hematologia aves y reptiles

7/28/2019 Hematologia Aves y Reptiles

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Haematology and clinical biochemistry

of exotic animals

univ. prof. Zdenek Knotek, DVM PhD.

Zora Knotkova, DVM, PhD.

Univ. of Veterinary and Pharm. Sci. Brno

Czech Republic


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Reptiles

Cheloniansdorsal coccygeal vein

Luer 25G x 1,5 needle

jugular vein

insulin set

Crocodilesventral coccygeal vein - Luer 18G x 1,5needle

inserted at an angle 30 - 60

then advanced at an angle 60 - 90

Lizards + Snakesventral coccygeal vein - Luer 25G x 1,5 needle

inserted at an angle 30 - 90


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Anticoagulant ...

EDTA red blood cells hemolysis

HEPARIN optimal (no risk of hemolysis)

for plasma

Na-Heparin 50 I.U./ml one drop

Li-Heparin


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Laboratory reptiles and birds

PCV centrifuge

Haemoglobin concentrations by cyanmethemoglobin method

RBC and WBC performed manually, using a hemocytometer with

Natt and Herrick stain (solution 1:100 dilution)

Blood smears Diff-Quick

Pappenheim method of biphasic staining

May-Grnwald and Giemsa-Romanowski

Wrights Giemsa


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RBC + WBC

Natt Herrick Solution and Stain

Sodium chloride (NaCl) 3.88 g

Sodium sulfate (Na2SO4) 2.50 g

Sodium phosphate (Na2HPO4) 1.74 g

Potassium phosphate (KH2HPO4) 0.25 g

Formalin (37%) 7.5 ml

Methyl violet 0.10 g

+ to 1 000 ml with distilled water + filter


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Staining methods

Wrights Stain(for Malaria) (Lane 1991)

Wrights stain, powdered 0.30 g

Glycerol, neutral 3.00 ml

Methyl alcohol, pure 97.0 ml

Giemsa Stain(for Malaria) (Lane 1991)

Giemsa stain, powdered 0.75 g

Glycerol, pure 35.00 ml

Methyl alcohol, pure 65.00 ml


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Staining methods

pH of the buffered water (Lane 1991)

distilled water pH = 5.5

tap water pH = 6.0

optimal buffered water pH = 7.2 (7.0 7.5)

Solution A Disodium Phosphate (Sigma # S 0876)(Na2 HPO4) 9.50 g

Distilled water to 1.0 l

Solution B Monosodium Phosphate (Sigma # S 0751)

(NaH2PO4 . 2 H2O) 9.20 g

Distilled water to 1.0 l

optimal buffered water pH = 7.2 A (72.0 ml) + B (28.0 ml)

+ distilled water 900.0 ml


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Iguana iguana

(orderSquamata, suborderSauria)

~ 40 75 % patients of the class Reptilia

~ 80 % patients suffering from nephropathy,hepatopathy, osteopathy (Brno)


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Iguana iguana

Iguanaiguana n = 12

8 males, 4 females

4 breeding goups (1,0 + 1,0 + 0,1)

air temperature 24 - 35 C

air humidity 60 - 80 %

light-regime 12 h/12 h

samples v. coccygea ventralis1. sample (age 10 msc

2. - 6. samples (intervals of 30 days)

PCV, erythrocytes, WBC, differential counts


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Erythrocytes + PCV

RBC 0,9 - 1,9 (T/l) (1012/l)

oval shape, eosinophil. cytoplasma

intracytoplasmatic inclusions (?)

oval to round nucleus

erythroblasts (


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WBC

9,1 - 25,1 (G/l) (109/l)

granulocytes

heterophils eosinophils

basophils

lymphocytes

monocytes + azurophils (granulocyty)


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LymphocytesLymphocytes

4,8 - 14,0 (G/l) (109/l)

36,8 - 81,0 %

the biggest subpopulation of leucocytes

round nucleus

blue cytoplasma lymphocytes x thrombocytes

PAS negative


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Thrombocytes

different size and shape

round to oval nucleus

cytoplasma

blue to grey PAS positive


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Heterophils (granulocytes)

0,9 - 10,2 (G/l) (109/l)

9,5 - 43,8 %

nucleus with segments

cytoplasmatic granules

rods orange to red


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Eosinophils (granulocytes)

0,2 - 1,5 (G/L) (109/l)

1,5 - 6,5 %

nucleus round to oval


round blue !!!


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Basophils (granulocytes)

0,2 - 1,7 (G/l) (109/l)

3,0 - 9,0 %

nucleus round to oval


round dark blue


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Monocytes

0,2 - 1,7 (G/l) (109/l)

1,2 - 7,2 %

cells of different size

nucleus oval to lobates

cytoplasma grey-blue

with vacuoles


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Azurophils (granulocytes)

0,1 - 4,9 (G/l) (109/l)

1,8 - 12,2 %

nucleus oval to round

situated lateraly to the center

cytoplasmatic granules round

azurophilic


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Basic plasma biochemistry profiles in reptiles

Parametr Units Crocodiles Chelonians Lizards Snakes

TP g/l 53,0 46,0 56,0 65,0

Glucose mmol/l 5,5 10,0 12,1 8,9

ALP kat/l 2,5 5,5 2,6 1,8ALT kat/l 0,6 0,7 0,8 0,5AST kat/l 1,1 1,6 1,2 0,4

Uric Acid mol/l 80,0 160,0 89,3 250,0Ca mmol/l 2,7 3,0 3,3 2,3

P mmo/l 1,0 1,5 1,6 1,3


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FEMALES

POOS POFS

MALES

Mean Mean Mean

TP g/l 59,3 56,8 49,3

Gluco mmol/l

12,7 12,3 12,7

UA mol/l

151,1 320,6 90,5

CHOL mol/l

8,2 9,1 5,1

ALT kat/l 0,4 0,3 0,5AST kat/l

0,8 0,8 0,3

Cammol/

l 2,1 6,9 2,8

Pmmol/

l 3,7 3,5 2,1


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Iguanas - renal disease Healthy iguanas

Boyer et al. (1996) Knotek et al.

(2002)

Knotek et al. (1999)

Mean Mean Mean

TP g/l 63,00 53,51 22,0 78,2

Gluco mmol/l

9,55 10,07 8,3 16,5

Uric

acid

mol/l

428,26 306,70 70,2 145,3

Creat mol/l

52,20 61,07 8,8 66,3

ALT kat/l 3,52 0,74 0,1 1,2AST kat/l

3,11 5,02 0,1 1,6K mmol

/l6,10 4,18 1,3 5,2

Ca mmol/l

1,95 2,06 2,2 3,5

P mmol

/l

7,69 5,95 1,4 3,1


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Agrionemys horsfieldi- plasma biochemistry

Wasting tortoises (n=60) Healthy tortoises (n=20)

min. max. mean min. max. mean

Total protein g/l 20.0 49.3 30.8 25.0 68.5 42.8

Glucose mmol/l 2.9 9.5 6.6 8.9 12.5 11.0

Uric acid mol/l 86.8 1 594.0 580.6 56.0 152.0 82.0ALP kat/l 3.1 12.8 5.3 2.6 8.8 4.5

ALT kat/l 0.8 3.4 1.3 0.5 1.9 0.8

AST kat/l 0.7 2.8 1.9 0.5 1.8 1.1

GMT kat/l < 0.1 < 0.1 < 0.1 < 0.1 < 0.1 < 0.1

Cholest. mmol/l 2.6 9.1 7.4 3.2 6.8 4.2

Na mmol/l 139.0 145.0 143.0 139.0 145.0 143.0K mmol/l 3.4 5.0 4.5 2.9 4.6 4.3

Ca mmol/l 0.8 2.2 1.0 1.9 2.8 2.5

P mmol/l 0.9 8.9 5.3 1.2 2.5 1.5


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Serum bile acid concentrationsin red-eared terrapin females

with active folliculogenesis.


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Liver diseases in chelonians

Acute ALT, AST ...

Chronic ?


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Serum concentration of BA

indirect control of liver functionduring the reproductive season

in captive red-eared terrapinfemales


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Mean serum BA concentration

8.69 5.11 mol/l

(99 % interval 6.16 11.22)


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The yolk protein precursors vitellogenins- are formed exclusively in the liver

(Hubert, 1985) under stimulation byestradiol (Ferguson, 1985).

Enlarged liver of pale colour appears tobe a normal physiological phenomenonrelated to metabolism in female reptilesduring vitellogenesis (McArthur et al.,2004).


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Determination of serum concentrations ofBA as the indirect method for monitoring

the hepatic function should be used inresearch focused on reptilian reproduction

and metabolism.


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Haematology - reptiles

Round to oval, kidney

shaped

Grey, like monocytes, but with

red-purple granulesAzurophils

Large, round to oval,

kidney shaped

Grey, pale blue to greyMonocytes

Round to oval, often is

not visible

Dark blue granulesBasophils

Lobed or unlobedGranules red, oval

Blue in some species(Iguana, Tupinambis)

Eosinophils

Unlobed, eccentric,Oval to round,

segmented

Granules bright red/ orangeoval, round, spindle.

Cytoplasma blue to grey

Heterophils

NucleusCytoplasmaCell type


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Haematology - birds

Dark blue, roundBlue to dark blue

Limited space

Lymphocytes

Large, round to oval,kidney shaped

Grey, pale blue to greyMonocytes

Round to oval, often is

not visible

Dark blue granulesBasophils

Lobed or unlobedGranules red, oval

Blue in some species

(Amazonia)

Eosinophils

Unlobed, eccentric,Oval to round,

segmented

Colourless, Granules bright red/orange

oval, round, spindle, rods

Heterophils

NucleusCytoplasmaCell type


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Variability

High in summer, sex, low in winter

Increase post hibernation

0 80Lymphocytes

High in young reptiles0 - 30Monocytes

Azurophils

High in terrestrial turtles0 40Basophils

Lowest in summer, highest during

hibernation, high in turtleslow in snakes

0 20Eosinophils

Season, sex, age,up to 40Heterophils

InfluencePortion (%)Cell type


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Changes

Inflammation, trauma, reproductionLymphocytosis

Chronic necroses, infectionsMonocytosis

Azurophilia

Suspect parasites (blood) , chronic infectionBasophilia

Inflammation, trauma, no direct correlation with

parasitesEosinophilia

Bacterial infections, inflammation, reproductionHeterophilia

Association with problemsCell type


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Birds

Blood samples collection

vena jugularis (right)

vena ulnaris

vena metatarsalis media

vena tibialis caudalis

vena brachialis


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Anticoagulant ...

EDTA only short contact with sample !

HEPARIN optimal for plasma

Na-Heparin

Li-Heparin


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Basic plasma biochemistry profiles in birds

Parametr Units Toucan Ostrich Common Crow Snowy Owl

TP g/dl 2.80 4.40 2.4 5.3 3.7 5.3

Albumin g/dl 1.4 2.4 1.1 2.3 1.8 1.4

Globulin g/dl 1.4 2.2 1.4 3.1 1.8 2.4

Glucose mg/dl 222 - 363 164 - 330 366 339.1

ALP IU/l 14 - 88 - 262 75

LDH IU/l 180 - 319 408 - 1236 291 337

AST IU/l 141 - 340 226 - 547 318 252

Uric Acid IU/l 2.4 - 14 1.0 15.0 12.1 0.5 (mol/l)

Ca mg/dl 8.8 11.8 8.0 13.6 8.2 9.4


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Basic plasma biochemistry profiles in birds

Parametr Units Amazon Parrot Cockatiel African Grey Macaw

TP g/dl 3.0 5.0 2.2 5.0 3.0 5.0 3.0 5.0

Glucose mg/dl 220 - 350 200 - 450 190 - 350 200 - 350

Uric Acid IU/l 2.0 10.0 3.5 11.0 4.0 10.0 2.5 11.5

AST IU/l 130 - 350 100 - 350 100 - 350 100 - 280

LDH IU/l 160 - 420 125 - 450 150 - 450 75 - 425

Ca mg/dl 8.0 13.0 8.5 13.0 8.0 13.0 9.0 13.0

K mg/dl 3.0 4.5 2.5 4.5 2.6 4.2 2.5 4.5

Na mg/dl 136 152 132 150 134 152 136 - 155


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Small mammalsFerret

vena cava cranialis

v. jugularis

Rat

vena saphena lateralis

vena cava cranialis

Rabbit

vena auricularis

vena saphena lateralis

Guinea pigvena cava cranialis

v. brachialis

v. saphena lateralis


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Anticoagulant ...

EDTA feasible anticoagulant

HEPARIN feasible anticoagulant

Na-Heparin

Li-Heparin


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Laboratory small mammals

PCV centrifuge

Haemoglobin concentrations by cyanmethemoglobin method

RBC and WBC performed manually, using a hemocytometer with

Trk and Hayem stain solutions

automat cell counter

Blood smears Diff-Quick

Pappenheim method of biphasic staining

May-Grnwald and Giemsa-Romanowski

Wrights Giemsa


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RBC + WBC

Hayems Stain (RBC)

HgCl2 0.25 g

NaCl 0.50 g

Na2SO4 2.50 gDistilled water to 100.0 g

Trks Stain (WBC)CH3COOH 1 ml

Methyl violet 1% water sol. 1 ml

Distilled water to 100.0 ml


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Staining methods

May-Grnwald sol.

eosine + methylene blue 1.00 g

Methyl alcohol 100.0 g

Glycerol 50.0 g

Giemsa-Romanowski sol.azureosin II 3.00 g

azur II 0.80 g

Glycerol 250.0 g

Methyl alcohol 0.35 g

Diluted before use (1 + 9 with H2O)

Distilled water (buffered)


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Basic plasma biochemistry profiles in mammals

Parametr Units Ferret Guinea pig Rabbit Rat

TP g/l 53.0 72.0 46.0 62.0 54.0 75.0 56.0 76.0

Glucose mmol/l 5.2 11.4 3.3 6.9 4.1 8.7 2.8 7.5

ALP IU/l 30.0 120.0 66.0 74.0 < 120 70.0 132.0

ALT IU/l 30.0 100.0 39.0 45.0 < 100 26.0 37.0AST IU/l 15.0 40.0 46.0 48.0 < 100 40.0 53.0

Creatinin mol/l 35.5 79.5 53 - 194 70.7 159.0 17.5 70.8BUN mmol/l 1.7 7.5 1.5 5.2 2.8 3.9 0.8 3.5

Bilirubin mmo/l 4.0 - 17.1 5.1 15.4 4.3 12.6 3.4 10.3

Ca mmo/l 2.0 2.9 1.3 2.8 1.4 3.1 2.6 3.2

P mmo/l 1.3 2.9 1.0 2.4 1.3 1.9 1.7 2.7Na mmo/l 137 - 162 152 - 162 138 - 155 143 156

K mmo/l 4.5 7.7 6.8 8.9 3.7 6.8 5.4 6.4


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Literature

Beynon PH, Lawton, MPC, Cooper, JE (Eds.) Manual of Reptiles. BSAVA, Cheltenham, 1992. Meredith A, Redrobe S (Eds.) BSAVA Manual of Exotic Pets. Quedgeley, 2002.

Fowler ME, Miller RE (Eds.) Zoo and Wild Animal Medicine. Saunders, St. Louis, 2003.

Jacobson ER (Ed.) Biology, Husbandry, and Medicine of the Green Iguana. Krieger Publ. Comp., Maabar,2003.

Flecknell P (Ed.) Manual of Rabbit Medicine and Surgery. Gloucester; BSAVA, 2000.

Harkness JE, Wagner JE (Eds.) The Biology and Medicine of Rabbit and Rodents. 4th ed., Baltimore;

Williams & Wilkins, 1995. Quesenberry KE, Carpenter JW (Eds.) Ferrets, Rabbits and Rodents. Clinical Medicine and Surgery. 2nd

ed. St. Louis; W.B. Saunders, 2003.

Harcourt-Brown F (Ed.) Textbook of Rabbit Medicine. Oxford; Reed Educational and ProfessionalPublishing Ltd., 2002:410.

Knotkova Z, Doubek J, Knotek Z, Hajkova P Blood Cell Morphology and Plasma Biochemistry in RussianTortoises (Agrionemys horsfieldi). Acta Veterinaria Brno, 2002, 71:191 - 198

Knotek Z, Hauptman K, Knotkova Z, Hajkova P, Tichy F Haemogram and Plasma Biochemistry in GreenIguanas with Renal Disease. Acta Veterinaria Brno, 2002, 71:333 - 340

Knotek Z, Knotkova Z, Doubek J, Pejrilova S, Hauptman K Plasma Biochemistry in Female GreenIguanas (Iguana iguana) with Calcium Metabolism Disorders. Acta Veterinaria Brno, 2003, 72:183 189


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Pejrilova S, Knotkova Z, Knotek Z, Vrbas J Age-Related Changes of the HaematologicalProfile in Green Iguana (Iguana iguana rhinolopha). Acta Veterinaria Brno, 2004, 73: 305 31

Jekl V, Hauptman K, Jeklova E, Knotek Z Blood sampling from the cranial vena cava in theNorway rat (Rattus norvegicus). Laboratory Animals 2005, 39:236 239

Knotkova Z, Mazanek S, Hovorka M, Sloboda M, Knotek Z Blood profile of Bornean river turtlessuffering from shell necrosis and haemogregarine parasites Vet. Med. Czech., 2005, 50:421 426

Knotkova Z, Pejrilova S, Trnkova S, Matouskova O, Knotek Z Influence of ReproductiveSeason upon Plasma Biochemistry Values in Green Iguanas Acta Vet. Brno, 2005, 74: 515-520

prof. Zdenek Knotek, DVM, PhD.

Avian and Exotic Animal Clinic

Faculty of Veterinary Medicine

Univ. Vet. and Pharm. Sci. Brno

Palackeho 1-4

CZ-612 42 Brno

Czech Republic

+420 5 4156 2381

hematologia aves y reptiles

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