Reticulocyte count

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<ol><li> 1. RETICULOCYTE COUNT Prabin Shah BScMLT, MSc(Biochemistry) </li><li> 2. RETICULOCYTES Reticulocytes are young , premature, non nucleated red blood cells, contain reticular material (RNA) that stain gray blue. Reticulum is present in newly released blood cells for 1-2 days before the cell reach its full mature state. </li><li> 3. RETICULOCYTE STAINS Reticulocytes are visualized by supra-vital staining (such as new methylene blue, Brilliant Cresyl Blue, Pure azure blue) that precipitate the RNA and organelles, forming a filamentous network of reticulum On Wright stain. the Reticulocyte appears polychromatophilic or as a Macrocytic blue red cell. </li><li> 4. PRINCIPLE Whole blood is incubated with supra-vital staining (new methylene blue). The vital stain causes the ribosomal and residual RNA to coprecipitate with the few remaining mitochondria and ferritin masses in living young erythrocytes to form dark-blue clusters and filaments (reticulum). Smears of this mixture are then prepared and examined. The number of reticulocytes in 1000 red blood cells is determined. This number is divided by 10 to obtain the reticulocyte count in percent. </li><li> 5. SPECIMEN Whole blood that is anticoagulated with either EDTA or heparin is suitable. Capillary blood drawn into heparinized tubes or immediately mixed with stain may also be used. Red blood cells must still be living when the test is performed therefore it is best to perform it promptly after blood collection. Blood may be used up to 8 hours after collection. Stained smears retain their color for a prolonged period of time. </li><li> 6. REQUIREMENTS 1. Commercially prepared liquid new methylene blue solution. It should be stored in a brown bottle. If precipitate is a problem on the smear, the stain should be filtered prior to use. 2. Microscope slides 3. Microscope 4. 10 x 75 mm test tubes 5. Pasteur pipets (with bulb if pipets are glass) 6. Capillary tubes 7. Miller ocular (if available) </li><li> 7. PROCEDURE Preparation of smears 1. Add 3-4 drops of new methylene blue solution to 3-4 drops of thoroughly mixed EDTA anticoagulated blood to a glass 10 x 75 mm test tube. 2. Mix the contents by gently shaking and allow to incubate at room temperature for a minimum of 10 minutes. 3. At the end of 10 minutes, gently mix the blood/stain solution. 4. Using a capillary tube, place a drop of the mixture on each of three slides near the frosted edge as you would when making a peripheral smear. 5. Using the wedge smear technique, make acceptable smears not too thick or thin. 6. Label the slides with patient name, ID# and date. 7. Allow to air dry. (Do not blow to hasten to drying.) </li><li> 8. COUNTING PROCEDURE Place the first slide on the microscope stage and, using the low power objective (10x), find an area in the thin portion of the smear in which the red cells are evenly distributed and are not touching each other. Carefully change to the oil immersion objective (100x) and further located an area in which there are approximately 100 red cells per oil immersion field. Do this by finding a field where the cells are evenly distributed and mentally divide the field into 4 quadrants. Count the cells in 1 quadrant. If there are about 25, you are in a good area. There will be a lot of open space between the red cells. </li><li> 9. 2. Be sure to count all cells that contain a blue-staining filament or at least 2 or more discrete blue aggregates of reticulum in the erythrocyte. 3. Count 1000 red cells in consecutive oil immersion fields. Record the number reticulocytes seen. 4. You may count 500 cells on two slides each. They should agree within 15% of each other. If they do not, repeat the reticulocyte count on the third smear. 5. Calculate the result as follow: %Retix= Reticulocyte counted/10 </li><li> 10. MILLER DISCS METHOD 1. Use a 100x objective and a 10x ocular secured with a Miller disc. The Miller disc imposes two squares (one 9 times the area of the other) onto the field of view. Find a suitable area of the smear. A good area will show 3-10 RBCs in the smaller square of the Miller disc. 2. Count the reticulocytes within the entire large square including those that are touching the lines on the left and bottom of the ruled area. Count RBCs in the smaller square whether they contain stained RNA or not. A retic in the smaller square should be counted as an RBC and a retic. Record RBC # counted and retic # counted separately. 3. Continue counting until a minimum of 111 RBCs have been observed (usually 15-20 fields). This would correspond to 999 RBCs counted with the standard procedure. </li><li> 11. The Miller disc may be placed in one of the ocular lenses to aid in the counting of the reticulocytes. %Reticulocyte= total reticulocyte in square A*100 total RBC in square B*9 </li><li> 12. REFERENCE VALUES RBCs life span ~ 100 days, 20 days Reticulocyte ~ 1 day in peripheral blood, Then the B.M. replaces approximately 1 % of the adult red blood cells every day. Normal value : 0.5 to 1.5/100 red blood cells (or, 0.5 to 1.5%) Absolute count : 25 to 75 X 109/L Newborn (0-2 weeks): 2.5-6.0% Normal Reticulocyte Index : 1-3% </li><li> 13. RETICULOCYTE </li><li> 14. REPORTING PATTERN Absolute Reticulocyte Count (ARC): is the actual number of reticulocytes in 1L of whole blood. This is calculated by multiplying the reticulocytes % by the RBCs count and dividing by 100. Corrected Reticulocyte Count is calculated based on a normal hematocrit of 45%. Reticulocyte Production Index (RPI) = Corrected retic count (%) / # Days (Maturation time) </li><li> 15. CORRECTED RETICULOCYTE COUNT In states of anemia, the reticulocyte percentage is not a true reflection of reticulocyte production. A correction factor must be used so as not to overestimate marrow production, because each reticulocyte is released into whole blood containing few RBCs - a low hematocrit (Hct) - thus relatively increasing the percentage. The corrected reticulocyte count my be calculated by the following formula: </li><li> 16. RETICULOCYTE PRODUCTION INDEX(RPI) The RI is a measurement for reticulocytes when anemia is present Estimating RBC production by using the corrected reticulocyte count may yield erroneously high values in patients when there is a premature release of younger reticulocytes from the marrow (owing to increased erythropoietin stimulation). The premature reticulocytes are called stress or shift reticulocytes. These result when the reticulocytes of the bone marrow pool are shifted to the circulation pool to compensate for anemia. The younger stress reticulocytes present with more filamentous reticulum. The mature reticulocyte may present with granular dots representing reticulum. Normally, reticulocytes lose their reticulum within 24 to 27 hours after entering the peripheral circulation. </li><li> 17. The premature stress retics have increased reticulum and require 2 to 2.5 days to lose their reticulum, resulting in a longer peripheral blood maturation time. The peripheral blood smear should be reviewed carefully for the presence of many polychromatophilic macrocytes, thus indicating stress reticulocytes and the need for correction for both the RBC count and the presence of stress reticulocytes. The value obtained is called the reticulocyte production index (RPI). </li><li> 18. Maturation Time Hematocrit % 1 day 45 1.5 day 35 2 day 24 3 day 15 </li><li> 19. INTERPRETATION The Reticulocyte count is an important diagnostic tool: The number of Reticulocytes is a good indicator of bone marrow activity, because it represents recent production. It is used to differentiate anemia's caused by bone marrow failure from those caused by hemorrhage or hemolysis. It used also to check the effectiveness of treatment in prenicious anemia and folate and iron deficiency. To assess the recovery of bone marrow function in aplastic anemia and to determine the effects of radioactive substance on exposed workers. A low reticulocyte count may mean a need for a bone marrow biopsy. This can tell if is a problem with how new reticulocytes are made by the bone marrow. </li><li> 20. Reticulocytosis (Increased RBC Production) Reticulocyte Index &gt;3%, Reticulocyte Count &gt;1.5% 1. Acute blood loss or hemorrhage 2. Post-Splenectomy 3. Acute Hemolytic Anemia (Microangiopathic Anemia) 4. Hemoglobinopathy Sickle Cell Anemia Thalassemia major 5. Post-Anemia Treatment Folate Supplementation Iron Supplementation Vitamin B12 Supplementation </li><li> 21. Reticulocytopenia (Decreased RBC Production) Reticulocyte Index</li></ol>


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