1. Filter 2–3 drops of the stain into a small tube or vial.
2. Add about 4 drops of EDTA anticoagulated blood or capillary blood and mix well.
The amount of blood used is not critical. Use at least twice the volume of blood to stain if the patient is severely anaemic.
3. Incubate at room temperature for 20 minutes or 10–15 minutes at 35–37 C.
4. Mix gently to resuspend the red cells and using a capillary or plastic bulb pipette, transfer a drop of the stained blood to each of two slides. Spread to make two evenly spread thin films. Wave the slides back and forth to air-dry the films. Protect
the films from dust and insects until the count can be performed.
5. Count the reticulocytes microscopically. Use the 10 objective (with reduced condenser iris diaphragm) to check the distribution of the red cells. Select an area where the red cells can be seen individually, add a drop of immersion oil,
and examine using the oil immersion objective (open more the condenser iris diaphragm).
6. Count systematically (i.e. consecutive fields), 500 red cells (1 000 if there are very few reticulocytes), noting the number that are reticulocytes.
Calculate the percentage of reticulocytes.
Appearance of reticulocytes
Reticulocytes appear as pale green-blue stained cells containing dark blue-violet inclusions in the form of small granules, distributed irregularly. Mature red cells stain pale green-blue.
Counting reticulocytes:
A convenient method of counting reticulocytes is to reduce the size of the microscope field by inserting in each eyepiece a circular piece of black (opaque) paper which has
a punched out hole of about 5 mm.
To calculate % of reticulocytes:
– Using a hand tally counter, count a total of 500 red cells, noting on paper the number of cells that are reticulocytes (alternatively use two hand tally counters or a white cell differential counter).
– Multiply the number of reticulocytes counted by 2.
– Divide the figure by 10 to obtain the percentage figure.
See also:
Lablink 
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