2.3.3.1. Sample collection

The most commonly used sites for blood collection in birds include:

• the jugular veins

• cutaneous ulnar veins

• tibiotarsal/metatarsal veins

Volumes needed rarely exceed 1 ml, and should not exceed 1% of the animal's body weight in grams.

Phlebotomy

Collection of such small volumes require the use of small needles (25g, 26g, 27g), and small receptacles (e.g. Microtainers, hematocrit tubes, unopettes). Avian blood clots very quickly and should be collected and transferred to the appropriate receptacle as quickly as possible.

Microtainers

2.3.3.2. Complete blood count

Avian CBCs are often performed in-house, but may be sent out to a diagnostic laboratory. To perform a CBC, you will need at least one smear (differential), one unopette (WBC count), and one hematocrit tube(PCV, TSP). Ideally a full CBC is performed on 0.2 ml EDTA blood (Microtainer).

Avian RBC's are big and nucleated!

Avian blood smear

WBC counts are accomplished using eosinophil unopettes. The unopette comes with its own pipette and a fixed volume of diluting fluid, which allows for an accurate dilution of the blood sample. One disadvantage is that the phloxine diluent stains only granulocytes (heterophils, eosinophils, and basophils); therefore, the count obtained must be corrected, since mononuclear cells (lymphocytes and monocytes) are not included. A hemacytometer is used to produce the granulocyte count. A white blood cell differential count must then be done to accurately complete the WBC count:

TOTAL WBC/ul EQUALS hemacytometer count (10 squares)X 32* divided by the percentage of granulocytes/100 (decimal)

*dilution factor of unopette

Differentials require practice and experience on the part of the veterinarian or technician performing the analysis. Several good references exist to help develop proficiency in identifying the various white blood cells. The following characteristics are typical of the different cell types:

Heterophils Heterophils are the most common leukocyte in the peripheral blood of most avian species. They tend to be round cells with a colorless cytoplasm containing eosinophilic or basophilic rod-shaped granules (the granules may be spherical in some species). Heterophil granules usually have adistinct central body that appears refractile. Mature heterophils have a lobed nucleus (usually two or three lobes) with a coarse, clumped chromatin that stains purple. The nucleus is often partially hidden by the cytoplasmic granules.

Heterophils

Eosinophils

Avian eosinophils are typically round but have a greater tendency toward irregularity than do heterophils. The cytoplasm stains a clear, pale blue (in contrast to the colorless cytoplasm of heterophils) and contains round eosinophilic granules (or rod-shaped in some species). In most birds, the granules of eosinophils have different staining qualities from heterophil granules from the same blood smear. The eosinophil granules may appear duller or brighter than the heterophil granules. Eosinophil granules tend to lack the central body found in heterophil granules. Eosinophil nuclei are lobed with coarse, clumped chromatin that stains purple, and the eosinophil nucleus often stains bluer and is usually more noticeable than the heterophil nucleus.

Basophils

Avian basophils tend to be round cells with a round, centrally located nucleus. The nucleus stains a light blue and is often hidden by the cytoplasmic granules. The cytoplasmic granules are deeply basophilic and may dissolve or coalesce in Wright’s stain (or other alcohol-solubilized stains). We rarely see basophils because they may dissolve or coalesce in Wright's stain(or other alcohol-solubilized stains like Hema 3).

Lymphocytes Lymphocytes are the most common leukocyte found in the peripheral blood of some avian species. They can be classified into three groups according to cell size (small, medium, and large). Most normal mature lymphocytes in the peripheral blood are small or medium. Lymphocytes are typically round cells but often show irregularity due to molding around adjacent cells in the smear. The nucleus is usually round (occasionally indented slightly) and centrally located. The nuclear chromatin is densely clumped or reticulated. The nucleoplasm is often colorless in lymphocytes with a reticular chromatin, and dark in those with dense chromatin clumping. The amount of cytoplasm varies from a narrow band surrounding the nucleus in small lymphocytes to a moderately wide band in medium and large lymphocytes. A high nuclear to cytoplasmic ratio is typical of lymphocytes. The cytoplasm is homogenous and weakly basophilic. Occasionally, lymphocytes have irregular cytoplasmic projections or blebs.

Lymphocytes

Monocytes

Monocytes are large leukocytes that are irregular in shape. The nuclei vary from round to bi-lobed and often have a delicate, lace like reticular chromatin. Chromatin clumps may be present but are few in number. The cytoplasm of monocytes stains blue-gray with a finely granular appearance and occasionally contains vacuoles. The cytoplasm may contain two distinct zones; a light-staining area and a more deeply stained area. Monocytes occasionally contain fine (dust like) eosinophilic cytoplasmic granulation.

Thrombocytes These cells can be confused with lymphocytes, so care must be taken not to count them as part of the WBC differential count. Mature thrombocytes are oval cells that are smaller and more rounded than mature erythrocytes. The thrombocyte nuclei are larger in relation to the amount of cytoplasm and more rounded than erythrocyte nuclei. The nuclear chromatin is dense and clumped. The cytoplasm is clear but not homogenous and often has a reticulated appearance. Avian thrombocytes frequently contain one or more red granules at the poles of the cell. Thrombocytes tend to clump in peripheral blood smears, which aids in their identification.

Thrombocytes and Heterophils

(Descriptions courtesy of Carolyn Corsiglia; Good reference: Campbell, Avian Hematology)

2.3.3.3. Chemistry analysis

Micro chemistry techniques are required for such small volumes and are available, but not with every diagnostic pathology laboratory. You must locate a lab that can perform the tests you want on the volumes you want (see APPENDIX for suggested diagnostic laboratories). There are no special "avian" requirements for general chemistry tests (including thyroid), and tests can be performed as they would on dog or cat serum. Individual tests using dry chemistry techniques can often be run on 10μl (0.01 ml) of serum.

Note that in birds, uric acid is used as an indicator of renal function, and in psittacines, AST is most predictive of hepatic insult although still not a very specific indicator of hepatocellular damage. Bile acids are now being used more and more in the diagnosis of liver disease in birds.

"Big 3" - A quick and easy emergency monitoring test using only 2 hematocrit tubes of blood can predict PCV, TSP, and blood glucose. Note that the azostick test has been dropped from the usual "Big 4".

2.3.3.4. Normal Hematologic and Biochemical Parameters of some species of birds

2.3.3.5. Serology, protein electrophoresis and other specific diagnostic tests

Various serology tests are available for chlamydophila, giardia, aspergillus and many viral diseases. These tests are discussed in sections dealing with the specific disease. Protein electrophoresis in conjunction with serologic tests and other diagnostic parameters has been used to help determine a diagnosis of some of these diseases, however, the accuracy of this strategy is in question. Protein electrophoresis is routinely offered in conjunction with other chemistries or serology by the University of Miami diagnostic laboratory.

2.3.3.6. Sex determination

Blood sampling for sex determination

Most psittacine species (especially South American) are sexually monomorphic. Individual owners as well as breeders often want to know the sex of their bird. The traditional method for sexing has been surgical (laparoscopy, discussed later), however less invasive techniques are now available.

DNA sexing through a blood sample is the technique which has been most successful. This technique requires only a drop of blood providing sufficient DNA to perform the test with a specially developed DNA probe. The laboratory that originated this test (ZOOGEN ) is also now keeping DNA records on every bird as a method of identification (DNA fingerprinting) for future reference.

Laparoscopy is still performed as a reproductive tool to better evaluate the exact condition of the reproductive organs and to predict breeding readiness, problems, etc.

2.4.1.1. Evaluation of hydration status

• History: anorexia, diarrhea, polyuria, dyspnea/hyperventilation, heat stress

• Physical appearance: dull sunken eyes, tacky oral mucous membranes, increased skin turgor

• PCV, TSP - may be helpful and/or misleading

• Body weight monitoring (once or twice daily)

2.4.1.2. Estimation of hydration needs

Estimation of hydration needs

• Daily maintenance requirements of

  • 60-100 ml/kg/day

  • OR 1 to 1.5 x MEC in ml/day = 1 to 1.5 x (78 X BW_kg^0.75)

• PLUS estimate of dehydration (percent)

• PLUS continuing losses

2.4.1.3. Choice of fluids

Choice of fluids to use for maintenance and supplementation is made based on the same principles used in mammals and include normal saline, 5% dextrose, lactated Ringers, and the various colloidal solutions such as dextrans and hetastarch.

2.4.1.4. Methods of fluid administration

Methods of administration can involve any of the following, depending on the situation at hand. The colloidal solutions must be given intravenously or through an intraosseous catheter.

• Oral fluids - gavage

• Subcutaneous fluids - inguinal, lumbar, patagium (wing). The use of subcutaneous fluids is the most common technique and is very successful unless rapid fluid delivery is required (shock, severe dehydration, acute blood loss, hypovolemia).

• Intravenous fluids - cutaneous ulnar/brachial v. jugular vein, medial metatarsal vein; a 24 ga. catheter may be placed in most sites in any bird larger than a cockatiel

• Intraosseous fluids - distal ulna or proximal tibiotarsal bone

IO Catheter