Reticulocytes are immature red cells, originated from bone marrow. Reticulocytes stay in circulation for about 24 hours and mature into erythrocytes.
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Reticulocytes have RNA and cytoplasmic remains, which pick up supravital stain. Reticulocyte cannot take Romanowsky stain because it contains methyl alcohol, which destroys RNA.
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Principle:
Blood is mixed with stain. The stain is allowed to penetrate the cells in living condition. The RNA in the cell is stained as dark blue network inside the cells.
The blood smear is made with a drop of blood and stained. Since direct count is not possible, relative count is taken out of red blood cells. Reticulocytes are expressed as % of RBCs.
Requirement:
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1. Blood specimen:
EDTA anticoagulated or heparinized blood is used. The test should be performed within 2 to 3 hours of blood collection.
2. Clean grease free slide.
3. Test tube.
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4. Microscope.
5. Stain—Most commonly, brilliant cresyl blue stain is used.
Composition:
Brilliant cresyl blue – 1.0 gm
Sodium citrate – 0.4 gm
Sodium chloride solution (0.85%) – 100 ml
Filter the stain and store in a plastic container. It should be stored in refrigerator at 2 to 8°C.
Methylene blue can also be used for staining.
Procedure:
Filter a small amount of stain. In a test tube, add two drops of blood and two drops of stain by using separate pasture pipettes, mix it thoroughly. Cover the tube with rubber cap and keep it as it for 15 to 20 min. Take a drop of mixed solution of blood and stain on a clean grease free slide.
Prepare a smear of this mixture by using spreader slide. Air dry the smear. First observe the smear under low power objective (10×) and locate tail portion of the smear where red cells are evenly distributed.
Now, under oil immersion objective count both, red cells as well as reticulocytes. Reticulocytes are observed as fine deep violet cell having network of RNA. The red cells stain pale blue. Observe for about 15 fields and count both the cells.
Calculation:
The % reticulocyte can be calculated as
Reticulocyte Count (%) = No. of Reticulocyte counted / No. of RBC counted × 100
Normal value:
i. In adults – 0.2 to 2% of RBC
ii. In infants – 2 to 6% of RBC
Importance:
The number of reticulocyte in peripheral blood is a reflection of red cell activity (erythropoiesis). Increase in their number indicates increased activity of bone marrow. This is known as reticulocytosis.
Decreased count of reticulocyte indicates bone marrow suppression, as observed in case of aplastic anaemia.