Psittacine Blood Collection and Hematology:  Basics for the Veterinary Practitioner

Kelly M. Phillips

Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602 USA

Abstract: Due to its intrinsic properties, avian blood is unable to be processed through the inexpensive automated hematology equipment that private practitioners use for mammalian blood samples. Therefore, veterinarians frequently rely on commercial diagnostic laboratories for evaluation of hematological parameters of their companion bird patients, instead of making their own assessment. This paper will describe simple methods for appraising companion bird blood samples in the private practice.

Key Words: Avian, Psittacine, Hematology, Blood, Leukocyte

Companion psittacine birds are becoming increasingly popular, and as a result, veterinary practitioners are experiencing an increase in the number of clients presenting their birds for medical evaluation. Unfortunately, our understanding of companion bird medicine lags behind that of domestic mammals. Not only are we faced with an entirely different class (Aves) of animal, but within that class we are forced to struggle with differences between genera as well as species. There exists no generic bird. However, as practitioners, we need to start somewhere.

Many veterinarians have selected to send avian blood samples to independent laboratories for hematologic analysis. Common reasons for this include perceived time constraints, or a lack of confidence in technique. Unfortunately, the patient, the client, and the practitioner may all be adversely affected by this decision. Due to the length of time required for shipping and processing, a patient may be improperly diagnosed and therefore improperly treated, blood cell death and lysis during shipping may affect laboratory results, and samples may be lost or damaged en route to the laboratory. The cost of shipping and laboratory fees must be passed on to the client, and in some cases may prove prohibitive. To the avian veterinarian, shipping samples may result in decreased potential income, but more importantly, it precludes many opportunities to learn.

When these factors are considered, in-clinic evaluation of psittacine hematologic parameters can only make sense. With some practice and a good reference source, the avian practitioner can be rapid and reliable, which results in better service to the client and the patient. Given some routine cytologic stains, a hemacytometer, and a microscope, a great amount of information about a patient can be collected and treatment can be directed appropriately. This discussion will focus on the evaluation of erythrocytes and leukocytes in psittacine bird blood samples.

Avian blood volume is approximately 10% of body weight.^1,2,9,12,13 A healthy psittacine bird can lose up to 10% of its blood volume and suffer no ill effects. As practitioners, we therefore limit ourselves to that 10% of blood volume for collection and analysis. Thus, 4 mL (1% of body weight) could safely be taken from a 400 gram amazon and would provide more than enough blood for hematology as well as chemistry evaluation. Conversely, withdrawing an equal percentage of blood from a 35 gram budgerigar would only provide 0.35 mL, thereby restricting the scope of testing that would be possible.

A variety of locations have been described for blood collection from avian species,^1,2,9,12 although not all are appropriate for companion psittacines. The method preferred by the author is jugular venipuncture. The right jugular vein of psittacines is usually easily visualized and cannulated with a 22 or 25 gauge needle. Smaller birds can be restrained and manipulated in one hand for phlebotomy, whereas the large macaws and cockatoos may require an assistant for patient restraint. Blood collection in this manner is rapid, thus sample clotting is less likely. Hematoma formation is uncommon with direct pressure on the venipuncture site.

The cutaneous ulnar (basilic) vein and the medial metatarsal vein can also be used for venipuncture. The cutaneous ulnar vein lies across the medial surface of the humero-radioulnar joint, and the medial metatarsal vein courses along the medial side of the tibiotarsus. These sites are considered second choice by the author, due to their increased degree of difficulty in small birds, their need for an assistant for restraint, and their tendency to form a large hematoma or bleed through the skin puncture even after the application of direct pressure.

Other methods of blood collection in the literature should be rarely utilized due to their inherent hazardous nature, or their resulting in inaccurate analysis results. The first category includes cardiac puncture and occipital venous sinus puncture. These locations should never be used in companion birds unless they are immediately euthanized.^1,2 Toenail clipping can be residually painful, often results in skewed cell distributions, and limits the amount of blood that can be collected. When no method of venipuncture is successful, then a toenail clip may be indicated. Finally, some authors mention skin puncture or incision as a method of blood collection. In birds too small for venipuncture, the vein (cutaneous ulnar, medial metatarsal, or external thoracic) is incised through the skin and blood collected from the wound. Again, this method allows only a small sample to be taken, and the risk of sample contamination is high.

For the measurement of hematologic parameters, anticoagulated blood is preferred. However, some authors advocate the use of freshly drawn blood without anticoagulant for the preparation of blood smears for staining,^12,14 or short term contact with EDTA.^1,2 Options for avian blood anticoagulants include EDTA and sodium citrate. Heparin (sodium, ammonium, or lithium) is also used, but has been observed to interfere with the staining of the blood cells and may cause cell clumping.^2,11 Additionally, although the EDTA microtainer tubes are convenient and prevent sample dilution, the EDTA does cause cell lysis and can complicate the differential count. With large volume samples, the author prefers the use of sodium citrate, although dilutional errors must be factored in when making final calculations. Appropriately diluted sodium citrate samples must have all of their final hematologic values multiplied by 1.1 to account for the dilution.

Blood smears should be made immediately after blood collection, even if the rest of the blood evaluation cannot be performed. Multiple methods exist for making smears: coverslip to coverslip, coverslip to slide, and slide to slide. The preparation of smears that are of optimum thickness and are sufficiently feathered at the edge can take practice. Stains for assessing avian blood smears include the Romanowsky stains such as Wright’s and Giemsa, as well as the rapid cytology stains like Diff Quik and Hemacolor. Since the cell appearances will vary from stain to stain, being consistent with the type of stain used may prevent confusion. The evaluation of reticulocytes requires the use of a vital stain such as new methylene blue. Excellent descriptions of these stains, as well as others, and their techniques can be found in the literature.^1,2

Once the blood smear(s) have been stained, the evaluation of the anticoagulated sample follows. A PCV can be obtained from blood filled microhematocrit tubes spun at approximately 12,000 G for five minutes. The red blood cell count can be determined easily using the erythrocyte Unopette system, or the Natt and Herrick’s method. Both of these techniques require a Neubauer-ruled hemacytometer for enumerating the stained red blood cells. Once the blood is diluted and stained, it can be applied to the hemacytometer and allowed to settle for five to ten minutes. At that time, the hemacytometer is placed on the microscope stage, and the red cells counted according to the standard enumeration procedure. The total count is multiplied by a factor of 10,000 to obtain the number of erythrocytes per microliter of blood.

Stained erythrocytes appear oval or oblong in shape, with pale orange to pink cytoplasm. The erythrocyte nucleus is also typically oval, and sits in a central location within the cell (Fig. 1). The nucleus is uniformly basophilic, becoming more condensed as the cell ages. Polychromatophilic erythrocytes are more basophilic, and their nuclear chromatin is less condensed (Fig. 2). The degree of polychromasia in psittacines and other avian species is usually 5% or less.^1,10,11 The average lifespan of an avian red blood cell is 28 days.

Fig. 1. Wright's stained blood smear from a Caique. Normal erythrocytes.	Fig. 2. Wright's stained blood smear from an Eclectus parrot. Multiple polychromatophilic erythrocytes.

Various abnormalities may be seen in avian erythrocytes. True abnormalities must be differentiated from staining artifacts and/or improper smear preparation, such as smudge cells and perinuclear rings. The author has observed an increased frequency of smudge cells in EDTA anticoagulated blood versus blood placed in sodium citrate. There are differing opinions on which cell lines are most likely to become smudged.^6,12

Basophilic stippling of the erythrocyte cytoplasm may be the result of a regenerative response or, rarely, heavy metal toxicity.^1,2,11,14 Abnormally shaped erythrocytes may be seen occasionally. Round red blood cells with oval nuclei are reflective of accelerated erythropoiesis. Psittacine birds may be infected with hemoparasites of both the erythrocytes and leukocytes, such as Haemoproteus, Leucocytozoon, and Plasmodium spp.

Although some practitioners exclusively estimate total leukocyte counts directly from a blood smear, the results are variable and not very accurate. When no other method is available, a total leukocyte count can be guessed by counting the number of leukocytes per high power field (40x) in ten different fields, averaging them, and multiplying by 2,000 to get the number of leukocytes per mL of blood.

Better methods of total leukocyte count include the indirect Unopette 5877 eosinophil system, or the direct Natt and Herrick’s method. As with the total erythrocyte count, both of these methods require the availability of a hemacytometer. Use of the Natt and Herrick’s allows for enumeration of both leukocytes and erythrocytes simultaneously. With the Unopette 5877 eosinophil system, only heterophils and eosinophils are directly enumerated. The resulting number is then easily mathematically adjusted, based on cell counts in the differential, to produce a final total leukocyte count.

For example, the following values are from a blood sample collected in EDTA from an eclectus parrot:

Unopette 5877 Average Cell Count = 324

Since 11,520 heterophils and eosinophils per mL = 66 + 2 = 68% of the cells, then

Had the sample been anticoagulated in sodium citrate, the total count would need to be multiplied by a factor of 1.1: 1.1 x 16,942 = 18,636 leukocytes per mL.

Modern technology has not yet developed a method for evaluating leukocyte differentials automatically, due to the inability of cell counters to separate nucleated erythrocytes and thrombocytes from avian leukocytes. This is complicated by the size similarity between thrombocytes and small lymphocytes, as well as between large lymphocytes and monocytes. In addition, there are slight interspecies differences in the appearance of some cell lines, such as eosinophils.^1,2,11 Consequently, avian medicine relies upon the manual identification of leukocytes by practitioners, laboratory technicians, and board certified pathologists with varying degrees of skill and experience. The practitioner just starting out with avian differentials needs to keep this in mind Much of the science of avian hematology is truly an art. The importance of practice cannot be overemphasized. Stain a smear of every avian blood sample taken in your clinic, even if it is only for DNA sexing. The "normal" smears are as important as the abnormal ones.

Types of psittacine leukocytes include the granulocytes, which are heterophils, eosinophils, and basophils, and the agranulocytes or mononuclear cells (monocytes and lymphocytes). Much of our understanding of the immune cells of psittacine birds is based on assumption and from a few studies in poultry. In that aspect, sometimes enumerating the differential is easier than the interpretation of those results. Regardless, properly identifying the avian cell lines in a blood smear is of great significance.

Heterophils function similarly to the mammalian neutrophil.^2,14 In some avian species they are the most common peripheral leukocyte. They are typically round, with colorless cytoplasm and many eosinophilic, rod shaped to spherical granules which may be observed to have a central refractile region. The shape of the granules is occasionally difficult to discern, and variations in staining may affect their eosinophilic properties. These granules may partially obscure the nucleus, which usually has two or three lobes and has very coarsely aggregated, purple chromatin.^1,2,12   Immature heterophils are rarely seen in psittacine blood smears, but are identified by their increased cytoplasmic basophilia, decreased nuclear lobation, and immature granules. Examples of mature peripheral heterophils from various psittacine species are shown below.

Fig. 3. Diff-Quik stained blood smear from a cockatoo. Mature heterophils.	Fig. 4. Wright’s stained blood smear from an Eclectus parrot. Mature heterophils.

Fig. 5. Wright’s stained blood smear from an Eclectus parrot. Mature heterophils. Note the distinct rod-shaped granules.	Fig. 6. Wright’s stained blood smear from a Lesser Sulfur Crested cockatoo. Mature heterophil.

Fig. 7. Wright’s stained blood smear from an African Grey parrot. Mature heterophil and thrombocyte.	Fig. 8. Diff-Quik stained blood smear from an African Grey parrot. Mature heterophil.

Fig. 9. Diff-Quik stained blood smear from a Moluccan cockatoo. Mature heterophils. Note the large smudge cell.

Psittacine eosinophils also tend to be round, although there may be more variability in shape than in the heterophil.¹ They have slightly basophilic cytoplasm and numerous round, intensely eosinophilic granules. The granules typically lack a central refractile region, and are brighter than the heterophil granules on the same smear. Cells containing large, round, colorless granules have been identified in the blood smears of various bird species, including psittacines. These cells are presumed to be eosinophils due to the presence of normal heterophils, basophils, and lymphocytes as well as a lack of typical eosinophils in the blood film. Eosinophil nuclei stain purple, and have similar lobation and chromatin clumping as heterophils,^1,2 although the nucleus may be more blue and obvious. The functions of eosinophils in psittacine birds are not well understood. There does not appear to be an increase in these cells due to the presence of parasites as is found in mammals.² Eosinophils are rarely seen in the peripheral blood of psittacines,¹⁴ although they may be common in other birds. See Figure10 below.

Fig. 10. Diff-Quik stained blood smear from a Screech Owl. Mature eosinophils.

Basophils in psittacine blood smears are not commonly seen. These cells resemble and function identically to mast cells.² They can be easily identified by their large, round, deeply basophilic granules and typically unlobed nucleus. The nucleus may be centrally located or eccentric, is light blue, and can be obscured by the granules. Some peripheral mature basophils are shown below.

Fig. 11. Wright’s stained blood smear from a cockatoo. Mature basophil.	Fig. 12. Wright's stained blood smear from an Eclectus parrot. Mature basophils.

Fig. 13. Wright’s stained blood smear from a Lesser Sulfur Crested cockatoo. Mature basophil.	Fig. 14. Wright’s stained blood smear from an African Grey parrot. Mature basophil.

Fig. 15. Diff-Quik stained blood smear from a Scarlet macaw. Mature basophil with heterophil.

Of the mononuclear cells, the lymphocyte is by far the most common. In fact, in some psittacine species (eg: Amazons, Eclectus) it is the most common leukocyte in the peripheral circulation.⁷ Lymphocyte identification can be difficult for several reasons: lymphocytes may be present in three different groups according to size, small lymphocytes may be confused with thrombocytes, and large lymphocytes may resemble monocytes. Normally, most circulating lymphocytes are small or medium in size, round in shape, with a centrally located nucleus, sometimes indented, having densely aggregated chromatin. Medium and large lymphocytes may have obvious reticulated chromatin. The amount of cytoplasm present is typically scant in the small lymphocytes, with progressively more in the larger cells. A general rule for all lymphocytes is a high nuclear to cytoplasmic ratio.^1,2,11,12 The cytoplasm tends to be only slightly basophilic, although some cells will demonstrate a deeper basophilia at the periphery of the cell membrane. The cells occasionally have pseudopodia or membrane blebs. See the following figures.

Fig. 16. Diff-Quik stained blood smear from a cockatoo. Medium to large lymphocytes. Note the reticulated nuclear chromatin.	Fig. 17. Wright’s stained blood smear from an Eclectus parrot. Small lymphocyte with pseudopodia. Compare with nearby thrombocytes.

Fig. 18. Wright’s stained blood smear from an Eclectus parrot. Medium lymphocyte with reticulated chromatin.	Fig. 19. Wright’s stained blood smear from a Lesser Sulfur Crested cockatoo. Small lymphocyte adjacent to thrombocyte.

Fig. 20. Wright’s stained blood smear from an African Grey parrot. Medium to large lymphocyte with pale blue cytoplasm.	Fig. 21. Wright’s stained blood smear from an African Grey parrot. Small lymphocyte.

Fig. 22. Diff-Quik stained blood smear from an Umbrella cockatoo. Small lymphocytes.	Fig. 23. Diff-Quik stained blood smear from an Umbrella cockatoo. Reactive lymphocyte.

Fig. 24. Diff-Quik stained blood smear from an Amazon parrot. Small lymphocytes.	Fig. 25. Diff-Quik stained blood smear from an Amazon parrot. Medium to large lymphocyte plus mature heterophil.

Fig. 26. Diff-Quik stained blood smear from an Amazon parrot. Multiple small lymphocytes.

At this time, there is no readily available method for distinguishing between psittacine T-lymphocytes and B-lymphocytes. On occasion, reactive lymphocytes may be seen, which can be identified by their increased size, strongly basophilic cytoplasm, and homogenous, unclumped chromatin. A perinuclear clearing, or Golgi zone, may be observed. The nucleus is typically central. These cells appear as a result of antigenic stimulation, such as inflammatory diseases, and indicate an immune response. Plasma cells are activated B-lymphocytes, and may also be seen as part of an immune response. They are large cells, round or slightly oval in shape, with a large amount of deeply basophilic cytoplasm. The nucleus is eccentric, and has the smooth, homogenous chromatin pattern similar to reactive lymphocytes. They also have an easily identifiable Golgi zone.²

Monocytes are mononuclear cells that are normally less common than lymphocytes in psittacine blood. They are large cells having a wide variety of shapes, ranging from round to rhomboid. The nucleus also varies in shape, and may be round, kidney shaped, or bilobed. They may very closely resemble large lymphocytes, although monocyte cytoplasm tends to be finely granular, darker in color, and may contain vacuoles. Additionally, two zones may be identified in the cytoplasm, including a perinuclear light staining area and a dark staining area adjacent to the cell membrane. The chromatin is less reticular or clumped compared to lymphocytes, as well.^1,2,11 Some examples of monocytes can be seen below.

Fig. 27. Wright’s stained blood smear from a Lesser Sulfur Crested cockatoo. Monocyte with cytoplasmic vacuole.	Fig. 28. Wright’s stained blood smear from an African Grey parrot. Monocyte with several cytoplasmic vacuoles.

Fig. 29. Diff-Quik stained blood smear from an Umbrella cockatoo. Monocyte.	Fig. 30. Diff-Quik stained blood smear from a Moluccan cockatoo. Monocyte.

Once hematologic or other data are obtained from a patient, what happens next? In order to evaluate the data, we must compare them with other data from similar animals that are clinically healthy. The data for comparison are generally arranged into a reference range, which accounts for both the low and high extremes of what values are found in healthy animals. The reference interval is a statistical narrowing of the reference range.  The terms "reference range" and "reference interval" are preferred over the term "normal values" when describing a distribution of hematologic or biochemical parameters from a sampled group. This is due to the fact that it is difficult, if not impossible, to definitively determine a "normal" value for anything in a biological system such as an animal, and in addition, we know that statistically only 95% of samples from clinically healthy animals will fall within a normally distributed reference interval or range. That means that 5% of clinically healthy, or "normal" animals qualify as "abnormal" when evaluated by that particular test.

How are reference intervals established? Typically, a clear idea of parameters that must be met in order to qualify as a clinically healthy animal is needed. These criteria may include species, age, sex, pregnancy status, medications taken, sampling site, and sample storage conditions as well as others.⁵ The method of evaluating the sample and the statistics used to calculate the reference interval must also be considered. The more diverse the sample population, the wider the resulting interval will be. Likewise, the more homogenous the population sampled, the narrower the reference range.

This methodology brings about some inherent problems. For example, can we apply a reference interval that is derived from samples obtained from a population that lives in the southeastern U.S. to an animal that lives in the Pacific northwest? Or is it appropriate to use ranges established by one laboratory to evaluate samples processed in a different laboratory? What about using ranges established for one species to assess another? Only a handful of studies have established hematologic reference intervals for free ranging psittacine birds in their natural habitats.^8,10 Can we assume that those values are useful to the avian veterinarian as well?

These questions are all applicable when we consider companion bird medicine. They are issues that we deal with on a regular basis. There is no easy answer at this point – we are bound by the restricted information available about many different species, and are generally unaware of how reference ranges are established at the laboratories that we use routinely. In fact, many of the avian reference ranges published in veterinary texts refer back to the same two or three sources of (assumed) captive bird information.^2,3,4,9,11 Some laboratories that process avian blood do not even provide reference intervals with their results. Therefore, just like the practitioner evaluating hematology in-house must utilize ranges provided by other laboratories (in texts), many practitioners are forced to do the same even when sending their samples elsewhere for analysis. There is currently no way around this. Simply recognizing the problem is a significant step when making correlations to individual patients.

In summary, avian hematology can easily be incorporated into the routine testing done by practitioners at their clinic. The time and effort put into learning the fairly simple techniques, as well as the differentiation of blood cells, will be far outweighed by the benefits of rapid and accurate results. All members of the practitioner-client-patient relationship benefit from this service.

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2. Campbell TW. Hematology. In Ritchie BW, Harrison GJ, Harrison LR (Eds.): Avian Medicine: Principles and Application. Wingers Publishing Inc., Lake Worth, FL, 1994, pp176-198.

3.  Clubb SL, Schubot RM, Joyner K, et al.: Hematologic and serum biochemical reference intervals in juvenile cockatoos. J Assoc Av Vet 5(1): 16-26, 1991.

4.  Clubb SL, Schubot RM, Wolf S: Hematologic and serum biochemical reference intervals for juvenile macaws, cockatoos and eclectus parrots. In: Schubot RM, Clubb KJ, Clubb SL (Eds.): Psittacine Aviculture: Perspectives, Techniques and Research. Avicultural Breeding and Research Center, Loxahatchee, FL, 1992, pp 18.1-18.20.

5.  Duncan JR, Prasse KW, Mahaffey EA. Veterinary Laboratory Medicine: Clinical Pathology, 3^rd ed. Iowa State University Press, Ames, 1994, pp 231-232.

6.  Fudge AM. Blood testing artifacts: interpretation and prevention. Seminars in Avian and Exotic Pet Medicine, 3(1): 2-4, 1994.

7.  Fudge AM. Problem-oriented approach to blood panel interpretation. Proceedings of the Association of Avian Veterinarians, St. Paul, MN, 1998, pp 285-299.

8.  Garcia del Campo AL, Huecas V, Fernandez A, Puerta ML. Hematology and blood chemistry of macaws, Ara rubrogenys. Comp Biochem Physiol A 100(4): 943-944, 1991.

9.  Johnson-Delaney CA. Exotic Companion Medicine Handbook for Veterinarians. Wingers, Lake Worth, FL, 1996, pp 11-16.

10. Karesh WB, del Campo A, Braselton WE, Puche H, Cook RA: Health evaluation of free-ranging and hand-reared macaws (Ara spp.) in Peru. J Zoo Wildl Med 28(4): 368-377, 1997.

11. Rosskopf W, Woerpel R: Diseases of Cage and Aviary Birds, 3^rd Ed. Williams and Wilkins, Baltimore, 1996, p1059.

12. Rupley AE. Manual of Avian Practice. WB Saunders Co., Philadelphia, 1997, pp 345-360.

13. Sturkie PD. Blood: Physical characteristics, formed elements, hemoglobin, and coagulation. In: Sturkie PD, (Ed.): Avian Physiology. Springer-Verlag, New York, 1976, pp 53-75.

14. Van der Heyden N. Evaluation and Interpretation of the Avian Hemogram. Seminars in Avian and Exotic Pet Medicine 3(1): 5-13, 1994.