RETICULOCYTES SOP.

DOC: 321 REV 7

1. PURPOSE

1.1 To describe the procedure for performing a Reticulocyte count.

2. SCOPE

2.1 To all registered qualified and student personnel in the discipline of

Haematology or Clinical pathology.

3. PROCEDURE

Principle

Reticulocytes are transitional red cells between nucleated red cells and the so-called
mature erythrocytes; they are also called juvenile red cells. When stained with a
supravital dye, for example New Methylene Blue, it contains stainable nucleic acids
(i.e., cellular RNA).

The number of Reticulocytes in the peripheral blood is a crude measurement of

Erythropoietic activity. During normal red cell production, there is a controlled
mechanism in place with regard to the Reticulocyte release from the bone marrow,
remaining in blood circulation for a normal period of time and then finally maturing as
an erythrocyte. In times of increased Erythropoietic stimuli as encountered during the
different degrees of anaemia, there is a premature release of Reticulocytes into the
circulation and the compromised time frame for Reticulocytes to mature in the bone
marrow.

The Reticulocyte count can be expressed as:

 % of Erythrocytes
 Corrected % of Erythrocytes
 Absolute count
 Reticulocyte index (RI)
 Reticulocyte production index (RPI)

3.1 Specimen Required

3.1.1 K3EDTA blood

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3.2 Specimen Storage

3.2.1 It is recommended that the reticulocyte count be performed promptly

after the collection of the blood specimen, or alternatively, that the
specimen be stored in such a way that it remains stable until the test is
performed.

3.2.2 When the original specimen is kept at room temperature, reticulocyte

counting should be done within six hours after blood collection.

3.2.3 With most reticulocyte methods, there is an apparent in vitro

maturation and subsequent disappearance of some of the
reticulocytes. This maturation is both time- and temperature-
dependant and can be expressed as the number of reticulocytes that
mature and are therefore not counted after the delay in analysis. This
loss can be up to 20% per 24 hours at room temperature.

3.2.4 If sample analysis is delayed, the sample should be refrigerated.

Sample storage at 2 - 8°C may be stable for up to 72 hours.

3.2.5 Specimen storage requirements may differ for automated counting

methods and the instrument manual should be consulted.

3.3 Controls

Automated method:
Laboratories running automated Reticulocytes must run commercial
controls designed for the specific counting system being used

3.4 Materials, Instrumentation and Equipment Requirements for Manual

Retic counts.

3.4.1 Test tubes (10 or 12 x75mm)

3.4.2 Automated pipette (adjustable volume)
3.4.3 Plastic Pasteur pipettes
3.4.4 Glass slides
3.4.5 37ºC Waterbath/Incubation unit
3.4.6 Microscope, oil lens x 100, objective

3.5 Reagents Required

3.5.1 The supravital dye, New Methylene Blue is the stain of choice. Before
use, an appropriate volume must be filtered through filter paper to
remove any precipitated dye and any other particulate matter.

3.5.2 Azure B, a purified component of polychrome methylene blue, has

been found to be equally acceptable as reticulocyte stain. It has some
advantages of purity and the dye does not precipitate on the blood film.

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3.5.3 Brilliant Cresyl Blue may also be used, but is found to not stain the
reticulo-filamentous material as deeply and uniformaly as New
Methylene Blue.

3.5.4 Ensure that the label and/or package insert of the commercial control
material clearly states which staining reagent it contains. A label
simply stating “Retic Stain” is not acceptable.

3.5.5 New Methylene Blue and Azure B staining reagents may be prepared
using the following method:

Preparation of Citrate-Saline buffer:

Dissolve 3g of Tri-Sodium Citrate in 100 ml distilled water.

Add 20 ml of the dissolved Tri-Sodium Citrate to 80 ml 9g/l NaCl.

Preparation of New Methylene Blue or Azure B Stain Reagent:

Dissolve 0.1gram of New Methylene Blue (CI 52030) or Azure

B (CI 52010) in 100 ml of Citrate-Saline buffer.
Solution should be shaken at intervals over 24 hours.
Filter the solution.
Store in a brown glass bottle at 2 to 8°C. At this temperature,
the stain solution has a shelf life of about one month.

3.6 Test Procedure

3.6.1 A specimen of whole blood in EDTA is mixed by gentle inversion ten

times or until the complete resuspension of cells is accomplished
3.6.2 To a test tube (plastic or glass) place 3 drops (approximately 150µl) of
the reagent of choice e.g. New Methylene Blue stain or Azure B stain.
3.6.3 Add 3 drops (approximately 150µl) of patient EDTA blood to the stain
solution.
a) If the patient is anemic use 4 drops (approximately 200 uL)
instead.
b) If the patient is polycythemic use 2 drops (approximately 100
uL) instead.
3.6.4 Stain at ambient temperature or 37ºC between for 15 minutes.
3.6.5 Mix the contents of the test tube gently after the incubation period and
use the stained blood to make 2 blood films.
3.6.6 Allow to air dry and then examine without fixing or counterstaining.

3.7 Counting the Reticulocytes

3.7.1 Examine slide under low power to ensure the uniform distribution of
the red cells.
3.7.2 Choose an area of the blood film behind the tail where the red cells are
closely spaced but not touching or overlapping. If satisfactory, the film
is examined with an x100 oil immersion lens moving from field to field
in a battlement pattern.

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3.7.3 Count ±1000 RBC’s by counting 10 fields of a 100 red cells. Ensure
that each field contains more or less 100 cells by verifying that there
are about 25 red cells in one quarter of each of the 10 fields.
3.7.4 Reticulocytes are identified as slightly larger red cells that contain at
least two or more distinct blue-staining granules (or one granule linked
to a filamentous thread) that are visible without requiring fine focus
microscope adjustment on the individual cell to confirm their presence.
The remnants of nuclei material may appear as either punctate (tiny
dots evenly throughout the red cell) or aggregates (large clumps of
reticular material). The granules should be away from the cell margin
to avoid confusion with Heinz bodies.

Fig 1: Reticulocytes stained with New Methylene blue stain

4. CALCULATION OF RETICULOCYTES

4.1 Reticulocyte Percentage

Reticulocyte % = Number of reticulocytes counted x 100

Total no. of red cells counted

Example: Counted 15 retics in 10 fields of 100 red blood cells each.

Reticulocyte % = 15 x 100
1000

= 1.5%

4.2 Absolute Reticulocyte Count

Absolute Retic Count (x 109/L) =RBC count (x1012/L) x Reticulocyte Count (%)
100

4.3 Corrected Reticulocyte %

Calculation A:
Corrected Reticulocyte % = Patient red cell count x Reticulocyte %
Normal mean red cell count

Please take in consideration the age and gender of the patient and use the
appropriate RCC for that age group.

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Calculation B:
This is the preferred calculation for anaemic patients.

Corrected Reticulocyte % = Reticulocyte % x patients PCV

Y

where y is the average of the PCV(haematocrit) reference range for the age
group.

Example:
The normal Hct reference range for an 85 year old female is 0.35 – 0.45.

Therefore:
Y = (0.35 + 0.45) ÷ 2
= 0.40

4.4 Reticulocyte index (RI)

RI = Observed reticulocyte% x Measured Hb or PVC

Appropriate normal Hb or PCV

4.5 Reticulocyte Production Index (RPI)

The reticulocytes in the peripheral blood are dependant on one of three

variables:

· The rate of release of reticulocytes from the bone marrow

· The degree of immaturity of the freshly released reticulocyte
· The rate of disappearance of reticulum.

Bone marrow maturation for normal reticulocytes takes 48 to 72 hours of

which the reticulocytes spend 24 hours in the blood circulation. In periods of
intense erythropoietic stress, the reticulocyte maturation time in the marrow
may shorten to ≤24 hours, while the reticulocyte maturation time in the
peripheral blood shortens with correspondingly lengths.

There are 2 formulas in use to calculate the Reticulocyte Production index

A.
Reticulocyte Production Index = Corrected reticulocyte count
Maturation Time (days)

B. Reticulocyte Index
Reticulocyte Production Index = Maturation Time (days)

Please take into consideration the age of the patient and use the appropriate
RCC for that age group.

The maturation time of the reticulocytes in the peripheral blood.

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Patient PCV (HCT) Maturation in Peripheral Blood (Days)

>0.45 1.0

0.35 to 0.45 1.5

0.25 to 0.34 2.0

0.15 to 0.24 2.5

<0.15 ≥3.0

5. ENTERING RESULTS INTO THE COMPUTER

NB Patient’s Haematocrit (PCV) and Red Cell count must be available.

Percentage (%) Enter only %

Corrected % Computer will calculate
RPI Computer will calculate
Absolute count (x109/l) Computer will calculate

If the test was not done (e.g. insufficient blood):

Percentage (%) ND
Corrected % NP (No extra fee)
RPI NP (No extra fee)
Absolute count (x109/l) NP (No extra fee)

6. EXPECTED VALUES

6.1 Elevated uncorrected reticulocyte % may result from the release of immature
red cells into the circulation in the absence of the need for an increased
production of red cells. This occurs primarily in infiltrate marrow disorders with
prominent extramedullary erythropoeisis.

6.2 Anaemia with a prominent Reticulocytosis, that gives an elevated Reticulocyte

count after correcting for Polychromasia, suggests the following possibilities:

· Haemolysis
· Acute blood loss
· Response to specific replacement therapy e.g. Iron, Folate or Vitamin
B12 replacement therapy,

Decreased Reticulocyte counts can be due to decreased or ineffective

Erythropoiesis e.g. Aplastic anaemia or Myelodysplasia.

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7. Measurement of Uncertainty

7.1 The subjective and variable interpretation by individual technologists on the

definition of a Reticulocyte.
7.2 The size of the sample of red cells counted and evaluated
7.3 The type of blood film evaluated i.e. the variability in red cell distribution as
prepared by different slide film techniques example spun slides vs. wedge film
slides.

7.4 The use or the lack of use of a standardised area reduction device e.g. a
Miller’s ocular as a counting aid.
7.5 The corrected retic count, RPI and absolute count calculations all remove the
possible inaccuracy of the retic % caused by variations in the RBC count. The
presence of haemolysis, leading to fewer RBC’s, does therefore not invalidate
these calculated parameters.

8. REFERENCES

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