RETICULOCYTE COUNT

RETICULOCYTE COUNT

RETICULOCYTE COUNT:- Reticulocytes, the young red cells that have lost their nuclei but not the cytoplasmic RNA, appear in the peripheral blood as polychromatic red cells when stained with a Romanowsky stain. However, reticulocytes are demonstrated with a supra-vital stain, when the RNA appears as a network of strands or granules.

Staining Solution for Supra-vital staining

Brilliant cresyl blue

Brilliant cresyl blue lg 

3 % sodium citrate 20 ml 

0.9 % sodium chloride 80 ml

Mix to dissolve and filter before use.

OR

New methylene blue

3 % sodium citrate 20ML

0.9% sodium chloride 80 ML

Mix to dissolve

20 ml 80 ml

Note:- Sodium citrate in the staining solution prevents coagulation when capillary specimen is used, and sodium chloride provides an isotonicity to preserve the red cells.

Specimen:- Capillary blood, or anticoagulated venous blood taken into EDTA or sodium citrate.

Technique

1. Mix two drops of blood and two drops of the staining solution in a test tube.

2. Allow to stand at room temperature for 15 TO 20 minutes or at 37°C for 10 minutes.

3. Gently tap the tube to remix the contents.

4. With a drop of the mixture, prepare a blood film in the same manner as for peripheralsmear.

5. Allow the film to dry completely.

6. Examine under the oil-immersion objective (X 100). The reticular material (RNA) is precipitated by the dye and is stained blue-black. Mature red cells appear pale green

Counting:- Select an area on the smear where the red cells do not overlap. Count the number of red cells and reticulocytes in each field. At least 10 successive fields should be counted, preferably with a total of 1000 red cells. To minimise the difficulty of counting 1000 red cells in a large field, the microscopic field can be reduced by inserting an adjustable diaphragm or a circular piece of paper with a small square cut in the centre.

CALCULATION =