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Veterinarian Technician August 2007 (Vol 28, No 8) Focus: Exotics

Identifying Avian Blood Cells

by Drew Bickford, AS, CVT

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    Hematology is a vital tool for the avian practitioner. Avian patients are well known for disguising signs, and they often show only subtle changes to indicate illness. By evaluating blood samples, veterinary technicians can help to assess the overall health of an avian patient. Often, abnormalities in the blood can be identified before the patient exhibits any outward signs of disease. This benefit makes hematologic assessment an important part of the routine health examination of avian patients.

    Key Points

    • Hematologic assessment is an important tool in diagnosing illness in avian patients.
    • Technicians should be able to correctly identify different cell types.
    • Proper sample handling and staining will ensure the most accurate results.

    Samples Collection

    Because avian species vary greatly in size and structure, venous access sites also vary greatly. Most companion birds are psittacine birds, for which two main sites are used when collecting blood samples: the medial metatarsal vein and the jugular vein.1-3 The jugular vein — more specifically the right jugular vein, which usually is larger than the left — is the site most commonly used for drawing blood in smaller birds, such as conures, cockatiels, and budgerigars. Most birds have a featherless tract called an apterium that lies on each side of the neck and allows easy visualization of the jugular vein. In small birds, this vein may be the only site from which an adequate sample can be obtained. In medium and large birds, the other option is the metatarsal vein, which runs along the medial aspect of the tibiotarsus and distal along the tarsometatarsus. Occasionally, owners report that their bird limps slightly after blood has been drawn from this areaa; however, limping can be minimized or avoided if proper technique is used.

    In several species, including raptors, the easily visible basilic vein and cutaneous ulnar vein are good sites for blood collection. The basilic vein runs along the humerus on the ventral aspect of the wing and then branches; the cutaneous ulnar vein, which is one of these branches, crosses the ulna just distal to the elbow. The basilic vein is commonly used to collect blood from raptors, waterfowl, and ratites.2 In practice, the ulnar vein is the more commonly accessed vessel. Very little soft tissue surrounds these veins, and as a result, hematoma formation is common. Also, because it is difficult to apply a bandage to this area, a cotton ball must be held over the vessel until the bleeding stops. For these reasons, blood is usually not collected from these veins in pet birds. Cutting a toenail into the quick is not recommended because of the poor sample quality, potential contamination, and patient discomfort.b

    Sample Handling

    As with mammalian blood, proper sample handling is important to ensure accurate test results and cell identification. Forcing blood through a needle during either collection or transfer can rupture or distort the cells. Only limited suction should be applied when collecting the sample, and if transfer to a container (e.g., Vacutainer [BD, Franklin Lakes, NJ]) is necessary, the needle should be removed. Because of the fragile nature of avian blood and blood vessels, Vacutainers should not be used during a blood draw. Microtainers (BD) can be used for collecting small samples so that the sample is not diluted with EDTA or heparin.

    Ideally, blood slides should be made immediately after the sample is taken and before an anticoagulant is added because the addition of any anticoagulant will alter the cell morphology with time. If anticoagulant is added to the sample before the slide is made, rapid analysis of cell morphology is important. The slide or coverslip used to make the smear must be high quality, free of debris and oils, and manufactured with a smooth edge. Technique is very important for making blood smears because white blood cell (WBC) distribution on the smear may be affected by poor technique, thus affecting estimated WBC counts.1,c

    Anticoagulants

    Several anticoagulants can be used for avian blood samples. The Raptor Center typically uses a technique of filling the hub of a needle with sodium heparin and then drawing the sample. However, problems can arise with this technique when an adequate sample cannot be obtained, when the sample is overdiluted with anticoagulant, and when the sample is exposed to the anticoagulant for a prolonged period. Heparin can also cause improper staining of cells, which may in turn cause erroneous leukocyte counts, and it can cause poor cellular morphologic features and clumping of leukocytes and thrombocytes, which results in inaccurate cell counts.1 EDTA has been used successfully in avian blood samples; however, drawbacks to its use include altered leukocyte morphology with prolonged exposure.4 In hematologic specimens from some species (e.g., crows, jays, magpies), prolonged exposure to EDTA may cause erythroly­sis and viscosity changes.4 For very small birds and patients that have anemia or poor peripheral circulation, a technique can be used in which a plain needle is inserted into the vessel and the blood sample is collected from the needle hub with EDTA or heparin capillary tubes. If blood chemistries are to be conducted, only lithium heparin is recommended. In the author's experience, lithium heparin is more commonly used with avian samples because a small sample can be used for both hematology and chemistry. Regardless of the anticoagulant used, blood counts should be performed as soon as possible to minimize cell changes caused by prolonged anticoagulant exposure.1

    Staining Techniques

    Staining techniques vary based on individual preference. The main consideration is consistency in the technique. Because the evaluation of many cells is based on color variation, it is important to know what normal cells look like using the chosen staining technique. Romanowsky stains, including Wright's and Diff-Quik, are the most typically used stains because most practices have them readily available. Some manufacturers have designed avian-specific stains to aid in differentiating WBCs.

    Avian Red Blood Cells

    Characteristics

    In avian species, mature red blood cells (RBCs) are oval cells with a central nucleus that stains dark blue with Diff-Quik stain. The cytoplasm normally stains a pink-orange color. Young RBCs begin as round cells that have a medium blue nucleus and light blue cytoplasm. It can be easy to confuse these immature RBCs with medium lymphocytes; however, the two cell types can be differentiated by the position of the nucleus. RBCs have a centrally located nucleus, whereas medium lymphocytes usually have a nucleus that is eccentrically placed. As an RBC matures, it takes on a more oval shape and the cytoplasm color shifts from blue to orange. It is normal to have some young RBCs in peripheral circulation.1,2,5,6

    Evaluation

    Four main factors are used to evaluate avian RBCs: polychromasia , hypochromasia, anisocytosis, and poikilocytosis. A small amount (1+, or 1% to 5%) of polychromasia (variation in cell color) is normal in most avian species.1,2,5 This number system is an example of how to quantify the degree that a factor is present in the RBCs. Alternatively, some veterinary staff may use the terms slight, moderate, marked, and severe to describe the degree a factor is present in the RBCs. Normal cell color variation is up to 5% polychromatic cells (also called 1+ polychromasia); if this value is greater than 5%, the subjective scale is used. Increases in polychromasia indicate RBC regeneration, which may be a response to anemia. Hypochromatic (abnormally pale) cells are usually seen in emaciated or anemic patients. Anemia may be the result of conditions such as lead toxicosis or nutritional deficiency; it may also be associated with infectious disease or parasitism.7 Both the nucleus and the cytoplasm can be hypochromatic, and the cytoplasm can contain decreased levels of heme.1 Care must be taken to ensure that variations recorded are not the result of poor staining. It is common to see a blotchy pattern in hypo­chromatic cells in which areas appear void of cytoplasmic or nuclear material.

    Anisocytosis (size variation) usually accompanies polychromasia as part of a regenerative response. Younger RBCs, in addition to being rounder than mature cells, are usually smaller. Poikilocytosis (variation in cell shape) can sometimes be difficult to determine because it can be caused by poor slide technique rather than disease. One way to distinguish the cause of poikilocytosis is to examine the surrounding cells: If they are all deformed, slide technique may be to blame. Common shape variations seen in birds include spikes (crenation), teardrop or ­spindle shapes, and ballooning of one side of the cell. ­Crenated and distorted cells are often seen with lead toxicosis, and excess numbers of teardrop-shaped cells can indicate septicemia.5,6,8

    Avian White Blood Cells

    Morphologic changes in WBCs can be a valuable indicator of health in birds, and special attention should be paid to these changes.

    Characteristics

    Heterophils

    The heterophil is the avian equivalent of the mammalian neutrophil, and the function of the two cell types is thought to be similar. In many avian species, the heterophil is the most common WBC seen. Although size is usually uniform, there are slight variations in the appearance of heterophils between species.1 Most heterophils have a bilobed or trilobed nucleus that stains dark blue. More lobes develop as the cell ages. The cytoplasm is clear; however, it is usually not visible because the cell is packed with brick-red granules. Granule shape and size vary among species. In most species, heterophil granules are rod shaped. The exact shape of the granules can sometimes be hard to see because they are often densely packed into the cell. Numbers of heterophils increase for the same reasons that neutrophils increase in mammals.1,9 Reasons for increased heterophils include inflammation, stress,2,9 physiologic response, infection, tissue destruction or necrosis (thrombosis and infarction), and drug administration (corticosteroids, estrogen).4

    The presence of immature forms of heterophils (myelocytes) in circulation, known as a left shift, indicates a very severe condition and an increase in intensity or acuity of the inflammatory process. Only severe left shifts can be easily identified.4 The number of available mature heterophils is usually adequate to fight infection, and often, even when WBC counts are two or three times normal (or higher), only mature heterophils are present. Younger stages of heterophils (myelocytes, metamyelocytes) have a nonlobed nucleus and granular-appearing cytoplasm that consists of unformed granules in many shades of red and dark blue. These are the only immature stages seen in peripheral blood that can be easily identified. A third immature stage, the band heterophil, is distinguishable from the mature heterophil only by the shape of the nucleus, which is nonlobed.1 However, because the cytoplasm of band heterophils is packed with granules that can obscure the nucleus, the identification of these cells should be limited. Band heterophils can be identified by using hematoxylin stain, which does not stain the granules, thereby allowing proper evaluation of the nucleus.1,4,9 Toxic changes in heterophils can be identified by using a subjective scale of 1+ to 4+1; alternatively, the types of toxic changes can be indicated. In early stages of toxicity, heterophils start to show mild degranulation along with slight darkening of the cytoplasm. Darkening of the cytoplasm can be difficult to assess in many instances because of the number of heterophil granules. As heterophils become more toxic, increased degranulation, clumping of the granules, and degeneration of the granules and nucleus are seen. Other findings in toxic heterophils include cytoplasmic vacuolation and cellular swelling.1

    Eosinophils

    Eosinophils probably have the most variation in appearance among avian species. In most species, eosinophils have a bilobed nucleus and contain round granules. The granules range in size from very small to large, depending on the species. The nucleus stains dark blue, similar to the nucleus of the heterophil, but the granules usually stain an orange-red color that is clearly different from the heterophil. However, in some species, the granules stain dark blue. In some avian species, the only obvious difference between a heterophil and an eosinophil is the color. Depending on the species, refractivity, cytoplasm color, rod shape, and granulation may be observed.1,6 Increased numbers of eosinophils are often loosely associated with parasitism.

    Basophils

    The appearance of avian basophils is usually different from that of mammalian basophils, especially when the cells are stained with Diff-Quik. Diff-Quik usually causes the granules of avian basophils to dissolve or fall out, leaving the cell with a "Swiss-cheese" appearance. The nucleus usually has a single lobe and is eccentrically placed. The function of basophils in birds is not fully clear, although it is believed to be similar to that of basophils and mast cells in mammals. The cytoplasmic granules contain histamine, and the cells participate in acute inflammatory hypersensitivity reactions.1,4

    Monocytes

    Monocytes are large cells with medium-blue, foamy cytoplasm. Most monocytes in birds do not have vacuoles. The nucleus varies from almost round to horseshoe shaped. In most cases, avian monocytes resemble their mammalian counterparts and their function — including the destruction of foreign organisms and defense against infectious agents — is the same.4

    Lymphocytes

    Lymphocytes tend to be the most difficult WBCs to identify because of their variations in size and shape. Most lymphocytes have a round nucleus and pale blue cytoplasm; however, the nuclear:cytoplasmic ratio varies in birds and is not fully reliable for identification.1,6 In some avian species, lymphocytes will be the most common WBC seen.4 The function of these cells in the immune response is similar to that of their mammalian counterparts.2

    The biggest key to identification of a lymphocyte is the pale blue cytoplasm. Small lymphocytes usually have a small amount of cytoplasm and can sometimes be confused with thrombocytes (platelets), which usually have a more centrally located nucleus and more abundant clear cytoplasm. Medium-sized lymphocytes can be confused with young, immature RBCs.

    Large lymphocytes can be confused with monocytes. Size and nuclear shape are not reliable identifying characteristics for these cells because both types can have round to slightly horseshoe-shaped nuclei.6 Therefore, cytoplasm color is important. If a large lymphocyte and a monocyte are next to each other, the cytoplasm of the monocyte will be an obviously darker blue than that of the lymphocyte. It may be necessary to scan a slide before performing the differential count to see a normal monocyte and a large lymphocyte.

    Identifying reactive changes in lymphocytes is one of the most difficult aspects of avian hematology. Immature forms of lymphocytes are usually grouped into blast category. In the author's experience, blast cells are usually associated with neoplastic conditions or can be found during bone marrow evaluations. Darkening of the cytoplasm is the most common reactive change.2 Problems arise when this darkening crosses into a shade similar to that of monocyte cytoplasm. Because of the size range and nucleus shape of the avian lymphocyte, reactive lymphocytes can be easily confused with monocytes. Again, to limit confusion, it may be helpful to scan the slide to see what the normal monocytes and lymphocytes on the slide look like before evaluating morphologic features and performing a count. There can be normal lymphocytes and reactive lymphocytes on the same slide.

    Lymphocytes can also show some other notable morphologic changes. Azurophilic granules are commonly seen in lymphocytes and appear as medium-blue granules in the cytoplasm of the lymphocyte.1,6 Other reactive changes include scalloped edges and vacuoles.2,6 Conditions associated with reactive lymphocyte changes include acute viral infection5 and terminal viral disease2 (e.g., polyomavirus, herpesvirus).

    White Blood Cell Count

    Because avian RBCs are nucleated, automated machines that scan the nucleus of cells cannot be used to count WBCs in avian samples. There are two generally accepted methods for determining the WBC count in avian species — the Unopette system (BD) or an estimated count using a stained slide method. WBC differentials in birds should always be performed using the highest available microscope magnification to ensure proper identification.

    The Unopette system is a reliable method of determining the WBC count in avian species. The eosinophil kit (Unopette 5877) is recommended because it presents only heterophils and eosinophils in the counting chamber. Standard Unopette systems also present thrombocytes in the counting chamber, causing difficulty in obtaining an accurate count. However, because the Unopette eosinophil kit only counts heterophils and eosinophils, a differential using the stained slide method should also be conducted to determine the percentage of lymphocytes, monocytes, and basophils. Together, the values obtained from the stained slide differential and the Unopette kit equal the absolute WBC count.

    Alternatively, an estimated WBC count can be determined by using only the stained slide method. In this method, WBCs in eight to 10 monolayer fields are counted on 40x magnification.5 These numbers are added, and the total is divided by the number of fields counted to determine an average number of WBCs. This number is then multiplied by 2,000 to give an estimated WBC count.2,4

    The standard mean packed cell volume (PCV) for birds is 40%. If the patient is anemic (i.e., the observed PCV is 35% or less), the WBC count should be corrected by dividing the observed PCV by 40% and multiplying the result by the estimated WBC count. This correction method is used by The Raptor Center and has proven clinically useful and repeatable. To increase the accuracy of the differential, it is often recommended to perform counts of up to 400 rather than 100 WBCs.4

    Conclusion

    Competence in avian hematology takes time and practice. However, many of the principles used in mammalian hematology also apply to avian hematology. By gaining experience in identifying avian blood cells, technicians can provide even more useful information to the veterinarian and to clients with avian pets.

    1. Campbell TW, Ellis CK: Hematology of birds, in Avian Hematology and Cytology, ed 3. Ames, IA, Blackwell Publishing Professional, 2007, pp 3-50.

    2. Fudge AM: Avian blood sampling and artifact considerations, in Fudge AM (ed): Laboratory Medicine: Avian and Exotic Pets. Philadelphia, WB Saunders, 2000, pp 1-8.

    3. Samour J: Diagnostic value of hematology, in Harrison GJ, Lightfoot TL (eds): Clinical Avian Medicine. Palm Beach, FL, Spix Publishing, 2006, pp 587-607.

    4. Latimer KS, Beinzle D: Determination and interpretation of the avian leukogram, in Feldman BF, Zinkl JG, Jain NC (eds): Schalm's Veterinary Hematology. Baltimore, Lippincott, 2000, pp 417-430.

    5. Fudge AM: Avian complete blood count, in Fudge AM (ed): Laboratory Medicine: Avian and Exotic Pets. Philadelphia, WB Saunders, 2000, pp 9-18.

    6. Lucas AM, Jamroz C: Atlas of Avian Hematology. Washington, DC, United States Department of Agriculture, 1961.

    7. Fudge AM: Disorders of avian erythrocytes, in Fudge AM (ed): Laboratory Medicine: Avian and Exotic Pets. Philadelphia, WB Saunders, 2000, pp 28-34.

    8. Samour J: Clinical and diagnostic procedures, in Avian Medicine. London, Mosby Publishing, 2000, pp 28-42.

    9. Fudge AM, Joseph V: Disorders of avian leukocytes, in Fudge AM (ed): Laboratory Medicine: Avian and Exotic Pets. Philadelphia, WB Saunders, 2000, pp 19-27.

    aHellenga C: Personal communication, Winter Park Veterinary Hospital, Winter Park, FL, 2005.

    bFor more information on avian venipuncture, including technique, recommended blood volume, and proper restraint, see "Venipuncture in Psittacine Birds," which appeared on page 721 of our October 2004 issue.

    cFor more information on how to make a blood slide, see "Peripheral Blood Films — A Forgotten Art?", which appeared on page 616 of our September 2004 issue.

    References »

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