Central Philippine University

College of Medical Laboratory Science

Laboratory Activity No. 8

Reticulocyte Count
Learning Outcomes:
At the end of this activity the student must be able to:
1. Demonstrate accurately blood collection procedure through venipuncture technique.
2. identify correctly a reticulocyte under the microscope.
3. Execute correctly the method used in reticulocyte counting.
4. Describe and illustrate precisely the appearance of reticulocyte in a supravital stained smear.
5. List completely the normal values associated with reticulocyte counting both in conventional
and SI unit.
6. Perform correctly laboratory safety and waste management at all times.
Contents
01 Introduction

02 Procedure
Materials and Equipment
Specimen
Principle
Procedure

03 QA/QC

Reference Range
04 Physiologic and Pathologic Variations

05 CRC, RPI, Automation

Introduction

Reticulocyte Count Indications for a

• Reflection of the amount Reticulocyte Count
of effective RBC a. Investigation of anemia
production in the BM b. Monitoring the effect of
erythropoietin therapy
• Used to assess BM c. Monitoring bone marrow
erythropoietic activity regenerative capacity after
chemotherapy or bone marrow
transplantation
d. Therapeutic monitoring during iron,
vitamin B12 or folic acid replacement
Introduction

Reticuloyte
• non-nucleated immature red
cells containing residual RNA
• To be considered a reticulocyte,
RBC must contain >2 blue
staining particles
• Reticulocytes remains in the
BM for 2-3 days, and for 1 day
in the peripheral bloodstream
Procedure

Reagents and Equipment

1. Test tube 6. EDTA tube
2. Dropper 7. New methylene blue stain
3. Glass slides (5) 8. Oil immersion
4. Tally counter 9. Phlebotomy kit
5. Microscope 5

Specimen 9
Whole blood anticoagulated with EDTA

Principle
To detect the presence of cytoplasmic RNA, the red cells must be
8 7 6
stained while they are still living. This process is called supravital
staining. The supravital stains that are most commonly used for the
1
4
reticulocyte count are brilliant cresyl blue and new methylene blue.
Both dyes exhibit similar results; they stain the red blood cells a pale
grayish-blue and the RNA in the cells can be seen as dark blue 3 2
strands and dots. Frequently the RNA will form a dark mesh or
network, known as reticulum, hence, the term reticulocyte.
Procedure

1 Place three drops of filtered stain in a small

test tube.

2 Add three drops of a well-mixed whole blood

to the tube containing the stain.

3 Mix the tube and allow to stand at room

temperature or incubate at 37°C for 15
minutes.
Procedure

4 At the end of 15 minutes, mix the contents of

the tube well.

5 Prepare several wedge smears and allow to

air dry.
Procedure

6 Place the first slide on the microscope stage

and, using the low power objective (LPO),
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 (OIO) and further locate an area in
which there are approximately 100 to 200 red
blood cells per oil immersion field.
Procedure

7 Count all the red blood cells in the smear

using the cell counter while at the same time
enumerate the reticulocytes present.

8 Repeat the reticulocyte count in the same

manner described in step 7 on the second
reticulocyte smear. The two results should
agree within ±20% of each other.
Procedure

9 Average the two results and calculate the

reticulocyte count as shown below.
Example:
Slide #1 = 26
reticulocytes/1,000 RBCs
Slide #2 = 24
reticulocytes/1,000 RBCs
Average = 25
reticulocytes/1,000 RBCs

% Reticulocyte count
./
= 0,222 × 100
= 2.5%
Procedure

Miller disk
1. RBCs in square B are counted until a total
of 500 red cells have been counted.
2. At the same time, the reticulocytes in
square A are enumerated
3. Compute % reticulocyte as:
Disc consisting of 2 squares
placed inside the microscope
eyepiece
Area of the smaller square (B)
is one-ninth that of square (A)
Procedure

Sources of Error
1) Unequal volumes of blood and stain
a. An excess of blood causes the reticulum to understain.
b. An excess of stain usually obscures the reticulum
2) Crenated erythrocytes and rouleaux formation make an accurate
count difficult to perform.
3) Stain precipitated on erythrocytes causes them to appear as
reticulocytes.
4) Dirty slides cause uneven spreading.
5) Inadequate time to penetrate the cell and stain the reticulum.
QA/QC

1. New methylene blue is recommended by the NCCLS for use. The stain
should be filtered before use.
2. One technologist can count 500 cells on each of two slides, or two
technologists can each count 500 or 1000 cells independently. Results
obtained should agree with ±20%. If they do not, repeat the reticulocyte
count on the third smear.
3. On high reticulocyte counts, the percentage of reticulocytes should correlate
roughly with the number of polychromatophilic RBCs seen on the PBS.
Reference Range

Conventional units Factor Recommended SI units

0.5%–1.5% 0.01 Number fraction: 0.005–0.015

25,000–75,000 cells/μL 106 25–75 × 109/L

At birth: 2.0 - 6.0 %; falls to adult range by the end of 2nd week of life.
 Physiologic and Pathologic Variations

Decreased Reticulocyte Increased Reticulocyte

Bone Count Count
Rapid
marrow
bone
failure • Aplastic anemia • Hemolytic anemia marrow
• Pernicious anemia or folic • IDA after iron therapy activity
or
acid deficiency • Blood loss blood
• Radiation therapy regene
-ration
• Bone marrow failure
Rate slightly higher in women and
caused by infection or persons living in high altitude
cancer
• Severe kidney disease
Corrected Reticulocyte Count (CRC)

• Also reticulocyte index (RI) or hematocrit

concentration Reference Range
a. Patients with
• Calculated when anemia is present; allow normal HCT
correction for the degree of patients anemia = 1%
• % reticulocyte may may appear increased b. Patients with
because of early reticulocyte release or decrease HCT of 0.35
in the number of mature RBC
= 2 – 3%
c. Patients with
CRC = Patients Hct × Retic count HCT of 0.25
Normal Hct (0.45) = 3 – 5%
Reticulocyte Production Index (RPI)

• Also known as shift correction MATURATION TIME OF

RETICULOCYTE
• When marrow reticulocytes (prematurely
Maturation
released retics) are present RPI is calculated Hct (%)
Time (Days)

40 – 45 1.0
• Refinement of CRC; more useful then CRC 35 – 39 1.5

25 – 34 2.0
RPI = Corrected Retic .

15 – 24 2.5
# of days retic mature in blood
<15 3
Reticulocyte Production Index (RPI)

Interpretation of RPI
<1 • ineffective erythropoiesis

1 • normal in absence of anemia

≥2 • marrow is responding

≥3 • evidence of a hemolytic process

Reference: PER Handbook
Automated Reticulocyte Count

Flow Cytometry
• Sample is incubated with an RET
RNA-binding fluorescence stain RBC
and counted by flow cytometry
• Light scatter and
immunofluorescence of a RNA
specific dye, such as auramine-O PLT

Scattergram of the reticulocyte

measuring channel, Sysmex X-Class
References

• Brown, B. A. (1993). Hematology: Principles and procedures (6th ed.). Estados

Unidos: Lea & Febiger.
• Keohane, E. M., Smith, L. J., Walenga, J. M., Rodak, B. F. (2016). Rodak's
hematology: Clinical principles and applications (5th ed.). St. Louis, MO: Elsevier.
• Lo, R. W., Liu, E. B., Orillaza, M. A., Faundo, A. C., & J., D. C. (2012). PCQACL'S
Standardization and harmonization of complete blood count in the Philippines
(1st ed.). Quezon City: C & E Publishing.
• McPherson, R. A., & Pincus, M. R. (2011). Henry's Clinical Diagnosis and
Management by Laboratory Methods E-Book. Saunders.
• Stiene-Martin, E. A., Lotspeich-Steininger, C. A., & Koepke, J. A. (1998). Clinical
Hematology: Principles, procedures, correlations. Philadelphia: Lippincott-Raven.