Determination of Reticulocyte Count - Copy.pptx

Reticulocyte count

1. Determination of Reticulocyte Count

2. Apparatus Glass slides, brilliant cresyl blue, new methylene blue, incubator, anti coagulant, distilled water, Compound Microscope, Cedar wood oil and pricking apparatus

3. Theory Reticulocytes are juvenile RBC,( the cell stage immediately before the mature erythrocyte.) which contain large amount of ribosomes and RNA (basophilic nature). This cell spends 2 to 3 days maturing in the bone marrow before it is released into the peripheral circulation, where it spends an additional day of maturation ( size is about 8 μm ) Ribosomes react with dyes such as cresyl blue or methylene blue to form blue precipitate of granules or filaments. This reaction takes place only on supravitally stained unfixed preparations i.e. staining cells in living state at normal body temperature before they have been killed by fixation, drying or laps of time.

4. Theory The reticulocyte count is the most effective measure of ery hropoietic activity. Reticulocyte counts are a reflection of bone marrow health or injury The maturity of reticulocyte is inversely related to the quantity of ribosomal material present. Therefore most immature reticulocytes have largest amount of precipitable ribosomal material. On contrary in the least immature few dots or strands are seen. In lead poisoning, this stained material is present in clumps which appears as discrete material, known as punctate basophilia .

5. Vital Stains used for demonstration of Reticulocytes 1% brilliant cresyl blue in isotonic saline or methyl alcohol. 1 gm of new methylene blue is dissolved in 100 ml of iso -osmotic phosphate buffer, ph7.4 i.e.24.3gm/dl of NaH 2 PO 4 and 21.3gm/ dlof Na 2 HPO 4 for iso -osmotic buffer (it is superior as it stains the reticulo -filamentous material in the reticulocytes more deeply and more uniformly.

6. Procedure Take 2-3 drops of Vital Stain in a test tube and add equal no. drops of Blood. Keep the test tube in incubator at 37°C FOR 15-20 min. This is to stimulate the living conditions so that the stain may go into the cells. The RBCs are then re-suspended by gentle thorough mixing. Make 4-5 moderately thick smear of this mixture on glass slides, dry and see under oil immersion without being fixed or counter stained.

7. Observation In a successful preparation, reticulocytes can be identified by their large size( larger than surrounding RBC)with reticulo -filamentous material in the cytoplasm stained deep blue. The RBCs and non reticulated cells are stained with the diffuse shades of pale greenish-blue colour . Put a small piece of paper with a hole in the centre below the eye piece lens. This will restrict the microscope field. Use the mechanical stage to traverse the full length of the film For counting, an area should be chosen where the cells are undistorted and where the staining is good. Count the RBCs under oil immersion in different microscopic fields. Count 500 RBC and record the number of reticulocytes encountered in this cell preparation in the table shown

8. Table Field Number Number of Reticulocytes (1) Number of RBCs (2) Total Number of Cells (1+2) 1 2 3 4 - - 10

9. Express the reticulocyte percentage as: No. of Reticulocytes X 100 Total no. of RBC counted

10. Reticulocyte

11. Normal Values 1. Adult and children: 0.2-2% (average1%) Absolute count: 24000-84000/µl Infants: 2-6% New born: 30-50%, decreases to 1-2% in the first week of life

12. Limitations 1. Recent blood transfusion can interfere with accurate reticulocyte results. 2. Mishandling, contamination, or inadequate refrigeration of the sample can interfere and cause inaccurate test results.

13. Applied Inceased Reticulocyte count, Reticulocytosis Increased reticulocyte counts indicate increased erythropoietic activity usually as the bone marrow compensates in response to anemic stress Increased count associated with: Rapid blood loss High elevation Hemolytic anemias Medications such as levodopa , malarial medications, corticotrophin, and fever-reducing medications Pregnancy

14. Decreased Reticulocyte count (<0.5%) LOW reticulocyte counts indicate decreased erythropoietic activity Decreased count associated with: Aplastic anemia (where the production of either white or red cells or both is seriously impaired) Exposure to radiation or radiation therapy Chronic infection Medications such as chloramphenicol , methotrexate and other chemotherapy medications Untreated pernicious anemia/ megaloblastic anemia

15. Thank you