RETICULOCYTE_COUNT DAVIDMANYIELAGOK.pptx

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1. RETICULOCYTE COUNT 30/1/2019

2. OBJECTIVES Define reticulocyte . Explain the principle of reticulocyte count. Describe the procedure of reticulocyte count test. Interpret the results of the test. Describe quality assurance of the test.

3. DEFINITION Reticulocytes are juvenile/young red blood cells. They contain remnants of ribosomes and ribonucleic acids which were present in larger amounts in their nucleated precursors. Because the number of reticulocytes in the pheripheral blood is a fairly accurate reflection of erythropoetic activity, a reticulocyte count is one of the essential procedures diagnostic haematology . Ribosomes have the property of reacting with certain dyes ( e.g azure B or New methylene blue) to form a blue precipitate of granules or filaments.

4. This reaction will only take a place in supravitally stained, unfixed preparation. The most immature reticulocytes are those with the largest amount of precipitable ribosomal material, while the least immature only a few dots or strands are seen.

5. PRINCIPLE An isotonic solution of supravital stain(one that stain living material) such as new methylene blue or brilliant crystal blue is incubated with a few drops of blood. To detect ribosomal RNA in reticulocytes , the red cells must be stained while they are still alive(not fixed). A thin preparation is made and the reticulocytes counted microscopically. Reticulocytes are recognized by the violet-blue stained granules.

6. METHODS OF RETICULOCYTE COUNT Staining solution: 1.0gm of new methylene blue or Brilliant crestal blue is dissolved in 100ml of ctitrate -saline solution 1 part 3% tri-sodium citrate to 4 parts 0.85% saline) After dissolving the dye, the solution is filtered and is then ready for use. Store at 2-8˚C.

7. Staining method Deliver two or three drops of stain into a tube, and add approximately equal volume of patient’s blood(EDTA blood). If anaemic use a larger proportion of blood; use a smaller proportion of blood if blood is polycythaemic . Mix and leave in water bath or incubator at 37˚C for 15-20 minutes. At the end of this time, resuspend the red cells by mixing and make a thin film in the usual way. Dry the film without counter staining.

8. The reticular material should be stained deep blue and non-reticulated cells shades of pale greenish-blue.

9. COUNTING THE RETICULOCYTES Choose an area of the field where the cells are undistorted and the staining is good using X10 objective. Using the X100 oil-immersion lens, count the number of reticulocytes seen per 1000 red cells. Counting the red cells can be helped by inserting into the eye piece a paper or cardboard diaphram in the centre of which has been cut a small square to reduce the optical field. As long as the red cells are evenly distributed, the red cells need to be counted only in the small square.

10. CALCULATION For e.g , if there were 18 reticulocytes per 1000 red cells, the percentage of reticulocyte is 18/1000 x 100% = 1.8% If the RBCs = 4.5 x 10 12 cells/l absolute reticulocyte count = 1.8 x 4.5 x 10 12 / 1000 = 81 x 10 9 /l

11. Interpretation of results Reference ranges Infants at birth 2-5 % Children and adults 0.5 – 2.5% Incase RBC count is available, express the reticulocytes as absolute number as shown in the example. QUALITY CONTROL Filter the stain before use. Check the best staining time for the stain preparation if new batch is going to be used.

12. Always use duplicate counts(preferably using a different person for each count) the two counts should agree within 20% of each other. INCREASE IN RBC PRODUCTION OCCURS IN Haemolytic anaemias (with evidence of erythropoeisis Following acute blood loss. After iron therapy for iron deficiency anaemia or specific therapy for megaloblastic anaemia . Reticulocytes respond is higher in haemolysis than in haemorrhage .

13. Decreased counts are associated with ineffective Erythropoiesis or decreased production of red cells. Qn what is the significant of reticulocyte count test?