It is used as diluent for blood samples to count red blood cells under high power using a manual cell counter or cell counting chamber such as neubauer chamber cell count
Principle of Hayem’s fluid
- RBC dilution fluid is isotonic with blood, so there is no hemolysis. Normal saline solution or physiological solution may also be used. However, it causes a slight formation of red blood cells and allows the formation of Rouleaux
- The blood specimen is diluted 1:200 with the RBC diluting fluid and the cells are counted by means of a counting chamber under high power( 40x objective).
- The number of cells in the undiluted blood is calculated and the number of red cells per cubic millimeter of the entire blood is reported
Composition of Hayem’s fluid
|Mercuric chloride||0.25 gram|
|Sodium sulphate||2.50 gram|
|Sodium chloride||0.50 gram|
|Final pH at temperature 25°C||5.9±0.1|
Appearance Hayem’s Diluting Fluid
Hayem’s fluid is a colourless clear solution with no insoluble particles
Diluting Fluid (Hayem’s) R.B.C procedure
- Draw EDTA anticoagulated blood to exactly the 0.5 mark of the RBC pipette.
- Wipe the tip of the diluting pipette (thoma pipette), clean with a piece of dry gauge without touching the opening of the capillary and immerse in the freshly filtered diluting fluid.
- Do not insert the pipette in the bottle of counting solution.
- By gentle mouth suction, draw the diluting fluid steadily into the pipette to exactly the 101 mark past the bulb, rotating the pipette on its long axis to ensure thorough mixing of blood and diluent.
- Immediately mix the contents of the pipette thoroughly by placing the thumb over one end and shake for 1 minute.
- Diluted blood must be examined within 2 hours.
Observation and Interpretation
Count the cells in the center and in the four corner squares of the central ruled area under a high power magnification.
Precaution to take note while counting RBC
- Avoid overflow in the moats
- Avoid air bubbles and debris in the chamber area
- There should be no scratches in the ruled area of the chamber
- Pipettes used must be clean and dry