1. Fish Heath Management Lab 4: Hematology February 23 rd. 2009

2. Cold Blooded Vertebrates Primary site of hematopoiesis is anterior fourth of the kidney Primary site of hematopoiesis is anterior fourth of the kidney Spleen is a accessory area of hematopoiesis as well as the site for blood cell destruction Spleen is a accessory area of hematopoiesis as well as the site for blood cell destruction Bone marrow, spleen and lymph nodes serve this function warm blooded vertebrates Bone marrow, spleen and lymph nodes serve this function warm blooded vertebrates Various types of blood cells in head kidney: Various types of blood cells in head kidney: – Immature – Mature – Red Blood Cells and White Blood Cells

4. Blood Cell Types Hemocytoblasts- Stem Cell or blast cells, - Most abundant in hematopoietic tissue of kidney, rare in blood Erythrocytes- Red Blood Cells - Carries Oxygen Leukocytes- White Blood Cells - Blood Clotting - Phagocytosis - Inflammation Response

5. Erythrocytes Primary Function: Oxygen transport Elongated elliptical cell: 13-16  m long by 7-10 wide  m Nucleus centrally located, elliptical in shape Poly-chromatins- Immature erythrocytes- less elliptical with bluish-gray cytoplasm

6. Ghost Cells Artifact of Sampling Artifact of Sampling – Not defined – Breakdown and waste of cells necrosis necrosis

7. Leukocytes Immune Response Coagulation Hard to differentiate

8. Lymphocytes Usually sparse in the blood Spherical in shape, 7- 10  m Eccentrically located spherical nucleus- stains reddish-purple

9. Neutrophils (Polymorphonuclear Leukocytes) Only granulocytic leukocyte present in rainbow trout Origin and function of these cells not well understood Accounts for 1-9% of total leukocytes Average diameter is 9-13  m Multi-lobed nucleus (2-5 lobes)

10. Thrombocytes Occur in varying shapes and size: elongated, spiked, fusiform, and spheroid. Elongated - 5-8  m long Spiked – streaked cytoplasm on one end Fusiform- streaked cytoplasm on both ends Spheroid- dense nuclei, little or no cytoplasm

11. Monocytes and Macrophages Monocytes- large 9-25  m Indistinguishable from Macrophages once in tissue Cytoplasm often has granules and a large nucleus Macrophages not usually seen in blood

12. Kidney Imprint Various states of hematopoiesis

13. Blood Sampling Non-Lethal Mouth Catheterization Caudal Artery Heart Lethal Sever Caudal Peduncle Sever Gill

14. Blood Assessment Three Indexes: – Red Blood Cell Abundance Hemoglobin Hematocrit - Ratio of Packed RBC’s to total blood volume Variations in reading can be caused by species, sex, age, temperature, clotting, water quality, season

15. Hematocrit Readings Normal – Dependent upon species – 25-40% Low – Indicates anemia, gill damage, osmoregulation impairment – <15% High – Indicates stress, dehydration, hemoglobin concentrations – >40%

16. Lab Procedure Take blood from caudal artery Use one tube for Blood Smear Seal other 2 with CritoSeal at vacant end Measure % of both Tissue imprints

17. Tissue Imprints Cut a small piece of Tissue Using forceps, create imprints by touching tissue to slide in succession K L S Fish 1

18. Stain Using Diff Quick Fix slides in Solution A (Methyl Alcohol) for 10 min Dip slides in Solution B 25 times Dip slides in Solution C 25 times Wash with DDI water Dry and examine the slides