Principle
Reticulocytes are immature red blood cells that still contain some RNA. This allows them to be distinguished from mature red blood cells when stained with certain dyes. The reticulocyte count provides a measure of bone marrow production of new red blood cells. It is used to evaluate anemias and bone marrow function.
We Use Supravital Stain which contains New Methylene Blue Dye that will help us visualize the RNA on the surface of the Reticulocyte cell.
Equipments
1. Peripheral blood sample in EDTA tube
2. Glass microscope slides
3. Supravital stain (brilliant cresyl blue, new methylene blue)
4. Light microscope with 100x oil immersion objective
5. Immersion oil
Sample Type
whole blood sample by venipuncture into an EDTA tube.
Procedure
1. Collect a whole blood sample by venipuncture into an EDTA tube. Gently mix to prevent clotting.
2. Make a thin peripheral blood smear on a glass slide. Spread the drop of blood evenly across the slide using a spreader slide. Allow air dry.
3. Fix the blood smear by immersing it briefly in methanol. Allow to air dry again.
4. Apply the supravital stain (brilliant cresyl blue or new methylene blue) to the smear and let sit for 5-15 minutes.
5. Rinse the stained slide gently with buffer solution to remove any excess stain. Allow to air dry.
6. Examine the stained smear under the 100x oil immersion objective. The reticulocytes will appear blue-purple compared to the pink mature RBCs.
7. Count the number of reticulocytes per 100 RBCs. Count a minimum of 1000 RBCs for accuracy.
8. Calculate the reticulocyte percentage using the formula: (Number of reticulocytes/Total RBCs counted) x 100
9. Report the reticulocyte percentage along with the reference range (0.5-2.5%).
10. Clean immersion oil from microscope objective when done. Discard slides in biohazard container.
Calculations
Here are the steps to calculate the reticulocyte percentage from a reticulocyte count test:
1. Count the number of reticulocytes seen on the stained blood smear. Count at least 1000 red blood cells to ensure accuracy.
For example, if 65 reticulocytes are counted over 1000 RBCs:
Reticulocytes counted = 65
2. Count the total number of red blood cells evaluated. This should be at least 1000.
For example, 1000 RBCs evaluated
3. Divide the number of reticulocytes by the total number of RBCs.
For example:
65 reticulocytes / 1000 RBCs = 0.065
4. Multiply this number by 100 to convert to a percentage.
For example:
0.065 x 100 = 6.5%
So if 65 reticulocytes were counted over 1000 RBCs, the reticulocyte percentage would be:
6.5%
The normal reference range is typically 0.5-2.5% for adults.
#Higher values indicate increased bone marrow production of RBCs.
#Lower levels indicate decreased production.
in Summary To calculate the reticulocyte percentage:
Count the # retics
Count total RBCs (at least 1000)
Divide # retics by total RBCs
Multiply by 100 to get %
Calculation formula:
Normal Range
M. Sulieman
mohammad@mlsgaate.com
in Summary To calculate the reticulocyte percentage:
Count the # retics
Count total RBCs (at least 1000)
Divide # retics by total RBCs
Multiply by 100 to get %
