reticulocyte count.pptx

hematology

1. Reticulocyte count By dr Abdiasis Omar Mohamed MBBS

2. Introduction Reticulocytes are juvenile red cells; they contain remnants of the ribosomal RNA which was present in large amounts in the cytoplasm of the nucleated precursors from which they were derived. The number of reticulocytes in the peripheral blood is a fairly accurate reflection of erythropoietic activity assuming that the reticulocytes are released normally from the bone marrow and that they remain in the circulation for the normal period of time.

3. The ripening process is thought to take 2-3 days of which about 24 hours are spent in the circulation. When there is an increased erythropoietic stimulus as in hemolytic anemia there will be premature release of reticulocytes into the circulation as their transit time in the bone marrow is reduced, the so-called 'stress' or 'shift' reticulocytosis.

4. Principle of reticulocyte count The count is based on the property of ribosomal RNA to react with basic dyes such as new methylene blue or brilliant cresyl blue to form a blue precipitate of granules or filaments. Although reticulocytes are larger than mature red cells and show diffuse basophilic staining (polychromasia) in Romanowsky stained films, only supravital staining techniques enable their number to be determined with sufficient accuracy.

5. Staining Solution New methylene blue (1%) or Brilliant cresyl blue (1%). Better and more reliable results are obtained with new methylene blue than brilliant cresyl blue as the former stains the reticulo-filamentous material in the reticulocytes more deeply and more uniformly than does the latter.

6. Method Deliver 2-3 drops of the dye solution into 75 X 10mm glass or plastic tube using a Pasteur pipette. Add 2-4 drops the patient’s EDTA anticoagulated blood to the dye solution and mix. Stopper the tube and incubate at 37C for 10-15 minutes. The exact volume of blood to be added to the dye solution for optimal staining depends upon the red cell count.

7. A larger proportion of anemic blood and a smaller proportion polycythemic blood should be added than normal blood. After incubation, resuspend the cells by gentle mixing and make films on glass slides in the usual way. When dry, examine the films without fixing staining.

8. In a successful preparation, the reticulofilamentous material should be stained deep blue and the non-reticulated cells stained diffuse shades of pale greenish blue .

9. Counting An area of the film should be chosen for the count where the cells are undistorted and where the staining is good. To count the cells, the oil immersion objective and if possible eye pieces provided with an adjustable diaphragm are used. If such eyepieces are not available, a paper or cardboard diaphragm in the center of which has been cut a small square with sides about 4mm in length can be inserted into an eyepiece and used as a substitute.

10. The counting procedure should be appropriate to the number of reticulocytes as estimated on the stained blood film. Very large numbers of cells have to be surveyed if a reasonably accurate count is to be obtained when the reticulocyte number is small.

11. When the reticulocyte count is expected to be 10% a total of 500 red cells should be counted noting the number of reticulocytes. If less than 10% reticulocytes are expected, at least 1000 red cells should be counted. Reticulocyte count (%) = Reticulocyte number X 100 RBC number Absolute reticulocyte count = Reticulocyte count (%) X RBC count

12. An alternative method is based on the principle of 'balanced sampling' using a Miller occular. This is an eyepiece giving a square field in the corner of which is a second ruled square one-ninth of the area of the total square. Reticulocytes are counted in the large square and red cells in the small square in successive fields until at least 300 red cells are counted.

13. Reticulocyte count (%) = Reticulocyte number X 100 RBC number X 9

14. The Reticulocyte Production Index (RPI) In the presence of anemia the reticulocyte percentage does not accurately reflect reticulocyte production, since each reticulocyte released is being diluted into fewer adult red cells. A better measure of erythroid production is the reticulocyte production index (RPI).

15. The reticulocyte percentage is first corrected to a normal hematocrit of 0.45 (l/l). For example, a reticulocyte percentage of 10% in a patient with a hematocrit of 0.23 (l/l) would be equivalent to a percentage of 5% in a patient with a hematocrit of 0.45% (l/l). This is equivalent to calculating the absolute reticulocyte count in terms of red cell number.

16. The RPI is an approximate measure of effective red cell production in the marrow. A normal marrow has an index of 1.0. In hemolytic anemia with excessive destruction of red cells in the peripheral blood in a functionally normal marrow, this index may be 3-7 times higher than normal.

17. When there is marrow damage, erythropoietin suppression or a deficiency of iron, vitamin B12 or folic acid, the index is less than expected for the degree of anemia, i.e., 2 or less. Ineffective erythropoiesis, with intramedullary (marrow) destruction of erythroid precursors can be deduced if the marrow contains many normoblasts but the RPI is low

18. Sources of error in the reticulocyte count • Insufficient number of cells counted. • Confusion of reticulocytes with red cell inclusions like Pappenheimer bodies and Heinz bodies.

19. Interpretation of results Reference value 0.5 - 2.5% of total erythrocytes (or 25 - 85 X 109/l)

20. Increased numbers: Reticulocytosis This means that hyperactive erythropoiesis is occurring as the bone marrow replaces cells lost or prematurely destroyed. Identifying reticulocytosis may lead to the recognition of an otherwise occult disease such as hidden chronic hemorrhage or unrecognized hemolysis.

21. An increase in the reticulocyte number is seen in the following conditions: - Hemolytic anemias (Immune HA, Primary RBC membrane defects, sickle cell disease, RBC enzyme deficits, exposure to toxins). - Following hemorrhage - Following treatment of anemias - Physiologic increase in pregnancy and in infants

22. Decreased levels This means that the bone marrow is not producing enough erythrocytes. A decrease in the reticulocyte number is seen in iron deficiency anemia, aplastic anemia, radiation therapy, untreated pernicious anemia, tumor in marrow.

23. THE END