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ISRAEL JOURNAL OF

VETERINARY MEDICINE
MORPHOLOGICAL CHANGES IN RED BLOOD CELLS OF BIRDS AND
REPTILES AND THEIR INTERPRETATION

H. Pendl

ARCHEOPTERYX DIAGNOSTICSTM Hematology and Cytology in Birds and Reptiles. Eichenau,


Germany

SUMMARY

The value of the evaluation of cell morphology in a blood film


with special focus on avian and reptilian erythrocytes is
demonstrated. In combination with the packed cell volume (pcv)
this rapid and low-cost procedure gives useful information on the
patient’s condition and helps the practitioner to take first
diagnostic and therapeutic measures while awaiting results being
done by outdoor laboratories. General principles for the distinction
between regenerative and degenerative changes in avian and
reptilian red blood cells are explained in crisp tables, figures, and
photographs. They are intended for diresct use and should
encourage practitioners to include this useful tool in their daily
practice routine.

INTRODUCTION

Despite the technological progress in avian and reptilian hematology, time-


consuming manual techniques are still necessary to perform a complete blood
cell count, which is difficult to do in-house during daily routine. Valuable time is
lost with the processing of samples in laboratories, before adequate diagnostic
and therapeutic steps can be started. The timely onset of an adequate treatment,
however, is vital for the outcome of the case, especially in debilitated patients.
Therefore, simple indoor laboratory techniques for a first check up are desirable.
Evaluation of cell morphology in a blood film is a rapid and low-cost procedure.
In combination with the packed cell volume (pcv) it gives useful information on
the patient’s overall condition and helps the practitioner to take first measures
while awaiting further detailed results from the laboratory. This paper focuses on
the characteristics and interpretation of morphological changes in avian and
reptilian erythrocytes.
TECHNICAL ASPECTS

Preparation of a blood film

Blood smears from birds and reptiles should be performed without any
anticoagulant directly from the syringe or needle and immediately after collection
by applying the “wedge smear” or the “slide and coverglass” technique (Fig. 1).
Concerning the first method, the use of a bevel-edged slide is crucial to avoid
artificial cell damage. Ideally, the blood film shows a continuous thinning with a
vane at the end. For any evaluation, the monolayer (ML) is examined in a
meandering search as shown in figure 2a. It is the area, where the cells lie close
to each other without overlapping themselves and can usually be found between
the middle and the last third of the blood film. Frequent preparation mistakes
include inadequate volumes of blood and interruption of the smear movement
(Fig 2b-d), which result in the loss of the monolayer area. A routine preparation
of at least three smears per blood sampling is recommended to be prepared for
additional examinations, such as suspected parasitemia or failure of proper
staining. The air-dried smears are kept in a dustfree environment until they are
stained. Dust particles on the sample get stained together with the blood cells
resulting in poor quality of the blood films. True subcellular structures cannot be
differentiated from artificial soiling.
Fig. 1: Techniques for blood film preparation

Fig. 2: Ideal blood film and common mistakes; the mononlayer (ML) for
evaluation is found between the middle and the last third of an ideal smear

Fig. 3: Flow charts for the interpretation of avian and reptilian hemograms
Staining of a blood film
Stains of the Romanowsky type, e.g. Wright’s, Wright-Giemsa, and May-
Gruenwald-Giemsa are recommended for birds and reptiles in one- or multiple-
step-protocols1, 2, 3, 4. Unfortunately, the quality of the staining with traditional
protocols is inconsistent. The reason is unknown3. Another disadvantage for
daily practice is the need of a labor-intensive fresh preparation of Giemsa’s
solution. Extended experimental work in this field has been done by J.H.
Samour5 resulting in a Wright-Giemsa formula, which gives highly reproducible
results and does not need to be prepared freshly (Table 1). The staining protocol
is excellent for avian and reptilian blood.

Due to their aggressive nature fast staining methods like Diff Quick and
Hemacolor tend to overstain or destroy fine subcellular structures. This is
especially true for reptile blood cells. Nevertheless, they are useful as an addition
to Romanowsky stains for double check purposes: If a suspect finding is seen
both in a Romanowsky and a Diff Quick stained smear, artefacts due to staining
can be excluded. Dry techniques have been proven to be unsuitable for birds6,
corresponding data for reptile blood are not available.
Table 1: Wright-Giemsa Stain for avian and reptilian blood films
Staining Stain
• Flood smear, allow to stand for 3 min • 3 g Wright stain
• Add equal amount of buffer 6.84 powder1
• Mix gently by blowing using a pipette or a straw until • 0.3 g Giemsa stain
metallic green sheen forms on the surface powder2
• allow to stand for 6 min • 5 ml glycerol
• Rinse with buffer allowing to stand for 1 min for • 1000 ml absolute
differentiation methanol (acetone free)3
• Wash copiously with buffer • filtered and stored
• Wipe the back of the smear to remove excess stain
• Prop in rack until dry or use hair dryer

• Cover with Entellan5 and cover glass – gives better


quality than without cover

1
Merck No 1.09203.0025, 2 Merck No 1.09278.0025, 3 Merck No 1.06009.1000, 4
Merck No 1.11374.0100
5
Merck No 1.07961.0100

EVALUATION OF ERYTHROCYTE MORPHOLOGY

In contrast to most mammalian erythrocytes, avian and reptilian erythrocytes are


true nucleus-containing cells. Reptilian cells are larger, flatter, and thinner than
avian erythrocytes. The physiological morphology of both is characterized by a
more-or-less congruent oval-to-round shape of cytoplasm and nucleus. Mature
avian red blood cells display a bright orange colouration, whereas reptile
erythrocytes tend to be paler. Morphologic changes can affect colouration and/or
structure of cytoplasm and nucleus. In general, one has to distinguish between
regenerative and degenerative changes. A regenerative response, also named left
shift, is characterized by an increased amount of immature cells in the peripheral
bloodstream and indicates an increased erythropoietic activity. Degenerative
changes in contrast are changes which do not fit into the normal developmental
series of erythropoiesis. Therefore, the increased occurence of degenerated cells
is always a sign of a clinical problem. Frequent morphologic changes and their
interpretation3, 7, 8, 9, 10 are summarized in table 2. In avian and reptilian samples
the erythropoietic activity is evaluated by estimating the polychromatophilic cells
in Romanowsky stained blood films3. For this purpose a semiquantitative scale,
the polychromatophilic index (PI) has been established for birds by Dein11 and
can be applied also to reptile specimens with little modifications9, 10 (Table 3).
The flow charts outlined in figure 3 are intended as rough guidelines for the
interpretation of common erythrocytic findings.

Concerning reptile blood, the practitioner has to take into account that certain
cells remain pluripotent in the peripheral bloodstream. Thrombocytes, for
example, can transform into red blood cells, if there is an increased demand for
erythrocytes. In this case, the bloodfilm contains multiple cells which display
both erythrocytic and thrombocytic characteristics (Colour plate 1, fig. d).
Blood parasites are a common finding in reptile blood films, most of them being
intraerythrocytic. They are more sporadic in birds. Usually their clinical
significance is more academic than hazardous. Under certain circumstances,
however, a moderate to severe anemia develops and can exacerbate a
pathological condition of different etiology. Some genera such as Plasmodium
and Haemoproteus are relatively easy to recognize as parasites, but others, like
Babesia, Aegyptianella, and Rikettsia-like organisms are readily mistaken for
inclusions of different origin. Criteria for the differentiation of parasitic
developmental stages from non-parasitic structures include the regularity of
occurence, the exclusively intraerythrocytic appearance, and in many cases the
evidence of some granulation in the parasitic inclusion. The specific
identification of hematozoa requires special knowledge and should be left to
specialized laboratories. In case of suspected parasitaemia, additional blood films
should be fixed in absolute methanol for ten minutes and stored in a dust free
environment for future identification12.
INTERPRETATION OF MORPHOLOGICAL CHANGES

The reference range for the pcv in most bird species is approximately 35-55%
and 20-40% in most reptile species. The ability to judge the erythrocytic
morphology in a blood smear enables the practitioner to specify the findings for
the pcv and to refine his first diagnostic and therapeutic approach towards a case.

Its importance becomes clear with the following example: If a parrot displays a
pcv of 30% it makes a significant difference, whether the blood smear shows a PI
of 1, 3, or 5. The higher the PI score is, the closer the patient has to be monitored.
Serial sampling is recommended to follow up the development and to take further
steps in case the situation exacerbates. In the first scenario with a PI of 1, the bird
shows a depressive anemia. This is a common finding with chronic disease. In
these patients, erythropoiesis is decreased, as their catabolic state does not allow
normal cell production. The anemia itself does not require a specific therapy,
although the application Vitamin B6, B12, folic acid and iron has been proven to
be of benefit. Usually it will resolve with the correct treatment of the underlying
cause.
If the PI is scored 3 like in scenario two, the bird is suffering from a regenerative
anemia. In this case, the practitioner is well advised to look for any cause of
hemorrhage and take measures to prevent further blood loss. The differential list
for this scenario includes trauma (e.g. injury, blood-sucking parasites),
coagulopathies (e.g. factor deficiencies, hepatopathies, toxins, hyperestrogenism,
disseminated intravascular coagulopathy in septicemic and viral infections),
ulcerative hemorrhagic inflammation, and secondary tissue bleeding due to
neoplastic conditions.

The third scenario with a PI score of 5 has to be considered an emergency case


and is mostly seen with hemolysis or severe blood loss. The differential list for
hemolysis includes septicemias, blood parasites (Plasmodium spp), intoxications,
autoimmune reactions, and burns. In contrast to the first case, this anemia has to
be considered a primary problem for the patient, as sufficient gas exchange for
the maintenance of elementary body functions is not guaranteed. Rapid
exacerbation of the situation is common in these cases. Thus, immediate
measures have to be taken to stabilize the patient. In case the pcv falls below
20% transfusion of blood or oxyglobin has to be considered.

Basically, these considerations are also valid for reptiles, but seasonal influences
on the status and reactivity of the blood system have to be taken into account
additionally. During hibernation, blood cell poiesis is decreased significantly and
increased again after awakening. Thus, a low pcv and an increased
polychromatophilic index can be physiological in a reptile post hibernation, but
pathologic in seasons of full activity.
DISCUSSION

The purpose of this paper is to encourage practitioners to use a stained blood


film as a quick check technique to get a first impression on the red blood cell
panel of the case presented. It has been kept to a very condensed style. Special
emphasis has been laid on clearly arranged tables, figures and photographs. They
are designed for direct use in daily practice and should help the examining person
to quickly make a decision. Without doubt this condensed design involves the
danger of shortening, which may lead to false conclusions. It has to be
emphasized, that the method presented cannot replace a complete blood cell
count. Any specific disease or etiology mentioned has to be understood as an
example, not as a definitive diagnosis. Nevertheless, the evaluation of the cell
morphology in a blood film, is a rapid and low cost procedure and therefore
predestined for indoor use in daily practice. In combination with the packed cell
volume (pcv) it will give useful information on the patients overall condition and
help the practitioner to take first measures while awaiting results beeing done
outdoors. Even in case of performing a complete blood cell count, morphologic
changes of blood cells contain important additional information and should be
evaluated routinely. This is especially true for reptiles. Due to their ectothermic
nature cell numbers are subject to an increased physiologic variability. Results
from numeric parameters have to be evaluated extremely carefully2. In case of
exotic species, where reliable normative data is lacking, the presence of cells
with morphologic abnormalities is a more reliable index of a pathological change
than numeric results3.
References
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tripotassium EDTA on hematologic values of Hermann's tortoises (Testudo
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