This document discusses techniques for counting reticulocytes. It begins by defining reticulocytes as immature red blood cells containing remnants of ribosomal RNA. The number of reticulocytes in peripheral blood reflects bone marrow erythropoietic activity. Supravital staining is used to identify reticulocytes, as it stains cells while viable. The document outlines methods for supravital staining blood samples and counting reticulocytes, and calculating the reticulocyte percentage and production index. Increased reticulocyte counts generally indicate hyperactive bone marrow erythropoiesis due to factors like hemorrhage, hemolysis, or treatment of anemia.

1. CHAPTER 13: THE
RETICULOCYTE
COUNTING
TECHNIQUES

2. Acknowledgements
 Addisa Ababa University
 Jimma University
 Hawassa University
 Haramaya University
 University of Gondar
 American Society for Clinical Pathology
 Center for Disease Control and Prevention-
Ethiopia

3. Objectives
At the end of this chapter, the student will be able to:
 Define reticulocytes
 Explain the relationship between the number of
reticulocytes in the peripheral blood and erythropoietic
activity in the bone marrow
 Discuss the reticulocyte production index
 Define supravital dyes
 Prepare supravital dyes in the right proportion and stain a
sample of blood
 Perform reticulocyte count on a sample of blood

4. Objectives cont’d
 Indicate the normal range for reticulocyte count
 Discuss the clinical implications of the reticulocyte count
 Perform QC measures in reticulocyte counting
 List possible sources of error and their remedies

5. Outline
 Introduction
 RPI
 Supravital staining
 Counting of reticulocytes
 Clinical implication
 QC in reticulocyte count

6. 13.1.Introduction
 Reticulocytes (Retics) are juvenile red cells
 They contain remnants of the ribosomal RNA
 The most immature reticulocytes are those with the
largest amount of precipitable material
 In the least immature Retics, only a few dots or strands
are seen.

7. Introduction cont’d
Reticulocytes after counter staining with
Romanowsky dyes (left panel) and supravital
dyes (right)

8. Introduction Cont’d
 Supravital staining is staining of cells while they are
viable
 The number of reticulocytes in the peripheral blood is a
fairly accurate reflection of erythropoietic activity
 Complete loss of basophilic material probably occurs as
a rule in the blood stream after the cells have left the
bone marrow.
 The ripening process is thought to take 2-3 days, of
which about 24 hours are spent in the circulation.
 When there is an increased erythropoietic stimulus as in
hemolytic anemia there will be premature release of
reticulocytes into the circulation as their transit time in
the bone marrow is reduced, the so-called 'stress' or
'shift' reticulocytosis.

9. Clinical significance
 To assist physicians in the diagnosis, treatment or
monitoring of patients with various anemias
 The reticulocyte count is an index of bone marrow red
cell production
 It measures erythropoietic activity

10. Principle
 The count is based on the property of ribosomal RNA to
react with basic dyes such as new methylene blue or
brilliant cresyl blue to form a blue precipitate of granules
or filaments. Retics are larger than mature red cells and
show diffuse basophilic staining (polychromasia) in
Romanowsky stained films.
Note: polychromasia on Romanowsky stains are indicative
of Reticulocytosis
 Only supravital staining techniques enable their number
to be determined with sufficient accuracy.

11. Cont’d
 Better and more reliable results are obtained with new
methylene blue than brilliant cresyl blue, the former
stains the reticulo-filamentous material in the
reticulocytes more deeply and more uniformly than
does the latter
Staining Solution:
 1% New methylene blue (NMB)
 1% Brilliant cresyl blue.

12. Specimen type
 EDTA whole blood:
 Specimen can be stored at room temperature for 8
hours or refrigerated at 2-8oC for 24 hours

13. Method
1. Deliver 2-3 drops of the dye solution into 75 X 10mm
glass or plastic tube using a Pasteur pipette.
2. Add 2-4 drops of the patient’s EDTA anticoagulated blood
to the dye solution and mix.
3. Plug the tube and incubate at 370C for 10-15 minutes.
 The exact volume of blood to be added to the dye
solution for optimal staining depends upon the red cell
count.
 A larger volume of blood in anemic condition
 a smaller proportion in polycythemic blood should be
added than normal blood.

14. Method cont’d
4. After incubation, resuspend the cells by gentle mixing and
make films on glass slides in the usual way
5. Allow to air dry
6. Examine the films without fixing or counter staining.
 In a successful preparation, the reticulofilamentous material
should be stained deep blue and the non-reticulated cells
stained diffuse shades of pale greenish blue.
Pt
name-
Almaz
Card
no-
2453/09
Date
–
12/09/09
1% NMB stained
smear

15. Method cont’d
Counting
 An area of the film should be chosen for the count where
the cells are undistorted and where the staining is good.
 To count the cells:
 eye pieces provided with an adjustable diaphragm
could be used
 or cardboard diaphragm with a small cut in the center
can be inserted into an eyepiece and used as a
substitute

16. Method cont’d
 When the reticulocyte count is expected to be 10%, a
total of 500 red cells should be counted noting the
number of reticulocytes.
 If less than 10% reticulocytes are expected, at least
1000 red cells should be counted.
 Reticulocyte count (%) = Reticulocyte number X 100
RBC number
 Absolute reticulocyte count = Reticulocyte count (%) X RBC count

17. Cont’d
 An alternative method is based on the
principle of ‘balanced sampling’ using a
Miller ocular.
 An eyepiece giving a square field in the
corner of which is a second ruled square
one-ninth of the area of the total square.
 Reticulocytes are counted in the large
square and red cells in the small square in
successive fields until at least 300 red cells
are counted
Reticulocyte count (%) = Reticulocyte number X 100
RBC number X 9

18. 13.2. The Reticulocyte Production Index (RPI)
 In the presence of anemia the reticulocyte percentage
does not accurately reflect reticulocyte production,
 since each reticulocyte released is being diluted into
fewer adult red cells.
 A better measure of erythroid production is the
reticulocyte production index (RPI).
 The reticulocyte percentage is first corrected to a normal
hematocrit of 0.45 (l/l)
 Example: a reticulocyte percentage of 10% in a
patient with a hematocrit of 0.23(l/l) would be
equivalent to a percentage of 5% in a patient with a
hematocrit of 0.45%(l/l)

19. RPI Cont’d
 Another correction is made because erythropoietin
production in response to anemia leads to premature
release of newly formed reticulocytes and these stress
reticulocytes take up to two days to mature into adult
erythrocytes
 If many polychromatophils are seen on the stained blood
film, then a correction factor of 2 is divided into the
corrected reticulocyte percentage
 Example:
RPI = 10 x 23/45 (hematocrit correction) = 2.5
2.0 (maturation time correction)

20. RPI Cont’d
 Maturation factors from 1.0-2.0 are used
 the higher numbers if there is a great deal of
polychromatophilia in the peripheral blood film, and
 the lower numbers if there is little polychromatophilia
 The RPI is an approximate measure of effective red cell
production in the marrow
 A normal marrow has an index of 1.0.
 In hemolytic anemia with excessive destruction of red
cells in the peripheral blood and a functionally normal
marrow, this index may be 3-7 times higher than
normal.

21. RPI Cont’d
 the index is less than expected for the degree of
anemia, i.e., 2 or less
 When there is marrow damage
 erythropoietin suppression
 a deficiency of iron, vitamin B12 or folic acid
 If the bone marrow contains many normoblasts but
the RPI is low, this may indicate ineffective
erythropoiesis, with intramedullary (marrow)
destruction of erythroid precursors

22. Sources of Error in the Reticulocyte Count
1. Insufficient number of cells counted.
2. Confusion of reticulocytes with red cell inclusions like
Basophilic stippling, Howell-Jolly bodies, Pappenheimer
bodies and
3. Recent blood transfusion
4. Mishandling, contamination, or inadequate refrigeration
of the sample
Pappenheimer bodies Basophilic stippling

23. Interpretation
Reference value
 0.5 - 2.5% of total erythrocytes (or 25 - 85 X 109/l)
 Increased numbers (Reticulocytosis):
 Indicates hyperactive erythropoiesis in the bone
marrow
A. An increase in reticulocyte number is seen in the
following conditions:
I. hemolytic anemias
 Immune hemolytic anemias
 Primary RBC membrane defects
 Sickle cell disease
 RBC enzyme defect
 Exposure to toxins

24. Cont’d
II. Following hemorrhage
III. Following treatment of anemias where an increase in
the reticulocyte number may be used as an index of
the effectiveness of treatment.
e.g.
 After doses of iron in iron deficiency anemia, the
reticulocyte count may exceed 20%
 Proportional increase of Retic count when pernicious
anemia is treated by transfusion or vitamin B12
therapy.

25. Cont’d
IV. Physiologic increase in pregnancy and in infants
B. Decreased levels: means that the bone marrow is not
producing enough erythrocytes.
Conditions associated with low Retic count:
 Iron deficiency anemia
 Aplastic anemia
 Radiation therapy
 Untreated pernicious anemia
 Tumour in marrow

26. Quality control
 Use of commercially available controls
 Strict adherence to SOP (preparation, counting, etc)

27. Review Questions
1. What are reticulocytes?
2. How could the number of reticulocytes in the peripheral
blood be a fairly accurate reflection of erythropoietic
activity in the bone marrow?
3. Define supravital staining.
4. What is the difference between a Romanowsky and
supravital stains
5. How do you manage to count the number of
reticulocytes in each field of the microscope after you
stain the cells with supravital dyes?
6. How do you calculate the relative number of
reticulocytes in the patient sample?
7. Briefly discuss RPI.

28. Review Questions/Summary
7) What is the clinical interpretation of an increase in the
number of reticulocytes in the peripheral blood in
general terms?
8) list sources of errors in reticulocyte counting and their
remedies