Manual Reticulocyte Stain Procedure 

Principle

A reticulocyte is an immature (non-nucleated) erythrocyte containing residual ribonucleoprotein (RNA) in its cytoplasm derived from the primitive nucleated erythrocytes. In normal erythropoiesis, reticulocytes remain in the bone marrow for 2 to 3 days for further maturation before entering the peripheral blood stream. In the peripheral blood stream, they mature quickly, losing their reticulum in about 10-24 hours. This makes the presence and number of reticulocytes in the peripheral blood a good measure of erythropoiesis. To demonstrate the presence of this RNA, the red blood cells must be stained while they are still living. This process is called supravital staining. After the cells have been stained by the new methylene blue the precipated RNA is demonstrated within the red blood cell in the form of granules, strands or a diffuse network of fibrils. The number of reticulocytes in 1000 red cells is determined. This number is divided by 10 in order to obtain the reticulocyte count in percent.

Materials

• Reticulocyte Stain (Supravital Stain)
• Glass Tubes
• Parafilm
• Pipettes
• Incubator
• Glass Slides
• Applicator sticks

Sample Type

Whole Blood: A whole blood sample collected in a tube containing EDTA (Lavender tube)

Procedure

1. Mix the sample
2. Label the aliquot tube with the patient’s information
3. Add 500ul of reticulocyte stain and 500ul of well-mixed patient sample.
4. Cover the aliquot tube and mix well.
5. Incubate the aliquot sample for 15 minutes at 37 degrees Celsius.
6. Mix well
7. Prepare 4 slides and label with the patient’s information.
8. Allow the slides to air dry for at least 15 minutes.
9. Bring your slides to the light microscope.
   • Bring your slide into focus under 10x, then 50x.
   • Find a good morphology zone.
   • This is an area near the feathered edge of the smear where the cells are close but not overlapping one another.
   • Then switch to the 100x objective.
10. Normal erythrocytes appear a blue-green color, reticulocytes are blue-green with blue-black non-refractile network of granules or strands of nuclear material. (RNA)
11. Using a counter, count RBC and retics on the counter, keeping a tally on paper of just the retics in those 100 cells.
    • The counter will only count to 100 so you will have to repeat this 5 times for a total count of 500 RBC’s.
12. Repeat step 11 on a second slide to have a total of 1000 RBC’s counted.
    • The 2 counts must agree within 20% of one another.
13. Record your results on the patient report form.

Calculation

Reticulocyte Percentage:

• Count % = (Retics) / (RBC and RET) x 100
• Average 1st and 2nd counts = Actual Result
• Reticulocyte Absolute:
• RBC x Retic % x 10 = Retic #

Expected Range:

• 0.5 – 2 %

Procedural Notes

1. There are several RBC inclusions that are stained by the new methylene blue in addition to the RNA of the reticulocytes.
   • Howell-Jolly bodies appear as one or two, round, deep-purple-stained structures.
   • Heinz bodies stain a light blue-green and are usually present at the peripheral edge of the red cell.
   • Pappenheimer bodies are most often confused with the reticulocytes and are the most difficult to distinguish.
   • Pappenheimer bodies usually present as several granules in small clusters to the more filament type material of the reticulocytes.
2. It is very important that the blood and stain be mixed well prior to making smears.
   • The reticulocytes have a lower specific gravity than mature red cells and therefore settle on top of the red cells in the mixture.
3. If there is moisture in the air and poor drying of the smear the RBC’s may appear more refractile and be confused with reticulocytes.