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VETERINARY MEDICINE
MORPHOLOGICAL CHANGES IN RED BLOOD CELLS OF BIRDS AND
REPTILES AND THEIR INTERPRETATION
H. Pendl
SUMMARY
INTRODUCTION
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
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
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.
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