Introduction to Fish Hematology and Blood Collection Techniques

1
Introduction to Fish Hematology and Blood
Collection Techniques

• FNR 351Aquatic Sampling Techniques

2
Objectives

• Understand the importance of blood sampling in
  fishes
• Introduce concepts related to hematopoiesis
• Familiarize with most the important techniques to
  evaluate red and white blood cell status in
  fishes
• Understand basic interpretation of these values
  and what they mean

3
Why is blood sampling important?

• ________________
• ________________
• ________________
• ________________

4
Common sources of variation and error

• Many intrinsic and extrinsic factors are known to
  affect blood parameters in fish
• Usually, the combination of ______________________
  __________________________________________________
  _______________

5
Can we establish Normal hematological values in
fish?

• Fish are __________________
• Fish are always in an adapting state
• So can we talk about ________ hematological
  values in fish?

6
Hematopoiesis

• Definition of hematopoiesis
• Fish hematopoietic tissues
• ____________
• ____________
• ____________

7
Hematopoiesis

• Fish hematopoietic tissues
• Thymus
• Location and structure vary with species
• ___________________________
• Most cells found are lymphocytes

8
Hematopoiesis

• Fish hematopoietic tissues
• Spleen
• Usually found _____________________________
• Contains _____ and ______ pulp areas
• Contain cells involved in hematopoiesis
  (production of red blood cells, white blood cells
  and trombocytes)

9
Hematopoiesis

• Fish hematopoietic tissues
• Kidney
• Major blood-forming organ of bony fish
• Can de divided in two components
• Cranial kidney ? head kidney
• Site of _______________(erythropoiesis)
• Site of _______________(lymphopoiesis)
• Also involved in the production of antibodies

10
Hematopoiesis
11
Hematopoiesis
Heterophils
12
Hematopoiesis
Thrombocyte
Erythrocyte
Neutrophil
HEMOCYTOBLAST
Monocyte
Plasma Cell
Lymphocyte
Macrophage
13
Hematopoiesis

• Red Blood Cells

14
Hematopoiesis

• White Blood Cells

15
Hematopoiesis
16
Techniques Blood Collection

• Several techniques are available, depending on
  fish size and whether fish want to be maintained
  alive or not
• Small fishes (lt 8 cm TL), terminal
• Large fishes, non-terminal
• Large or Small, non-terminal

17
Techniques Blood Collection
18
Techniques Red Blood Cells

• RBC (or erythrocyte) status is usually described
  in terms of the following parameters
• a) Hemoglobin content (Hb)
• b) Hematocrit (Hct) or Packed Cell Volume (PCV)
• c) Red Blood Cell counts (RBC)
• d) Red Blood Cell Morphology

19
Techniques Red Blood Cells

• 1) Hemoglobin (Hb)
• Indicator of ____________________
• ______________________________
• Method of choice ______________________________
• Whole blood (20 µL) is mixed with cyanide
  reagents (e.g. KCN) and the amount of
  cyanomethemoglogin (Hb-CN-) measured
• Units g/100 mL whole blood

20
Techniques Red Blood Cells

• 2) Packed Cell Volume or Hematocrit
• Simple and rapid test used to evaluate the amount
  of RBCs
• Samples are centrifuging for 5-10 min in a
  microhematocrit centrifuge at 7,000 RPM
• Hematocrit is calculated as _________
• _______________________________
• Results are expressed as

21
Techniques Red Blood Cells

• 3) RBC Numbers cells/µL of whole blood

Hemocytometer
22
Techniques Red Blood Cells

• Hb, RBC numbers, and PCV are used to calculate
  the following indices
• Mean Corpuscular Volume (MCV)
• MCV PCV () x 10 reported in nm3
• RBC (106 mm3)
• Mean Corpuscular Hemoglobin (MCH)
• MEH __Hb (g/L) reported in µg/cell
  
• RBC (106 mm3)
• Mean Corpuscular Hemoglobin Concentration (MCHC)
• MEHC Hb (g/100 mL) reported in g/100 mL
• PCV ()

23
Techniques Red Blood Cells

• 4) Red Blood Cell Morphology Blood Smears
• Used to evaluate __________________and cell
  _____________________in peripheral blood
• Also used to examine for the presence of
  ________________ and ______________
• Thin smears consist of blood spread in a layer
  such that the thickness decreases progressively
  toward the feathered edge
• Slides are first air dried fixed in methanol for
  5 min washed with distilled water stained
  (Giemsa, Wrights, etc.) for 10-20 min washed
  again examined under light microscope (100X with
  immersion oil)

24
Hemolymph Smear
Bitter Crab Syndrome
Source www.afsc.noaa.gov/.../jfm04/images/RACE_fi
g4.jpg
A
B
Cells from hemolymph smears stained with Giemsa
stain modified (Sigma) at 250X magnification.  A.
Crab hemocytes.  B. Hematodinium sp (center
cell).
25
Techniques White Blood Cells

• 1) WBC Counts
• Similar method as described earlier for RBC
  counts
• 2) Differential WBC Counts
• Express the percentage distribution of each WBC
  type to the total WBC population
• This is done by staining blood smears and
  examining them under the microscope

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Interpretation of Values RBC

• Decrease in RBC numbers, sizes, and/or Hb content
  ? __________
• Increase in RBC numbers ?
• Real increase is called ________________(immatu
  re stages abundant)
• Artifact due to dehydration (cell morphology
  normal)

27
Interpretation of Values WBC

• Decrease in WBC numbers (leukopenia) ?
• Lymphopenia
• Heteropenia
• Increase in WBC numbers (leukocytosis) ?
• Heterophilia
• Lymphocytosis