VPP 322: Aquatic Animal Diseases, Health Care and Management (1+1)

HEMATOLOGICAL TECHNIQUES

The regular monitoring of fish blood is a very useful diagnostic tool in establishing the health status of fish farm stocks. Several techniques are used to investigate the various properties of fish blood.

• Haematocrit
• Hemoglobin determination
• Erythrocyte count
• Leucocyte count
• Differential cell counts

Haematocrit

• A haematocrit is a method which is used to determine of the volume of packed cells in the blood. The haematocrit will vary, depending on the health and physiological condition of the individual fish.

Haemoglobin determination

• Calculating the concentration of hemoglobin in the blood is a rapid method of detecting disease conditions in fish, including anemia.
• The precision of various methods for estimating haemoglobin concentration may differ considerably. The cyanohaemoglobin method has been the standard method used in haematological studies for a number of decades. However the alkaline haematin D-575 method has been adapted for studies in fish haematology and it has been reported that the cyanohaemoglobin method may yield lower hemoglobin concentrations than those obtained with the alkaline haematin D-575 method. It has been suggested that the cyanohaemoglobin method may have other disadvantages; primarily that the reagent contains cyanide which is toxic, and that the end-product is light labile. As cyanohaemoglobin method is the most widely used procedure in determining hemoglobin concentration in fish (and in other animals) it is described in this chapter.
• Anaemia: A condition characterised by a deficiency of functional haemoglobin, packed cell volume and/or erythrocytes. The more important types of anaemia in fish are: normocytic anaemia caused by acute haemorrhaging, bacterial and viral infections, and metabolic diseases resulting in red cell destruction; microcytic anaemia due to chronic haemorrhaging, e.g. caused by external parasites, iron deficiency and the deficiency of certain haematopoietic factors; and macrocytic anaemia (e.g. absence of juvenile cells; too many mature cells) resulting from an increase in haematopoietic activity in the spleen and kidney.

Erythrocyte count

• Obtaining a count of erythrocytes in fish blood is a useful tool because of the association of abnormal red blood cell counts with disease conditions. There is a great deal of variation between laboratories in the procedures employed to carry out blood cell counts. Diluting solutions which may be used include Hendricks diluting solution (1:200 dilution of blood), and formal citrate solution (1:200 dilution of blood). Blaxhall and Daisley’s method for red blood cell counts outlined here is widely used. The same method may be used to carry out leucocyte counts, although the procedure employed for counting and calculating occurrence of the cells is different.
• Make a 1:50 dilution of blood in Dacies fluid.
• Dacies fluid
  • Formaldehyde: 10 ml
  • Trisodium citrate: 31.3 g
  • Brilliant cresyl blue: 1.0 g
  • Distilled water 1 litre
  • Dacies fluid should be filtered immediately before use.

Leucocyte count

• There is a great deal of variation between laboratories in the procedures employed to carry out blood cell counts. Shaws counting solutions may be used to estimate leucocyte occurrence (making a 1:200 dilution of blood).
• Blaxhall and Daisley’s method outlined here for white blood cell counts is widely used. The same method may be used to carry out erythrocyte counts, although the procedure employed for counting and calculating occurrence of the cells is different. Make a 1:50 dilution of blood in Dacies fluid.
• Dacies fluid: As above.

Differential cell counts

• Microscopic examination of different cell types as revealed by differential staining can be carried out by making a blood smear. The purpose of this method is to obtain a single layer of cells on a microscope slide, which is then fixed and stained in the appropriate dyes. Different types of cell (erythrocytes, lymphocytes, etc.) and components within the cell, take up different stains, and this allows cells to be identified and counted.

Details of structure and function of various blood cells...