2.3.3.1. Sample collection

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

• the jugular veins (largest on the right)
• 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.

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.

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!

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 WBCcount:

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 peripheralblood of most avian species. They tend to be round cells with a colorless cytoplasm containing eosinophilic or basophilic rod-shaped granules (thegranules 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.

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 aretypically round cells but often show irregularity due to molding around adjacent cells in the smear. The nucleus is usually round (occasionallyindented slightly) and centrally located. The nuclear chromatin is densely clumped or reticulated. The nucleoplasm is often colorless in lymphocytes witha 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.

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 isdense 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 peripheralblood smears, which aids in their identification.

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

2.3.3.3. Chemistry analysis

Remember that micro-chemistry techniques preferred. Some suggested laboratories can be found in the supplementary material Avian/Wildlife Diagnostic Laboratories.

2.3.3.4. 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. 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. At Tufts a multiple test panel usually requires at least 0.3 ml of serum.

The most basic chemistry panel is suggested below (however a full chemistry is more desirable if possible ):

Avian Mini Panel

• Uric acid
• Total protein
• Calcium
• Phosphorus
• Glucose
• Aspartate transaminase (AST)

Note that uric acid is used as an indicator of renal function, and 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".

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

2.3.3.6. Serology, protein electrophoresis and other specific diagnostic tests

Various serology tests are available for chlamydia, giardia, aspergillus and many viral diseases. These tests will be discussed in sections dealing with the specific disease. Use of protein electrophoresis in conjunction with serologic tests and other diagnostic parameters has proved to be useful in reaching a diagnosis. This test is routinely offered in conjunction with other chemistries or serology by the University of Miami diagnostic laboratory.

2.3.3.7. 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 surgica l(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.

2.3.3.8. Radiology

Radiology is an essential diagnostic tool in pet bird medicine practice. It is often part of a routine workup, along with cultures and blood work. Radiographs provide supplemental information often missing from physical examination and allows more accurate assessment of the skeletal system (fractures etc.), respiratory system (lungs and air sacs), gastrointestinal system (size and position of esophagus, proventriculus,ventriculus, intestine, cloaca), and assessment of other internal organs -liver, spleen, kidneys, gonads and uterus.

Details of avian radiography will be covered in an upcoming lecture.

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

• 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

  Intraosseous catheter placement in the ulna