The document outlines the objectives and procedures for conducting a reticulocyte count test, which measures the number of juvenile red blood cells in the blood to assess erythropoietic activity. It details the staining methods, counting techniques, calculation of results, reference ranges, interpretation, and quality control measures. Additionally, it discusses the implications of reticulocyte counts in various medical conditions related to red blood cell production.

1. RETICULOCYTE COUNT
30/1/2019

2. OBJECTIVES
• Define reticulocyte.
• Explain the principle of reticulocyte count.
• Describe the procedure of reticulocyte count test.
• Interpret the results of the test.
• Describe quality assurance of the test.

3. DEFINITION
• Reticulocytes are juvenile/young red blood cells.
They contain remnants of ribosomes and ribonucleic
acids which were present in larger amounts in their
nucleated precursors.
• Because the number of reticulocytes in the
pheripheral blood is a fairly accurate reflection of
erythropoetic activity, a reticulocyte count is one of
the essential procedures diagnostic haematology.
• Ribosomes have the property of reacting with certain
dyes (e.g azure B or New methylene blue) to form a
blue precipitate of granules or filaments.

4. This reaction will only take a place in supravitally
stained, unfixed preparation.
• The most immature reticulocytes are those with the
largest amount of precipitable ribosomal material,
while the least immature only a few dots or strands
are seen.

5. PRINCIPLE
• An isotonic solution of supravital stain(one that stain
living material) such as new methylene blue or
brilliant crystal blue is incubated with a few drops of
blood.
• To detect ribosomal RNA in reticulocytes, the red
cells must be stained while they are still alive(not
fixed). A thin preparation is made and the
reticulocytes counted microscopically. Reticulocytes
are recognized by the violet-blue stained granules.

7. METHODS OF RETICULOCYTE COUNT
• Staining solution:
• 1.0gm of new methylene blue or Brilliant crestal blue
is dissolved in 100ml of ctitrate-saline solution 1 part
3% tri-sodium citrate to 4 parts 0.85% saline)
• After dissolving the dye, the solution is filtered and is
then ready for use. Store at 2-8˚C.

8. Staining method
• Deliver two or three drops of stain into a tube, and
add approximately equal volume of patient’s
blood(EDTA blood). If anaemic use a larger
proportion of blood; use a smaller proportion of
blood if blood is polycythaemic.
• Mix and leave in water bath or incubator at 37˚C for
15-20 minutes.
• At the end of this time, resuspend the red cells by
mixing and make a thin film in the usual way.
• Dry the film without counter staining.

9. • The reticular material should be stained deep blue
and non-reticulated cells shades of pale greenish-
blue.

10. COUNTING THE RETICULOCYTES
• Choose an area of the field where the cells are
undistorted and the staining is good using X10 objective.
• Using the X100 oil-immersion lens, count the number of
reticulocytes seen per 1000 red cells.
• Counting the red cells can be helped by inserting into the
eye piece a paper or cardboard diaphram in the centre of
which has been cut a small square to reduce the optical
field.
• As long as the red cells are evenly distributed, the red
cells need to be counted only in the small square.

11. CALCULATION
• For e.g, if there were 18 reticulocytes per 1000 red
cells, the percentage of reticulocyte is 18/1000 x
100% = 1.8%
• If the RBCs = 4.5 x 1012 cells/l absolute reticulocyte
count = 1.8 x 4.5 x 1012 /1000 = 81 x 109/l

12. Interpretation of results
• Reference ranges
 Infants at birth 2-5 %
 Children and adults 0.5 – 2.5%
• Incase RBC count is available, express the
reticulocytes as absolute number as shown in the
example.
QUALITY CONTROL
• Filter the stain before use.
• Check the best staining time for the stain preparation
if new batch is going to be used.

13. • Always use duplicate counts(preferably using a different
person for each count) the two counts should agree within
20% of each other.
INCREASE IN RBC PRODUCTION OCCURS IN
• Haemolytic anaemias(with evidence of erythropoeisis
• Following acute blood loss.
• After iron therapy for iron deficiency anaemia or specific
therapy for megaloblastic anaemia.
• Reticulocytes respond is higher in haemolysis than in
haemorrhage.
•

14. • Decreased counts are associated with ineffective
Erythropoiesis or decreased production of red cells.
• Qn what is the significant of reticulocyte count test?