ROUTINE Hematology LabORATORY

STUDENT MANUAL ; Volume 1

Class No. Score

Name: __________________________________ BSMT : III

Date: :___________________________________ Section / SET _________
EXERISE No.5
RETICULOCYTE COUNT

Objectives: The students will be able to:

1. Identify a reticulocyte on a blood smear that is stained supravitally.
2. Determine the supravital stains used in counting reticulocytes.
3. Calculate the results based on the actual counted retics using the given
formula.
4. Differentiate the normal values of reticulocyte count in adults and
newborn.

A reticulocyte is an immature erythrocyte that has lost its nucleus but

retains aggregates of ribonucleic acid (RNA) within its ribosomes. The amount of
RNA decreases as the erythrocyte matures. After the normoblast loses its
nucleus, the reticulocyte usually remains 2 days in the bone marrow and one day
in the peripheral blood before becoming a mature erythrocyte. The reticulocyte
count, with its associated corrections, can be used to asses’ bone marrow
erythropoietic activity.

Principle
The ribosomal RNA of reticulocytes must be stained supravitality; that is,
with the erythrocytes in the living state. A reticulocyte is defined as any non-
nucleated erythrocyte that contains two or more particles of blue-stained,
granulofilamentus material after new methylene blue (supravital) staining.

Materials
1. New methylene blue stain solution
New methylene blue 0.5 g
Potassium oxalate 1.4 g
Sodium chloride 0.8 g
Distilled water 100 ml
Mix for at least 15 minutes, filter, and store at room temperature.
Filter again in the day of use.
2. Glass slides
3. Microhematocrit tubes
4. Microscopes
 ROUTINE Hematology LabORATORY
STUDENT MANUAL ; Volume 1
5. Small test rubes

Procedure
1. Place three drops of filtered reticulocyte stain in a small test tube.
2. Add three drops of well-mixed whole blood to the tube containing the stain.
3. Mix the tube and allow standing at room temperature, or incubating at
37˚C, for 15 minutes this allows the reticulocytes adequate time to take up
the stain.
4. At the end of 15 minutes, mix the contents of the tube well.
5. Prepare several wedges or spun smears and allow to air dry.
6. Place the first slide in the microscope stage, and using the low power
objective (10x), find an area in the thin portion of the smear in which the
red blood cells are evenly distributed and are not touching each other.
Carefully change to the oil immersion objective (100x) and further locate
an area in which there are approximately 100 to 200 red blood cells per oil
immersion field.
7. As soon as the proper area is selected, the reticulocytes may be counted.
The red blood cells will be a light to medium green in color. The RNA
present in the reticulocytes stains a deep blue. The reticulum may be
abundant or sparse, depending on the cell’s stage of development.
8. Count all the red blood cells in the first field on one cell counter. At the
same time, enumerate the reticulocytes
in the same field with a second cell counter. To be considered a
reticulocyte the red cell must contain two or more blue staining particles.
Move the slide as described in the different cell count procedure, using the
cross sectional method, until all reticulocytes in 1000 red blood cells have
been counted.
9. Compute the reticulocyte count as shown below:
No. of reticulocytes counted
 ROUTINE Hematology LabORATORY
STUDENT MANUAL ; Volume 1
% Reticulocytes = ____________________________ x 100
No. of RBC counted (1000)
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ILLUSTRATION:
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Stained reticulocytes
 ROUTINE Hematology LabORATORY
STUDENT MANUAL ; Volume 1

Questions:
1. Give the two general methods of reticulocyte staining and their diluents.
Enumerate at least one specific method for each.

2. Factors that determine the number of reticulocytes in the circulation.

 ROUTINE Hematology LabORATORY
STUDENT MANUAL ; Volume 1

3. Briefly discuss on the following:

a. Reticulocyte production index

b. Corrected reticulocyte count

c. Absolute reticulocyte count

4. What is the importance of a Miller Disk?

 ROUTINE Hematology LabORATORY
STUDENT MANUAL ; Volume 1

5. Illustrate and briefly describe a Miller Disk