AVIAN HEMATOLOGY. MVDr. Zora Knotková, Ph.D. MVDr. Anna Hrdá, Ph.D.

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1 AVIAN HEMATOLOGY MVDr. Zora Knotková, Ph.D. MVDr. Anna Hrdá, Ph.D. Avian and Exotic Animal Clinic University of Veterinary and Pharmaceutical Sciences, Brno, Czech Republic

2 HEMATOLOGIC TESTS Form an excellent tool to evaluate the patient s general condition Monitor the progress of a disease Form a prognosis

3 HEMATOLOGIC EVALUATION Routine hematologic evaluation includes determination of Packed cell volume (PCV) Hemoglobin (Hb) concentration Red blood cell count (RBC) RBC indices (MCV, MCH, MCHC) Total white blood cell count (WBC) Leukocyte differential count (leukogram) Assessement of blood cell morphology

4 SAMPLE HANDLING In order to obtain objective results it is crucial to use appropriate methods of blood collection and handling with samples.

5 PREANALYTIC PHASE In this first phase, it is necessary: to prepare the patient and collection material collect and process the sample transport the sample and preserve it until analysis This phase have more than 50 % responsibility for correctness of results

6 BLOOD COLLECTION 0.2 ml sufficient for complete hemogram 1.0 ml/100 g body weight for healthy birds less in clinically sick patients be aware of iatrogenic blood loss due to hematoma formation after venipuncture

7 BLOOD COLLECTION Blood collection should be performed safely and quickly. Most birds, especially ill birds and small birds such as finches and canaries, are easily stressed when handled. The stress associated with capture and restraint of avian patient can further compromise the health of the patient and may affect the hematologic indices

8 VENIPUNCTURE v. jugularis dextra (jugular vein) v. cutanea ulnaris superficialis (wing vein) v. metatarsalis plantaris superficialis (caudal tibial vein) cardiac puncture puncture of the occipital venous sinus

9 ANTICOAGULANTS EDTA (ethylenediaminetetraacetic acid) HEPARIN (lithium heparin) artificial hemolysis due to EDTA reported in Corvidae, Gruidae, Strutionidae, Alcedinidae (Kookaburra)

10 ANALYTIC PHASE This second phase includes: preparation of the sample analysis itself Avian hematology is approached in a manner similar to the mammalian hematology, but a few differences in blood cell morphology require modification of the laboratory techniques

11 ANALYTIC PHASE Despite the technological progress in avian hematology, the evaluation of cell morphology and the interpretation of hematological results cannot be realized sufficiently by automated procedures.

12 ANALYTIC PHASE Major differences include the presence of nucleated erythrocytes, thrombocytes, and heterofil granulocytes in the peripheral blood of birds The modified procedures include the use of manual counting techniques to obtain accurate cell counts

13 Red blood cell count (RBC, /l) White blood cell count (WBC, 10 9 /l) Manual technique 4975 µl of Natt and Herrick s solution + 25 µl of blood (dilution 1:200) Bürker s chamber method Ery: 20 rectangles (1/100 mm 2 ) Leu: 50 squares (1/25 mm 2 )

14 PACKED CELL VOLUME (PCV) Centrifugation of blood in a heparinized micro-hematocrit tube at approximately g for 5 minutes

15 HEMOGLOBIN CONCENTRATION (g/l) Spectrophotometric analysis of cyanmethemoglobin at 540 nm Centifugation after red cell lysis necessary (1000 g for 10 min)

16 RBC INDICES Calculation from PCV, Hb, RBC Morphologic classification of anemias MCV (mean cell volume, fl) = PCV (l/l) 1000 / RBC (10 12 /l) MCH (mean cell hemoglobin, pg) = hemoglobin (g/l) / RBC (10 12 /l) MCHC (mean cell hemoglobin concentration, g/l) = hemoglobin (g/l) / PCV (l/l)

17 DIFFERENCIAL COUNT OF LEUKOCYTES (10 9 /l) Blood smear preparation Without anticoagulant directly from the syringe or needle immediately after collection Wedge smear technique (bevel edged slide!!) or Slide and coverglass technique

18 DIFFERENCIAL COUNT OF LEUKOCYTES (10 9 /l) Blood smear staining May-Grünwald/Giemsa-Romanowski stain, Wright-Giemsa stain fast staining methods, Diff-Quick, Hemacolor

19 DIFFERENCIAL COUNT OF LEUKOCYTES Recomended order for the evaluation of the blood smear: Differentiation of at least 100 leukocytes in meandering search for leukocytes from the central to the last third of the blood film (monolayer) expressing each type by a percentage (%) in relative percentage (l/l) calculation each type of absolute numbers

20 POSTANALYTIC PHASE This phase is based on appropriate interpretation of obtained results (in relation to reference values, other laboratory parameters, clinical findings) Intrinsic and external factors affecting the hemogram (age, sex, ambient environmental and season, captive versus wild birds, contamination of blood samples with lymph)

21 PACKED CELL VOLUME INTERPRETATION OF RESULTS PCV = g/l Ht < 0.32 anemia Ht > 0.58 dehydratation, erythrocytosis (polycytemia)

22 HEMOGLOBIN, MCV, MCH, MCHC INTERPRRETATION OF RESULTS Hb = g/l MCV = fl MCH = pg MCHC = g/l

23 ERYTROCYTES INTERPRETATION OF RESULTS Ery = 2,00 4, /l Polychromatophilic Ery = 1 5 % (normal) Birds with anemia Polychromatophilic ERY 5 % moderate regenerative response Polychromatophilic ERY 10 % marked regenerative response

24

25

26 MORPHOLOGIC CLASSIFICATION OF ANEMIAS RBC INDICES MCH normal MCH decreased MCV normal normocytic normocytic normochrome hypochrome MCV increased macrocytic macrocytic normochrome hypochrome MCV decreased microcytic microcytic normochrome hypochrome

27 ANEMIA Hemorrhagic anemia blood loss (traumatic injury, blood-sucking parasites, coagulopathies, hemorrhagic lesion of internal organs) Hemolytic anemia increased red cell destruction Depression anemia decreased red cell production (bone marrow damage)

28

29 HEMORRHAGIC ANEMIA The most common causes of this type of anemia include: traumatic injury blood-sucking parasites (ticks,mites,coccidia) coagulopathies (aflatoxicosis, coumarin poisoning, severe liver disease such as papovavirus) hemorrhagic lesion of internal organs (ulcerated neoplasm, gastric ulceration, rupture of the liver or spleen)

30 HEMOLYTIC ANEMIA This type of anemia can result from: parasitemias (blood parasites) bacterial septicemia (salmonelosis, spirochetosis) toxicosis (aflatoxins, certain plant chemicals-i.e. mustards, petroleum products)

31 DEPRESSION ANEMIA The most common causes of this type of anemia include: tuberculosis, chlamydiosis, aspergilosis chronic hepatopaties chronic nefropaties iron-deficiency, deficiency of vit. B 12 neoplasia

32 LEUKOCYTES Interpretation of results Leu = 3,50 15, /l Leukocytosis > 15, /l Leukopenia < 3, /l

33

34 HETEROPHILIA stress (corticosteroid-induced leukocytosis) bacterial infections, pox and herpesvirus infections, parasitic infections, fungal infections inflamations, i.e. egg yolk peritonitis lead/smoke intoxication hemorrhages, hemolysis

35 HETEROPENIA Bacterial and viral infections with bone marrow depression Inflammation with marked tissue necrosis Neoplasia leukemia, lymphosarcoma

36 LYMPHOCYTOSIS Chronic infections bacterial, viral, fungal, parasitic Lymphoid neoplasia lymphosarcoma, lymphatic leukemia

37 LYMPHOPENIA Acute stress Acute general infections Immunosuppression corticosteroids, radiation

38 MONOCYTOSIS Chronic granulomatous inflammation Chronic infections mykobacteriosis, chlamydiosis, aspergilosis Zinc deficiency

39

40 EOZINOPHILIA internal and external parasitism hypersensitive response type IV

41

42

43

44 BAZOPHILS The function of the avian basophile is not fully known. Avian basophils produce, store, and release histamin, so they may function in immediate hypersensitivity reactions, release mediators for thrombocyte activation, cause smooth muscle contractions, initiate edema, affect coagulation

45

46 THROMBOCYTES Interpretation of results /l thrombocytes / 1000 Ery (evaluation of blood smears)

47

48 THROMBOCYTOSIS Thrombocytosis associated with large thrombocytes has been reported in birds with chronic inflammation, which may be related to the thrombocytes phagocytic role in inflammatory disease

49 THROMBOCYTOPENIA Thrombocytopenia is usually the result of decreased bone marrow production or excessive peripheral utilization or destruction, and may be associated with severe septicemia and possibly diffuse intravascular coagulation (DIC)

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