Determination of Reticulocyte Count

AIM: Determination of Reticulocyte Count

Principle

• The supravital staining method is used for reticulocyte count.
• Blood is mixed with the stain, and the stain enters the cells in a living condition. 
• The RNA in the cells is precipitated by staining as a dark blue network or reticulum.
• Blood smear is made afterwards. Since a direct count is not possible, a relative count is taken against the number of red blood cells and expressed as a percentage of red cells.

 

---

Requirements

• Blood sample collected in an EDTA tube

• New methylene blue stain (or other supravital stain)

• Glass slides

• Coverslips

• Microscope with oil immersion objective 

• Pipettes

• Mixing tubes or slides for staining

 

---

Procedure

1. Mix the anticoagulated blood sample gently.

2. Mix equal volumes of blood and new methylene blue stain (for example, 2 drops of blood + 2 drops of stain) in a small test tube.

3. Cover the test tube with a cotton plug, keep at room temperature for 30 minutes to allow staining of reticulocytes.

4. Prepare a thin blood smear on a clean glass slide.

5. Allow the smear to air dry.

6. Examine the smear under an oil immersion objective (100x) under the microscope.

7. Count the number of reticulocytes and total red blood cells in several fields until at least 1000 RBCs are counted.

8. Calculate the percentage of reticulocytes.

---

Calculation

Reticulocyte %= (Number of reticulocytes counted / Total number of RBCs counted) × 100

---

Normal Range

• Adults: 0.5% to 1.5% 

• Infants: 2% to 6%

---

Clinical Significance

Increased Reticulocyte Count (Reticulocytosis):

• Indicates increased red blood cell production by the bone marrow.

• Seen in cases such as:

  • Hemolytic anaemia (due to increased RBC destruction)

  • Blood loss (compensatory increase in RBC production)

  • Response to treatment of anemia (e.g., after iron or vitamin therapy)

Decreased Reticulocyte Count:

• Indicates decreased bone marrow production of red blood cells.

• Seen in conditions like:

  • Aplastic anemia

  • Bone marrow failure or suppression

  • Nutritional deficiencies (severe iron, folate, or vitamin B12 deficiency)