Avian & Exotic Clinic of Palm City, Inc. and Animal Health Clinic, Inc., Palm City, FL, USA and Jupiter, FL, USA
Lecture outlines basic avian and reptile hematology, chemistry, and cytology. Similarities and differences in birds and reptiles will be illustrated and discussed. The avian and reptile veterinarian must know what tests are available, when to run them, and how to interpret them. If you can diagnose it or ID it, you can treat it!
Preanalytical Blood Collection and Sample Preparation1-4
Collect less then < 1% of a bird's or reptile's body weight. Use heparinized, size appropriate, pediatric Microtainer® or Capiject® tubes.
Use the larger right jugular vein, it is the best site for most birds and reptiles. Although the right jugular works well in all psittacines, most passerines, and other species. Columbiformes have a diffuse venous plexus rather than a jugular vein. Other sites include the basilic and medial metatarsal vein or a cleaned toenail clip when all other options are not possible or as a last resort. Many reptiles can be safely bled from the tail. Preanalytical slide preparation is important. Hematology and cytology slides are thinly and uniformly smeared in the center of the slide. Coverslips are best for hematology and are fast and easy to prepare. Standard push method blood smears are also acceptable; but for both methods, smears should be made as soon as possible after sample acquisition.
Hematology slides are stained with Diff-Quik; cytology and fecals can be stained with Diff-Quik, Wright-Giemsa, or Gram stain.
Hematology2-4
RBCs are oval, large, and nucleated in birds and reptiles. Normal hematocrit: 15–55% in reptiles and 35–55% in birds. Reptiles RBCs counts vary inversely with reptile size and RBC size is largest in turtles, intermediate in snakes, and smallest in lizards. Seasonal and sex differences may be present in birds and reptiles. Male reptiles have higher RBC counts than females. In hibernating reptiles, RBC counts are highest before hibernation and lowest after.
Heterophil: Equivalent of the mammalian neutrophil. Round, oval, or bilobed nucleus with pinkish rod-shaped granules in birds. Reptile granules are also pink, but can be rod, oval, or elliptical shaped or may appear fused, melted, or degranulated. Heterophils increase in stress hemograms, aquatic chelonians, and neoplasia. They decrease with leukopenia from infection, viral diseases such as inclusion body disease, and with hibernation.
Lymphocyte: Round nucleus with dense chromatin and high N:C ratio, generally smaller than heterophil unless reactive with a basophilic cytoplasm. They increase with inflammation, wound healing and parasitic infection. Except for radiated or desert tortoises, most reptiles are lymphocytic, with lymphocyte numbers higher in summer and in females. Malnutrition decreases lymphocytosis.
Monocytes and azurophils (in reptiles): Nucleus is round to kidney-bean shaped with light staining cytoplasm often with vacuoles. Monocytes increase with chronic disease. Azurophils, most common in snakes, are large mononuclear cells with a fine dusting of pink to magenta cytoplasmic granules. Monocytes and azurophils are similar.
Eosinophil: Lobed nucleus in round cell with pink, round, plump, distinct granules; can increase with allergy and parasitism. Some reptile species have granules that stain blue. Eosinophilia is lowest during hibernation. Some reptiles are eosinophilic (e.g., green sea turtle, prehensile tailed skink). Lizards have low numbers and some snakes lack them.
Basophil: Round to oval nucleus with deeply blue staining round granules. Basophils are rare in sea turtles, but high in Reeve's turtles, snappers, and cooters.
Chemistry1-4
General reference intervals are laboratory/method specific and species specific. The levels presented in the table are for generalization only.
|
Analyte
|
Bird general reference intervals
|
Reptile general references intervals
|
Uses
|
|
CPK
|
100–300 U/L
|
50–500 U/L
|
Muscle enzyme
|
|
AST
|
< 330 U/L
|
< 250 U/L
|
Indicator of liver disease; can also change with muscle so interpret with CPK.
|
|
LDH
|
70–550 U/L
Poor diagnostic value in birds
|
< 1000 U/L, > AST
Poor diagnostic value in reptiles
|
General measure of liver health. Generally nonspecific in birds and reptiles.
|
|
Alkaline phosphatase
|
10–350 U/L
|
36–650 U/L
Temperature/species dependent
|
Widely distributed, not an organ-specific test. Increases in the young and growing.
|
|
Uric acid
|
2–10 mg/dL
|
< 10 mg/dL
|
Indicator of renal function.
|
|
Phosphorus
|
2–10 mg/dL
|
1–5 mg/dL
|
Severe elevations in end-stage renal disease. Moderate elevations in the young and growing.
|
|
Glucose
|
200–400 mg/dL
|
60–100 mg/dL
|
Decreases with malnutrition and liver disease; increases with breeding and stress; very high levels with diabetes.
|
|
Calcium
|
8–12 mg/dL
Increase in females during folliculogenesis
2–4 x
|
2–5 mg/dL
8–20 mg/dL
Increase in females during folliculogenesis
2–4 x
|
Decreases with seizures, decreased bone density, chronic egg laying; increases with hyperovulation. Tortoises at lower reptile end.
|
|
Total protein
|
3.0–5.5 g/dL
|
1–8 g/dL
|
Decreases with malnutrition and chronic diseases; increases with dehydration and inflammation.
|
|
Bile acids
|
< 100 µmol/L
|
< 10
Poor diagnostic value in reptiles
|
Assesses liver function in birds.
|
Electrophoresis
Electrophoresis (EPH) is an adjunct diagnostic tool that divides protein into six fractions: prealbumin, albumin, alpha-1 and -2, beta- and gamma-globulins. Albumin, the largest fraction in healthy birds and reptiles, decreases with inflammation. Electrophoresis is the only method to accurately quantitate this protein. Inflammation or infection alter the globulin fractions - especially the beta and gamma globulins which are then reflected in a decreased A/G ratio. While not diagnostic, electrophoresis can be a valuable prognostic tool.
Cytology2-6
Evaluates cell morphology and differentiates the physiologic process of inflammation from cysts and neoplasia. With cytology a positive is positive. A negative means it wasn't present in the sample. Histopathology, unlike cytology, evaluates architecture of the lesion, not cellular morphology.
Inflammatory/Infectious Lesions
Avian and reptile inflammation is characterized by increased numbers of WBCs over the blood present in the background. In general, infectious agents of birds and reptiles include bacteria, fungi, parasites, Chlamydophila, and viruses.
Categories of Inflammation:
1. Suppurative-purulent-heterophilic inflammation characterized by > 85% of WBCs present being heterophils. An acute inflammation process includes innate immunity where heterophils are the first inflammatory cell to migrate from the blood to damaged tissue. Typically a reaction to bacterial pathogens, but can also be associated with neoplasia, autoimmune processes, or response to chemical irritation.
2. Pyogranulomatous, or mixed inflammation, is characterized by > 15% macrophages with the presence of heterophils and occasionally lymphocytes. Differentials include subacute bacterial infection, mycobacteria or Nocardia, fungal infection, foreign body or injection reaction, and furunculosis.
3. Granulomatous inflammation is characterized by > 85% macrophages. Rarely seen cytologically, except in seromas with low macrophages numbers.
4. Eosinophilic/basophilic inflammation is seen with parasitic migration and allergic or hypersensitivity reactions.
Bacteria, Fungi, and Parasites
Mycobacterial rods are refractile and unstained by Diff-Quik, Wright's, and Giemsa stains; but stain red with acid-fast techniques.
Macrorhabdus ornithogaster, a large fungus that infects the proventricular-ventricular junction, stains blue with Diff-Quik, dark blue with Gram, and bright pink with periodic acid-Schiff stain. The rod is elongated, bowed, with parallel sides and blunt ends.
Candida sp., visualized as single or budding yeast, may also form pseudohyphae. Stains blue with Diff-Quik and dark blue with Gram stain.
Aspergillus, true septate fungal hyphae, have thin parallel walls, septa, and right-angle branching. If well aerated, macroconidia and spores may be observed as rounded structures covered with oval to round fungal spores.
Cryptococcus neoformans, reported in the sinus of psittacines, is a round, thick-walled yeast, 5–20 µm in diameter with narrow-based budding. Its mucopolysaccharide capsule forms a halo effect when stained.
Chlamydia
Chlamydial organisms are obligate intracellular parasites, stain dark blue with Diff-Quik and Giemsa. Gimenez, the confirmatory stain, stains elementary bodies red and cells green.
Viruses
Pox virus - raised plaques or small masses on eyelids, palpebral conjunctiva, nares, beak, tarsometatarsus, and feet. Cytologically characterized by ballooning degeneration of epithelial cells, which are granular, slightly eosinophilic, with Bollinger bodies, and large, clear, cytoplasmic, vacuole-like inclusions. Smaller, rare Borrel bodies are round, pink, cytoplasmic inclusions representing viral glycoproteins.
Cystic Lesions
Cystic lesions with low cellularity, large amounts of debris or fluids, include feather and ovarian cysts and seromas.
Haemorrhagic Lesions
Characterized by erythrocytes with variable numbers of macrophages. Erythrophagia indicates acute haemorrhage differentiating it from blood contamination. Thrombocytes are not seen in haemorrhage after one hour. Siderophages may be seen and indicate more chronic haemorrhage. Haemorrhagic lesions include hematoma and neoplasia, especially haemangioma and hemangiosarcoma.
Neoplasic Lesions
When evaluating a cytologic sample, differentiation of inflammation from neoplasia is key. Assessment of the WBCs present is crucial. Simple tumors lack inflammation and have monomorphic cell populations. Determine tissue of origin then assess malignancy.3-5
Differentiate Inflammation From Neoplasia:
1. Cytologic components abnormal for site, uncharacteristic of inflammation, composed of distinct homogeneous cell populations. If so it's a neoplasm.
2. Differentiate benign or malignant neoplasms. Benign neoplasms have uniform cell populations with mature nuclei, little mitotic activity. Malignant neoplasms show > 3 criteria for malignancy:
a. Anisocytosis
b. Anisokaryosis
c. Pleomorphism
d. High or variable N:C ratio
e. Increased mitotic activity
f. Nucleoliosis
g. Clumped chromatin
h. Nuclear molding
i. Multinucleation
Cells may contain unusual granules, pigment, or express unusual phagocytic activity. Occasionally, cytologic morphology is not consistent with true malignancy.
3. Determine cell lineage based on characteristic morphologic features.
Neoplasia 3 Cell Types
Epithelial neoplasms/carcinomas exfoliate in clumps or sheets. Adenocarcinoma is likely with acinar and secretory cells.
Mesenchymal neoplasms/sarcomas are spindle cell tumours. Including fibromas, fibrosarcomas, hemangiomas, and hemangiosarcomas.
Round cell tumours, lymphomas, plasmacytomas, mast cell, and melanomas exfoliate well.
Melanomas characterized by melanocytes mostly in reptiles and some birds. Melanomas have mass and hyperpigmentation does not. Histopathology differentiates melanoma from benign hyperpigmentation.
Benign Neoplasms
These have uniform cytoplasm, nuclear size and shape, and nucleoli, and a low N:C ratio.
Xanthomatosis
Benign tumors, nodular or diffuse skin thickening seen on lipomas or after trauma. Xanthomas contain lipids, are firm-to-spongy mobile yellow masses that are friable, featherless, and well-vascularized. Oily gritty aspirates contain free lipid, cholesterol clefts, vacuolated macrophages, and multinucleated giant cells. Etiology unknown.
Lipomas
Benign greasy neoplasms of well-differentiated lipocytes that dissolve in methanol.
Uropygial Gland
The only cutaneous gland of birds, contains lipid sebaceous secretions that are spread over feathers by preening, making them waterproof. Gland is absent in ostrich, emu, cassowary, bustard, frogmouth, Amazon parrots, some pigeons, and woodpeckers and are highly developed in aquatic species. Differentials for uropygial gland inflammation include impaction, adenocarcinoma/carcinoma, adenoma, squamous cell carcinoma, bacterial infection, polyoma, and trauma.
Gastrointestinal Droppings
Normal avian FGS contain 100% Gram-positive bacteria seen as blue-staining rods and cocci. Abnormal Gram-negative rods stain red. Clostridium are abnormal large Gram-positive bacilli with clear round gas-containing spores. They also stain blue with Diff-Quik. Gastrointestinal parasites identified on wet mounts, floats, FGS, and Diff-Quik. Including round worms, Capillaria, Giardia, Hexamita, and Trichomonas.
Coelomic Cavity
Abnormal cytologic findings include cellular infiltrates with bacteria, fungi, egg yolk, or neoplastic cells. Egg yolk effusion is yellow-cream to clear or flocculent. Yolk is homogeneous to granular-globular, pink to blue, amorphous material containing macrophages, heterophils, mesothelial cells, and multinucleate giant cells. Carcinoma cells are found in coelomic cavity effusions of hens with ovarian, oviductal, or intestinal carcinoma.
Joint Aspirates
Inflammation in synovial fluid indicates arthritis and infectious agents such as Mycoplasma synoviae. Gout seen as chalky white material, or refractile uric acid crystals seen as needle-like configurations with polarized light.
Necropsy Sampling
Lung tissue imprints reveal Sarcocystis falcatula and lung mites.
Respiratory Parasites
Sternostoma sp./Cytodites sp. (tracheal mites); Cyathostoma sp., Syngamus sp., Serratospiculum sp. (nematodes); cryptosporidia; Trichomonas (sinuses, trachea); coccidia (Gruiformes); Sarcocystis sp.; trematodes.
References
1. Campbell TW, Ellis C. Avian and Exotic Animal Haematology and Cytology. 3rd ed. Ames, IA: Blackwell Publishing; 2007.
2. Heatley JJ. Evaluating reptile and amphibian hematology: a clinical perspective. In: Proceedings from the Association of Avian Veterinarians Annual Conference; 2014: 39–46. New Orleans, LA.
3. Harr K, Romagnano A. Avian cytology. In: Proceedings from the Association of Avian Veterinarians Annual Conference; 2013: 137–146. Jacksonville, FL.
4. Romagnano A. Avian clinical pathology. In: Proceedings from the British Small Animal Veterinary Association Annual Conference; 2014: 344–347. Birmingham, UK.
5. Reavill DR. Clinical pathology and sample collection. Vet Clin North Am Exot Anim Pract. 1999;2(3).
6. Schmidt RE, Reavill DR, Phalen D. Pathology of Pet and Aviary Birds. Ames, IA: Iowa State Press; 2003.