderleg-like pseudo-pods)
on the surface of the platelet. This phenomenon can often be found in
Giemsa-stained smears even in healthy animals. As a definition of pathologically
‘activated’ or ‘non-activated’ platelets can hardly be found in the literature, we
formed an arbitrary classification and considered the appearance of non-activated
platelets pathological if their number exceeded 50% of all platelets and if they si-multaneously
showed other morphologic abnormalities (e.g. hypochromasia, pres-ence
of vacuoles). In healthy dogs the ratio of activated platelets was less than 50%.
In control dogs activated and non-activated platelets were present in simi-lar
amounts. A very high ratio of non-activated platelets was found in dogs with
thymic haemorrhage in Group 2 (Fig. 2). A lower ratio of inactive thrombocytes
occurred in nephropathies and in some other diseases. Thick cytoplasmic proc-esses
– which were different from the well-known fine pseudopods – were found

on the surface of platelets in certain haemolytic disorders (IMHA, babesiosis) as
well as in hepatopathies, splenic haemangioma and sepsis (Fig. 5).
1 2
Fig. 1. Healthy macroplatelets with fine pseudopods
Fig. 2. Polychromasia. Non-activated hypochromic (Î) and hyperchromic (→) platelets
me
mi
ma
A B
Fig. 3A. Anisocytosis (me: medium-sized platelet, mi: microcyte, ma: macrocyte)
Fig. 3B. Giant, basophilic platelets (→)
4 5
Fig. 4. Polymorphism
me
me
Fig. 5. Thick pseudopods on an activated giant platelet (→); me: medium-sized platelet

344 HALMAY et al.
A B
Fig. 6A. Pseudonuclear formation (→)
Fig. 6B. Spot-like formation (→)
7 8
A B
Fig. 7. Vacuolisation (→)
Fig. 8. Ring-like formation (→)
Fig. 9A. PAS-positive concentrated (→) and PAS-negative (Î) platelets
Fig. 9B. PAS-positive concentrated (→) and diffuse (Î) platelets

Normal and pathologic granulation. The platelets of control (healthy)
dogs had fine, diffuse azurophilic granules in their cytoplasm (Fig. 1). In the dis-eased
group two types of characteristic pathological granulation were recorded:
– Pseudonuclear formation, i.e. a strong granulation in the centre of the cell that
looked like a well-stained nucleus (Fig. 6A).
– Spot-like formation near the periphery of the platelet. This was the presence of
one or more big, strongly stained granules localised eccentrically. Sometimes it
seemed to be stuck to the surface of the cell (Fig. 6B).
Mild to intense pathologic granulation was observed in almost all dogs of
Group 2. Spot-like formation occurred more frequently than pseudonuclear for-mation.
In Group 2, dogs with thymic apoplexy, diabetes mellitus or Cushing’s
syndrome had the highest number of abnormal, granulated platelets, followed by
dogs with IMHA, hepatopathy and certain tumours (e.g. intestinal neoplasm,
splenic haemangioma, and hepatic neoplasm). Pseudonuclear formation of gran-ules
was found in IMHA and in certain cardiac diseases. It also appeared in the
case of essential thrombocythaemia, babesiosis, hepatopathy and neoplasms. In
dogs with idiopathic thrombocytopenic purpura and skin diseases the platelets
did not show any pathologic granulations.
Vacuolisation of the cytoplasm. In healthy dogs we never met this phe-nomenon.
However, one or more vacuolated platelets were often found in Group
2 regardless of the disease. Vacuolisation was most frequent in thymic apoplexy
but its appearance was considerable in pancreatitis, diabetes mellitus and hy-peradrenocorticism
as well (Fig. 7).
Other findings. We found a previously unknown, most probably physio-logical
phenomenon in some platelets both in healthy and diseased dogs. It was a
fine, ring-like, azurophilic formation near the periphery of the cell (Fig. 8). It ap-peared
approximately in 1–2% of the platelets.
(2) PAS staining
On PAS-stained smears PAS-negative (Fig. 9A) and PAS-positive (granu-lated)
platelets were recorded in both groups in different proportions. These cy-toplasmic
granules were either concentrated at the periphery of the platelets (e.g.
strongly-stained granules near the cell membrane) or eccentric (similarly stained
granules somewhere in the cytoplasm) (Fig. 9A and B). In some cases several
fine or rough granules were diffusely scattered in the cytoplasm, providing a
strong, acidophilic character to the platelet (Fig. 9B).
Physiological granulation and morphologic characteristics of PAS-stained
platelets in healthy dogs. In the 20 healthy dogs most of the platelets (62%) were
PAS negative. Among the PAS-positive platelets the number of platelets contain-ing
concentrated granules exceeded that of platelets with diffuse granulation. The
granulation was usually mild.

346 HALMAY et al.
Differences between PAS-stained platelets of healthy and diseased ani-mals.
In dogs with haematological disorders an increased percentage (> 65%) of
PAS-positive platelets was found. In case of haemorrhagic and haemolytic disor-ders
very strong concentrated cytoplasmic granulation was observed, while in
immune-mediated thrombocytopenia the intracellular distribution of the PAS-positive
granules was diffuse.
In dogs suffering from pyometra and diabetes mellitus almost all platelets
were PAS positive, and the proportion of PAS positivity was also substantial in
dogs with sepsis, nephropathy and neoplasms. The number of platelets with
strongly-stained granules also increased in these disorders. However, while in
nephropathy there were mainly peripheral granulations, in sepsis and pyometra
the granules were situated mainly eccentrically somewhere in the cytoplasm.
Discussion and conclusions
The most frequent morphologic alteration in Giemsa-stained platelets was
polychromasia with the appearance of basophilic (hyperchromic) and mildly
stained (hypochromic) platelets. Basophilia refers to ineffective thrombopoiesis
or the presence of young platelets (Bessis, 1972). In our study, polychromasia
has been found for the first time in connection with some disorders (thymic
haemorrhage, nephropathy, diabetes mellitus and Cushing’s syndrome). Hy-pochromic
cells presumably correspond to aged or agranular platelets (Jain, 1993).
Anisocytosis is the sign of disturbed thrombopoiesis (Bessis, 1972). Inter-estingly,
we did not see anisocytosis in immune-mediated thrombocytopenia, most
probably because of the low platelet count and the few cases (n = 4) studied. The
presence of microthrombocytes usually refers to iron deficiency and immune-mediated
thrombocytopenia (Bush, 1991; Hoffbrand and Pettit, 1997; Day et al.,
2000). We found that some other disorders (nephropathy, hepatic neoplasm, diabe-tes
mellitus and Cushing’s syndrome) were also associated with microcytosis.
Probably an iron metabolism disorder and a secondary iron deficiency that accom-panied
that disorder were in the background. Giant platelets (also called stress- or
shift-platelets and megathrombocytes; Bessis, 1972) are always pathological in ca-nine
blood smears (Cowell et al., 1999). Rarely they appear in severe thrombocy-topenia
but more often in thrombocytosis and thrombocytopathy as a sign of inef-fective
thrombopoiesis or an increased demand for platelets at the periphery. It
may be a regenerative response of the bone marrow and the sign of forced throm-bopoiesis
as the healthy tissue is capable of fast compensation resulting in massive
haematopoiesis and even forced thrombopoiesis (Bessis, 1972; Jain, 1986; Bush,
1991; Cowell et al., 1999; Hoffbrand and Pettit, 1997; Day et al., 2000). The very
high number of giant platelets suggests a myeloproliferative disorder or myelofi-brosis
(Jain, 1986; Cowell et al., 1999; Day et al., 2000).

Various shapes of platelets (star, comma, cigar, tadpole and butterfly)
have already been described (Bessis, 1972; Hoffbrand and Pettit, 1997). The ap-pearance
of these irregular forms is probably indicative of bone marrow disor-ders
or defective thrombocyte function (Hoffbrand and Pettit, 1997; Cowell et
al., 1999). If platelets are large, abnormally shaped and there is simultaneously
thrombocytopenia, this suggests increased platelet destruction at the periphery
(Bush, 1991). It may develop also in thymic apoplexy. We supposed that a hy-pochromic,
spherical platelet was functionally impaired. Therefore, we regarded
smears with the dominance of hypochromic platelets as pathological.
The appearance of platelets with thin cytoplasmic processes (Hoffbrand and
Pettit, 1997; Cowell et al., 1999) is a physiological phenomenon. These cells might
have become activated during the blood collection procedure (Jain, 1986). It is dif-ficult
to distinguish between normally activated platelets (e.g. those activated dur-ing
blood collection) and those activated with hyperaggregability on blood smears
(Day et al., 2000). Unfortunately, one can hardly find any directions how to estab-lish
‘pathologic activation’ on smears in the references. We highly recommend
executing the blood collection as precisely and quickly as possible to avoid throm-boplastin
contamination and consequent arbitrary platelet activation on the smear.
In our investigation another new morphological observation has also been
described, i.e. the presence of one or several thick cytoplasmic processes on the
surface of platelets. This may be a sign of an extreme form of platelet activation.
The appearance of non-activated (supposedly non-functioning) platelets on
smears never occurred alone but was always accompanied by other morphologic
alterations as found by others (Bessis, 1972).
In this study we found two new granule formations of platelets (pseudonu-clear
and spot-like granule at the periphery) in Giemsa-stained thrombocytes.
Until now the pseudonuclear formation of granules has been described only in
the blood smears of healthy cats (Bessis, 1972; Hoffbrand and Pettit, 1997). In
the case of ineffective thrombopoiesis a few or more distinguished granules can
be seen in the cytoplasm of platelets (Bessis, 1972). Atypical granulation can be
found in the functional defect of the bone marrow (e.g. myelofibrosis; Hoffbrand
and Pettit, 1997; Cowell et al., 1999). Presence of pseudonuclear and spot-like
granules suggests a dysthrombopoietic background, including forced bone mar-row
activity and increased platelet turnover. This process might result in im-paired
cytoplasm maturation with pathologic granule distribution. The reason for
the special distribution of granules is unknown. The granules are absent from the
cytoplasm in disseminated intravascular coagulopathy (Bush, 1991).
Vacuolisation – which does not necessarily mean the functional defect of
platelets – was described in disseminated intravascular coagulopathy (Bush,
1991). We found platelet vacuoles in some other diseases (thymic haemorrhage,
pancreatitis, diabetes mellitus, and Cushing’s syndrome), too. We think that
vacuolisation might be of toxic or immune-mediated origin.

348 HALMAY et al.
Both in healthy and diseased animals we sometimes found a ring-like for-mation
near the periphery of the platelets that had not been observed before on
Giemsa-stained smears. This might represent the cell cytoskeleton consisting of
microfilaments and microtubules. In resting (non-activated) platelets they form a
ring under the cell membrane in humans (Bessis, 1972). This formation can only
be examined with an electron microscope and has not been described on rou-tinely
stained blood smears in animals so far.
It is known that young, functionally and metabolically active platelets con-tain
more glycogen than resting ones (Bessis, 1972; Hoffbrand and Pettit, 1997).
The cyclic adenosine monophosphate (cAMP) of platelets regulates their activ-ity.
During the change of shape, at the beginning of activation, the metabolism of
the platelet is growing, with increasing glucose oxidation. Every substance that
causes a rise in intracellular cAMP level increases the glycogen content of the
cell (Jain, 1986). Since glycogen is the main energy source of platelets, the in-creased
number of PAS-positive glycogen granules supposes a more active gly-cogenesis.
Certain disorders, such as septic diseases, may be accompanied by se-vere
thrombocytopenia. As the result of the response of the bone marrow, forced
thrombopoiesis occurs and the presence of many PAS-positive platelets is sup-posed
to be the sign of this response: the greater PAS positivity, the stronger gly-cogenesis
is supposed. The intracellular distribution of glycogen can be patho-logical
in several disorders. When glycogen granules form large clumps in more
than 10% of the platelets, this refers to impaired thrombopoiesis (Bessis, 1972).
In thrombocytosis and thrombocythaemia an increased number of glycogen par-ticles
was also observed (Bessis, 1972). The explanation of the presence and
changes in distribution of PAS-positive granules in several disorders is unknown.
Most probably electron microscopic studies are necessary to explain these phe-nomena.
We think that the decreased amount of glycogen particles in platelets is
not common. Most probably platelets with decreased glycogen are metabolically
less active than those giving stronger PAS positivity.
Finally, we concluded that alterations of platelet morphology do not char-acterise
exclusively any of the disorders studied in this work. It is interesting that
the most significant platelet abnormalities (polychromasia, polymorphism, and
presence of non-activated platelets, pathologic granulation and vacuolisation)
were observed in thymic apoplexy. This suggests increased platelet destruction
and utilisation. Several morphologic alterations were described also in diabetes
mellitus and Cushing’s syndrome. In immune-mediated thrombocytopenia plate-lets
do not have morphological abnormalities, only microcytosis occurred on
Giemsa-stained smears. In the case of forced thrombopoiesis basophilic, giant
platelets with or without thick cytoplasmic processes were often found. We sup-pose
that their appearance helps to decide whether or not the earlier thrombocy-topenia
was regenerative. This may be a new diagnostic observation, which, like
the evaluation of anaemia, proves the ability of the bone marrow to respond. Al-

though the morphologic evaluation of platelets by light microscopy is very diffi-cult,
there are some aspects during Giemsa- or PAS-stained blood smear evalua-tion
that may aid our practical diagnostic work in the future.
Since there were not enough patients in all of the subgroups, our results
should be considered preliminary and, thus, further examinations are necessary.
Acknowledgements
The authors express their thanks for the technical assistance provided by the co-workers
of the Pathophysiology & Diagnostic Laboratory of the Department of Internal
Medicine and to Róbert Kocsis, DVM, for his excellent contribution in the preparation of
the pictures.
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