Reticulocyte Count test Procedure

Reticulocytes are immature red blood cells (RBCs) with considerable quantities of ribosomal and mitochondrial RNA. Normally, there are a small number of reticulocytes in the peripheral blood which remain for 24-48 hours during maturation.

Also Known as: Retic Count, Reticulocyte Percent, Reticulocyte Index, Corrected Reticulocyte, Reticulocyte Production Index, RPI

Sample Required:

• The EDTA blood is the best sample.

Principle

The reticulocyte count is based on the property of ribosomal RNA to react with isotonic solution of a supravital stain such as New methylene blue or brilliant cresyl blue. Supravital stains are those which stain living material. Hence, for the detection of ribosomal RNA is reticulocytes, they should be fixed.
Blood is mixed with the stain and incubated. The RNA in the cell gets precipitated as dark blue network or reticulum. Blood smear is made and examined under microscope. As a direct count is not possible, a relative count is taken against the number of RBcs and expressed as the percentage of RBC.

Requirements

• Specimen: EDTA whole blood/capillary blood
• Reagent:
  1. New Methylene Blue
     • New methylene blue = 1.0 gm
     • Sodium citrate = 0.6 gm
     • Sodium chloride = 0.7 gm
     • Distilled water = 100 mlOR
  2. Brilliant Cresyl Blue
     • Brilliant cresyl blue = 1.0 gm
     • Sodium citrate = 0.6 gm
     • Sodium chloride = 0.7 gm
     • Distilled water = 100 ml

Procedure

1. Take 2-3 drops of dye solution in a test tube.
2. Add 2-4 drops of well-mixed blood sample and mix.
3. Stopper the tube and incubate at 37⁰C for 10-15 minutes.
4. After incubation, mix well and make a thin smear of stained blood.
5. When dry, examine the films without fixing or counterstain.
6. Count 1000 RBCs and note the number of reticulocytes among them. A dark-blue reticulum or network will present in reticulocytes.

Calculation

1. Retculocyte Percentage

This is the percentage of reticulocytes per 1000 RBCs.

2. Absolute Reticulocyte Count

The absolute reticulocyte count (ARC) is the actual number of reticulocytes in 1 L of whole blood.

3. Corrected Reticulocyte Count

In specimens with a low hematocrit, the percentage of reticulocytes may be falsely elevated because whole blood contains fewer RBCs. A correction factor is used, with the average normal hematocrit considered to be 45%.

Normal Values:

• Adult = 0.5 to 1.5 % of total RBCs counted.
• Newborn = 3 to 6 %.

Clinical Significances

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Possible References Used