Aim: To determine reticulocyte count of own blood.
Requirements: Microscope, glass slide, watch glass, glass test tubes, filter paper, sterile disposable needle (26G), 70% alcohol, or any other suitable marketed antiseptic and cresyl blue stain or methylene blue.
Principle: Reticulocytes are non-nucleated immature precursors of RBCs that contain residual RNA and organelles like mitochondria and ribosomes. These reticulocytes are visualized by staining with methylene blue or brilliant cresyl blue-like stains as shown in the figure. The stains cause precipitation of the RNA and above-mentioned organelles with the few ferritin masses present in erythrocytes and form dark-blue cluster filaments.
The reticulocyte count is a means of measuring the erythropoietic action (i.e. RBC regeneration) of the bone marrow. The number of reticulocytes is expressed as a percentage of the total number of erythrocytes counted.
- Take a clean and dry glass test tube and slides.
- Add 4 to 5 drops of methylene blue or cresyl blue stain in the test tube (instead of methylene blue stain any other reticulocyte stain can also be used).
- Sterilize the tip of the ring finger with the suitable antiseptic solution and gently prick the finger.
- Add 4-5 drops of blood from the pricked finger into the test tube.
- Mix the blood and stain by gentle shaking to get a purplish-gray-colored mixture.
- Keep the test tube undisturbed for 5-10 minutes in an incubator maintained at 20-24°C.
- Take 1-2 drops of the above mixture from the test tube to the glass slide with the help of a pipette.
- Draw the edge of another slide as a spreader on the mixture to make a thin smear and then allow it to air dry.
- Observe the slide under 10x or 45x or 100x for counting.
- Identify red cells (pale blue appearance) and reticulocytes (violet fragment granules network).
- Count minimum 1000 cells or minimum 10 fields of 100 cells each and estimate reticulocyte count using formula.
Note: Normal reticulocyte count – 0.2 to 2.0% (Absolute count – 20,000 to 90,000 mm3)
|Field Number||Number of reticulocytes (violet fragment granules network) (A)||Number of red cells (pale blue appearance) (B)||Total number of cells observed (A + B)|
Result: The percentage of reticulocyte count in the blood sample was …….
|Sr. No.||Reticulocyte count||Indications/Factors responsible|
|1.||Elevated||Hemolytic anemia, hemorrhage, erythroblastosis fetalis, renal complaints (increased level of erythropoietin) (Note: Count may be higher during pregnancy).|
|2.||Reduced||Aplastic anemia, Bone marrow infiltration (leukemia, lymphoma, carcinoma), nutritional deficiencies (low iron vitamin B12 level), disorders resulting in ineffective erythropoiesis, Bone marrow failure (commonly due to chemotherapy, radiation therapy, infection), Chronic kidney disease, Cirrhosis of the liver, hypopituitarism, and myxedema or hypothyroidism.|
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