Non-Mammalian Hematology

Question 1

describe blood collection methods in birds (3)

Answer 1

1. right jugular vein: common site used in most pet species (psittacines) and smaller birds (passerines)
2. basilic wing vein: used in medium to large sized birds when jugular isn’t possible
3. medial metatarsal vein: in waterfowl, chickens, and flightless birds

Question 2

describe blood collection in reptiles (generally, lizards, snakes, chelonians)

Answer 2

1. for all is blind venipuncture
2. lizards: use ventral coccygeal or jugular vein but use caution in species with tail autotomy (geckos, skinks)
3. snakes: use ventral coccygeal vein or cardiocentesis
4. chelonians: use jugular vein; other sites are at risk for lymph contamination

Question 3

what is lymph contamination?

Answer 3

1. grossly seen by a flash of clear yellow to pink fluid prior to blood during venipuncture
2. falsely decreases the PCV and increases lymphocyte counts
3. any reptile is susceptible depending on site of blood collection

Question 4

describe sample handling and storage (5)

Answer 4

1. need at least 200uL for a CBC but a properly prepared blood smear takes priority over CBC
2. storage: whole blood has short term stability at room temp so refrigerate and ideally use within 24 hours and MAKE and blood smear before storing
3. use EDTA or heparin anticoagulants for a CBC; EDTA is preferred in most birds but heparin is preferred in most reptiles
4. if having problems with cell preservation, use albumin in a 2:1 ratio (2 drops blood for 1 drop albumin)
5. use a heparin vial if running a chemistry, as EDTA can cause artificial changes

Question 5

describe the different ways to obtain a total leukocyte count in non-mammalian species

Answer 5

1. indirect counting method: use Pholxine B stain, causes lysis of RBCs and stains heterophils and eosinophils bright pink
2. direct counting method: use Natt and Herrick’s solution, stains all leukocytes dark blue
3. leukocyte estimates using blood smears: ALWAYS done whether direct or indirect; performed on a stained blood smear, should be considered a rough estimate because this is a variable method based on smear density and/or quality and user error

Question 6

describe and identify each of the avian and reptilian leukocytes, including species differences

Answer 6

1. heterophils (non-mammalian neutrophils):
   -birds, lizards, and most amphibians have segmented nuclei (2-3 lobes) with coarse chromatin
   -chelonians, crocodiles, and snakes have round, eccentric nuclei
   -generally, cytoplasm is clear with eosinophilic secondary granules, but birds have bright pink oval-spindle shaped granules and reptiles have orange-bright pink, round-spindle shaped granules
2. eosinophils: round to rod-shaped granules that are bright pink in most species
   -exception is that the eosinophil granules stain blue in psittacines, tegus, and iguanas
   -similar numbers in birds as in mammals, chelonians have much more and snakes have basically none
3. basophils: more similar to mast cells than mammalian basophils
   -round, non-segmented nucleus, may contain deep purple granules, present in low numbers in health
4. monocytes: morphology and function similar to mammals
   -oval to reniform nucleus with basophilic cytoplasm +/- vacuoles
   -in reptiles, may contain melanin pigment granules (melanomacrophages)
5. azurophils: unique to reptiles, commonly seen in lizards, snakes, and crocodilians
   -look similar to monocytes, but may contain many pale, blue-purple, dust-like granules that give them a pink-purple tinge
   -often counted together with monocytes in the leukocyte differential due to similar function, EXCEPT in snakes are more similar to mammalian neutrophils and should count separately
6. lymphocytes: predominant leukocyte in some birds and reptiles (lizards and snakes)
   -similar function and morphology as mammalian lymphocytes
   -round cells with round to oval nuclei and coarse chromatin and may contain a few red granules

Question 7

describe non-mammalian thrombocytes and contrast to platelets

Answer 7

1. non-mammalian version of platelets, nucleated with phagocytic activity
2. round-oval shape with clear, blebbed (foamy) cytoplasm
3. may appear activated, and will have basophilic cytoplasm with a few vacuoles and/or purple granules
4. lymphocytes versus platelets: lymphocytes have basophilic cytoplasm versus clear to foamy cytoplasm in thrombocytes; lymphocytes also have clumped chromatin versus the more deeply basophilic and condensed chromatin of thrombocytes, and thrombocytes frequently clump together

Question 8

describe and identify the common hemoparasites of non-mammalian species and understand their significance

Answer 8

1. hemoproteus:
   -avian
   -organisms do not displace nucleus (Hemoproteus Hugs the nucleus)
   -2 shapes: banana or cignet ring
   -nonpathogenic
2. leukocytozoon:
   -avian
   -originally thought to invade leukocytes (the name) but now we think they invade RBCs
   -spindle or oval shaped gametocytes that DISPLACE nucleus to periphery
   -most are nonpathogenic
3. hemogregarina:
   -reptilian
   -is a blanket term used to describe 4 different genera of morphologically similar organisms
   -seen within the cytoplasm of RBCs and appear as oblong organisms that may displace or wrap around the nucleus
   -nonpathogenic usually but if heavy load or immunosuppression or illness can cause hemolytic anemia
4. plasmodium:
   -in mammals, birds, and reptiles!
   -transmitted by mosquitoes and pathogenic! cause hemolytic anemia
   -signet ring shaped trophozoites
   -distinguish from hemoproteus and hemogregarina because Plasmodium Pushes the nucleus, but usually need PCR to confirm