SYSMEX EDUCATIONAL ENHANCEMENT

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SEED HAEMATOLOGY

The importance of reticulocyte detection

Production of reticulocytes History
All blood cells emanate from a stem cell. Under pronounced n 
1865 First description of reticulocytes by Erb, who
proliferation they differentiate into cells of the three blood discovered an intracellular reticulum using picric acid.
cell lines (erythropoiesis, granulopoiesis and thrombopoie- n 
1881 Using supravital staining, Ehrlich demonstrated
sis). Focusing on the red blood cell (RBC) line, we see that an intracellular network that was described as sub-
the life span of circulating erythrocytes is approx. 120 days. stantia reticulo-filamentosa.
Nearly 1 % of this total is lost daily and is replenished by new n 
1891 Smith identified reticulocytes as immature red
cells. Every second, about two millions of RBC are being blood cells.
produced. In the bone marrow the young erythroblasts eject n 1932 Classification of maturation stages by
their cell nucleus, becoming reticulocytes which then enter Heilmeyer (see Table 1).
the peripheral bloodstream. n 
1953 Seip quantifies the maturation stages with
reference ranges (see Table 1).
As a rule, the reticulocyte remains in the bone marrow for n 
1960 Counting of reticulocytes with methods based
a further three days and for one day in the peripheral blood- on fluorescence (acridine orange), developed by
stream. The name ‘reticulocyte’ originates from the web-like Kosenov & Mai.
structure (‘reticulum’ in Latin), which becomes visible after n 
1983 Tanke automates the measurement of reticulo-
staining with supravital stains, such as brilliant cresyl blue cytes by using the fluorescence method and flow
or new methylene blue (precipitation of ribonucleic acid cytometry.
fragments; Fig. 1). By removing the endoplasmic reticulum,
the reticulocyte develops into a mature red blood cell
within four days.
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Table 1 Maturation stages according to Heilmeyer and quantification according to Seip

Maturation stages according Morphological description Quantification according to Seip

to Heilmeyer (normal %)

Stage 0 Nucleus

Stage I Reticulum consists of dense clots < 0.1

Stage II Loosely arranged reticulum 7.0

Stage III Diffusely arranged reticulum 32.0

Stage IV Some scattered granulae 61.0

The maturation stages I and IV are often interpreted incor- If erythropoiesis is stimulated, the earlier level of matura-
rectly: stage I is sometimes described as an erythroblast and tion shifts into the peripheral blood (similar to a ‘left shift’
stage IV as a mature RBC as its low content of RNA is not in granulopoiesis).
detected. The correct interpretation of stage IV is especially
important, as this maturation stage is dominant in the Reticulocyte count
peripheral bloodstream. The normal fraction of reticulocytes in the blood depends
on the clinical situation but is usually 0.5 % to 1.5 % in adults
In 1986 the National Committee for Clinical Laboratory [3] and 2 % to 6 % in newborns [4]. The number of reticulo-
Standards (NCCLS) classified as a reticulocyte ‘any non- cytes is a good indicator of bone marrow activity because it
nucleated red cell containing two or more particles of blue represents recent production and shows the erythropoietic
stained material corresponding to ribosomal RNA’ [1]. The status of the patient and if the production is healthy or not.
International Council for Standardization in Haematology Therefore, a reliable count of the reticulocytes is needed.
(ICSH) also accepted this definition [2]. A comparison of different reference ranges as reported by
different authors can be found in a review article published
by Piva et al. [5]

Indications for counting reticulocytes

n Basic diagnostic work-up in all types of anaemias
n Therapeutic monitoring during iron, vitamin B12
or folate replacement
n Therapeutic monitoring under erythropoietin
treatment
n Monitoring during stem cell transplantation
n Newborns and paediatric patients
Fig. 1 Supravital stain of a smear of human blood with different stages
of reticulocytes

The reticulocytes reflect the regeneration of erythropoiesis. Determining reticulocytes from EDTA blood remains reliable
In a balanced system, > 90 % of the very mature stages of for up to 72 hours after blood sampling. The storage temper-
reticulocytes (stages III and IV) are present in the peripheral ature of +4 °C or 20 °C, respectively, has no significant effect
bloodstream. on the measured result [6].
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Manual count
(Materials: supravital stains, such as brilliant cresyl blue level of reproducibility of the results. In automated counts
or new methylene blue, a prepared microscope slide and the measurement signals of up to 30,000 red blood cells are
a microscope). evaluated. This results in both high count rates and a high
1. Whole blood is mixed with equal volumes of a supravital degree of precision. Compared to manual reticulocyte
stain. counting, automated counting results (Fig. 2) are available
2. After incubation the sample is smeared on a microscope much faster, actually in less than one minute.
slide and air-dried.
3. The reticulocytes are counted under the microscope at oil

Forward scatter
immersion magnification (1,000-fold magnification).
4. 1,000 red blood cells are counted. At 1,000-fold magnifi-
cation this corresponds to approx. five visual fields, each
consisting of approx. 200 red cells.
5. Reticulocyte counts are given per mill [‰] or per
cent [%].

Error rates for manual counting are quoted in the literature

at 25 – 50 % CV [7 – 8] and higher, depending on the number
of reticulocytes. Counting 1,000 cells is recommended
as standard. According to a recommendation by the ICSH
(1998) [9] on the counting rate in the reference range
of reticulocytes, at least 40,000 cells should be counted
to avoid exceeding a statistical error of 5 % (Table 2).
Fluorescence

Table 2 Effect of the number of counted RBC on the statistical error Fig. 2 Scattergram of the reticulocyte channel (RBC: mature red blood
in reticulocyte counts. The numbers quoted refer to a CV of 5 %. cells; RET: reticulocytes; PLT: fluorescence-optical platelets)

Reticulocyte count Number of cells to be counted

in blood (%) to achieve a CV of 5 % Reticulocyte count in the clinical diagnosis
1 39,600 The interpretation of the reticulocyte count is problematic

2 19,600 in severe anaemias. A moderately increased relative reticu-

locyte count in severe anaemia does not indicate a suffi-
5 7,600
ciently strong regeneration of erythropoiesis, but merely
10 3,600
indicates a shortened life span of the red blood cells. It is
20 1,600
preferable to report the absolute reticulocyte concentration
50 400 as reticulocytes/µL, as this provides a direct measurement
of erythropoietic performance.
Automated counting
To accurately measure reticulocyte counts, automated counters For example, a value of 20 ‰ reticulocytes is considered
use a combination of laser excitation, detectors and a fluo- increased. However, in severe anaemia with 2 million red
rescence marker that labels RNA and DNA (such as thiazole blood cells, 20 ‰ reticulocytes merely represent 40,000
orange or polymethines) [10]. reticulocytes/µL, a value within the reference range.

To measure the reticulocytes, the sample is incubated with The relative number of reticulocytes (‰ and %) gives
an RNA-binding fluorescence marker and counted by flow some information about the life span of the red blood cells,
cytometry. Automated reticulocyte counters use objective whereas their cell concentration (reticulocytes/ µL) reflects
thresholds for the classification of cells. This ensures a high the erythropoietic productivity of the bone marrow.
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should increase within 2 – 3 days in response to the loss of

With a simple formula, relative counts (%) can be RBC, and reach its peak in 6 – 10 days [11]. If that does not
converted into cell concentrations (RET/µL): happen, it could point out a defect of the erythropoietic
process in the bone marrow.
RET [%] x RBC [106/µL]
= reticulocyte concentration [106/µL]
100
In case of a pronounced red blood cell production, the mat-
The reference ranges – in percentage and absolute – uration of the reticulocytes shifts into the peripheral blood
of reticulocytes according to Cavill et al. [6] are the as the reticulocytes are passed into peripheral blood at an
following: earlier stage (the altered dwell time in peripheral blood is
called a ‘shift’). This leads to a pronounced increase in circu-
Relative reticulocytes: m/f 0.43 – 1.36 %
lating reticulocytes, but does not represent proof of eryth-
Reticulocyte count: f 17.0 – 63.8 x 109/L
m 23.0 – 70.1 x 109/L ropoietic performance. The maturation time of reticulocytes
in bone marrow is proportional to the haematocrit, i.e. it
It is necessary to remember that published reference ranges decreases with the haematocrit, and the maturation time
from other analysers may be used on newer analysers, but in peripheral blood increases. To give an indication of the
only after having validated them. Since there are also differ- efficiency of the bone marrow, the reticulocyte count is
ences between haematological reference intervals from dif- corrected by this haematocrit-dependent factor.
ferent populations, every lab will need to select which of the
published reference ranges fits better their own population. Table 3 Link between haematocrit and maturation time of reticulocytes
in peripheral blood

Reticulocyte-associated parameters
Haematocrit Reticulocyte survival in blood
Reticulocyte index (RI) = maturation correction
The relative portion (‰ and %) of reticulocytes may 36 – 45 % 1 day
increase, if either the reticulocytes are in fact increased or
26 – 35 % 1.5 days
the red blood cells are decreased. Corrective measures can
16 – 25 % 2 days
be carried out via the patient’s haematocrit by reference
15 % and below 2.5 days
to a normal haematocrit of 0.45 [L/L]. This type of correction
is recommended with anaemias:
The RPI should be between 0.5 % and 2.5 % for a healthy
RI = RET [%] x HCT [L/L] individual [11]. In case of anaemia, an RPI < 2 % indicates
0.45 [L/L] (standard HCT) inadequate production of reticulocytes, while an RPI > 3 %
indicates compensatory production of reticulocytes to
Reticulocyte production index (RPI) replace the lost red blood cells [12].
The RPI is an index, which helps to evaluate erythropoiesis
efficiency and thus the productivity of the bone marrow. The
physiological maturation of reticulocytes is divided into the
RET [%] HCT [L/L] (patient)
bone marrow maturation (about 8 – 10 days since the first RPI = x
RET maturation time 0.45 (standard HCT)
division of the proerythroblast) and about 1 – 2 days in in blood in days

the peripheral blood stream until they become mature red

Example:
blood cells.
Patient values: HCT= 0.25 L/L, reticulocytes = 20

The idea of the RPI is to assess whether the bone marrow

20 [%] 0.25
is producing an appropriate response to an anaemic state. RPI = x = 5.5
2 0.45
After an acute haemorrhage, the reticulocyte production
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Immature Reticulocyte Fraction (IRF) Reticulocyte maturation

The IRF value is an early marker for evaluating the regeneration In addition to conventional reticulocyte measurement, the
of erythropoiesis. Whereas the IRF percentage increases after fluorescence flow cytometry method allows the classifica-
only a few hours, the reticulocyte count increases after 2 – 3 tion of reticulocytes into three maturation stages. These
days. If the IRF value does not increase during the treatment maturation stages are defined by the RNA content of the
of deficiency anaemias with erythropoietin or vitamins, this reticulocyte, measured on the analyser as fluorescence
indicates a lack of response to therapy. In addition, it helps to intensity. The higher the RNA content, the less mature the
classify hypo-, normo- and hyper-regenerative anaemias. reticulocytes are.

Together, the IRF value and the reticulocyte count have Reticulocytes are fractioned according to their fluorescence
proven themselves as monitoring parameters for bone mar- intensity into the following three categories representing
row and stem cell transplants. With successful transplants, different maturity stages (Table 4):
in 80 % of the cases the IRF value reaches its 5 % mark earlier n LFR (low-fluorescence reticulocytes)
than the granulocytes their classic threshold of 0.5 x 109 – ‘mature’ reticulocytes
granulocytes/L [13]. n MFR (medium-fluorescence reticulocytes)
–‘semi-mature’ reticulocytes
An example of this utility can be seen in Fig. 3, where differ- n HFR (high-fluorescence reticulocytes)
ent pathologies are represented with different levels of – ‘immature’ reticulocytes
immature and mature reticulocytes. As an example, in the
case of aplasia, both immature (IRF) and total reticulocytes IRF is the sum of MFR plus HFR, i.e. the immature
are low. This type of anaemia affects the precursors of red reticulocytes, and is also referred to as the ‘reticulocyte
blood cells but not the ones of the white blood cell line. maturation index’.
The bone marrow ceases to produce red blood cells, and IRF = MFR + HFR
that is why neither immature nor mature reticulocytes can
be observed.

The reference range [14] of IRF is 1.6 – 10.5 %, for both men

and women.

Immature
reticulocytes
Acute Haemolysis
leukaemia
High

Polycyth.
Dyserythropoiesis vera

Normal
Normal

Low Aplasia Fig. 4 Scattergram of the reticulocyte channel

Low Normal High Reticulocytes

Fig. 3 The importance of reticulocytes for clinical diagnosis

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Table 4 Maturation stages of reticulocytes

LFR MFR HFR

Low-Fluorescence Reticulocytes Medium-Fluorescence Reticulocytes High-Fluorescence Reticulocytes

Little content of RNA Medium content of RNA High content of RNA

Mature reticulocytes Semi-mature reticulocytes Immature reticulocytes

Reference range: 86.5 – 98.5 % Reference range: 1.5 – 11.5 % Reference range: 0 – 1.4 %

Reticulocyte Haemoglobin equivalent (RET-He)

In addition to the quantitative determination of the blood Indication
n Classification of normochromic and hypochromic
count parameters (RBC, HGB, RET#/%, IRF, MCV), RET-He
offers a qualitative dimension. Red blood cells have a 120-day anaemias
n Monitoring the therapy of chronic infections or
lifetime. Therefore, using them for detecting iron deficiencies
and changes in the iron status of erythropoiesis is only tumours
n Monitoring erythropoietin therapy and iron
possible at a relatively late point in time. In contrast to that,
RET-He represents the HGB content of young RBC, the retic- substitution
n Determining the trend of the current iron status
ulocytes, and thus offers real-time information on iron supply
n Early marker for diseases. Together with RET#, it lets
to erythropoiesis.
clinicians draw conclusions on both the quality and

Measuring the haemoglobin content of the reticulocytes quantity of the young RBC fraction.

means you can look at the current iron supply to erythro-

poiesis and judge the ‘quality’ of the newly produced cells. The reference ranges of RET-He [14] are (for both men
This lets you detect changes in iron status far earlier than and women) 1,996 – 2,407 fmol or 32.1 – 38.8 pg.
through the haemoglobin content of mature red blood cells.

The determination of RET-He can be performed on the

haematology analyser together with the routine parameters RET-He alone provides information on the current bioavaila-
obtained during the whole blood test. A major advantage bility of iron – a low value means there is a lack of iron or
over the parameters ferritin or transferrin is that RET-He is iron is not bioavailable for erythropoiesis. It is often used
not affected by the acute phase reaction. These conventional together with ferritin – a high or normal ferritin value
biochemical markers are drastically disturbed e.g. during together with a low RET-He value can suggest functional
inflammation, pregnancy, or in the presence of many other iron deficiency while low ferritin values together with a low
severe diseases, so that a clinical interpretation of the RET-He suggest a classic iron deficiency. Since ferritin is
results would then be difficult or impossible. Measuring the falsely increased during the acute phase of diseases, pres-
haemoglobin content of the reticulocytes as a direct assess- ence of inflammation should be checked, e.g. by CRP.
ment of the iron actually used for the biosynthesis of hae-
moglobin can indicate even in these cases whether there is RET-He is used for monitoring erythropoietin (EPO) and/
enough iron available for erythropoiesis. or IV iron therapy. If the value increases it indicates the
therapy is having a positive effect.
The clinical usefulness of the RET-He parameter has been
proven and it is now an established parameter in advanced
haematological analysis.
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Thomas-Plot index Thomas plot can also be used to monitor the patients that
In 2006, Thomas et al. described a diagnostic plot model in are under treatment and see how they move from one quad-
order to differentiate iron-deficient states and predict those rant to another.
patients who will respond to erythropoietin therapy [15].
The balance of the iron in our body is regulated by the rate Conclusions
of erythropoiesis and the size of the iron stores. The rela- A reliable reticulocyte count including some more parameters
tionship between erythropoietic haemoglobin incorporation associated with reticulocytes (IRF, RPI, etc.) are important to
(RET-He) and iron stores (ferritin) can be described in a see if the bone marrow is working properly to develop new
diagnostic plot. This plot, called ‘Thomas plot’, allows the red blood cells, but the quantity aspect is not the only one
differentiation of classical iron deficiency from anaemia of that is important. We have seen that the reticulocyte hae-
chronic disease. The plot indicates the correlation between moglobinisation, RET-He, is also of high importance in order
the ratio sTfR/log ferritin (ferritin index), a marker of iron to define the quality of the newly produced reticulocytes.
supply for erythropoiesis, and the RET-He (Fig. 5) [16]. The
Reliable reticulocyte information, regarding quality and
quantity, helps in the differential diagnosis of anaemias as
well as in the monitoring of patients.

Fig. 5 Thomas plot to identify different phases of advancing

iron deficiency (ACD: anaemia of chronic disorders, ERF: end-stage
renal failure, IDA: Iron deficiency anaemia, ID: classic iron deficiency)

References
Koepke et al. (1986): Reticulocytes. Clin Lab Haematol 8, 169 – 179.
[1]  Peebles DA et al. (1981): Paediatric Haematology. Churchill
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 he Expert Panel on Cytometry of the International Council of
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Means RT et al. (2009): Anemia: Wintrobe’s Clinical Hematology,

[3]  [9] ICSH Expert Panel on Cytometry. (1998): Proposed reference
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Lilleyman JJ et al. (1992): Paediatric Haematology. Churchill

[4]  [10] Davis BH, Bigelow NC. (1994): ‘Reticulocyte analysis and
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d’Onofrio G et al. (1996): Indicators of haematopoietic recovery

[13]  Thomas C et al. (2006): The diagnostic plot: A concept for
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measurements. Clin Lab Haem 18(Suppl.1):45 – 53. response to epoetin therapy. Medical Oncology Volume 23, Issue 1,
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