HEMATOPATHOLOGY

Original Article

Clinical Significance of Immature Reticulocyte Fraction

Determined by Automated Reticulocyte Counting
CHUNG-CHE CHANG, MD, PhD, AND LAWRENCE KASS, MD

The Sysmex R-3000 (TOA Medical Electronics, Kobe, Japan) eval- (IRF >0.23) and increased ARC generally indicated an adequate

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uates maturation of reticulocytes by quantitating the fraction of erythroid response to anemia. All but three specimens with an
reticulocytes within low-, middle-, and high-fluorescence inten- IRF less than 0.23 showed an RPI of 2 or less. These specimens
sity regions. We defined the immature reticulocyte fraction (IRF) were from patients with underlying diseases known to lead to
as the sum of the fraction of high-fluorescence intensity regions decreased erythropoietic activity, predominantly chronic renal
plus the fraction of middle-fluorescence intensity regions. Then, insufficiency. S p e c i m e n s w i t h a s u b n o r m a l or n o r m a l ARC
we studied the clinical significance of IRF in the evaluation of (with a corresponding RPI <2) but with an IRF of more than
anemia by comparing the IRF with the absolute reticulocyte 0.23 were from patients with various u n d e r l y i n g conditions,
count (ARC) and with the reticulocyte production index (RPI) i n c l u d i n g acute i n f e c t i o n , iron deficiency a n e m i a , h u m a n
and by r e v i e w i n g p e r t i n e n t clinical i n f o r m a t i o n a b o u t t h e immunodeficiency virus infection, sickle disease with crisis,
patients. In the study, 132 specimens from 102 patients undergo- pregnancy, and myelodysplastic syndrome. Our results indicate
ing evaluation of anemia were analyzed. By using simple regres- that an IRF of 0.23 or less in patients with anemia reflects bone
sion analysis, our results showed that the IRF has a weak but sig- m a r r o w t h a t is n o n r e s p o n s i v e or u n d e r r e s p o n s i v e to t h e
nificantly positive correlation with ARC and with RPI, indicating anemia. Patients with an increased IRF (IRF >0.23) may require
that IRF is an additional useful parameter to evaluate the erythro- further examination to clarify the cause of the anemia. (Key
poietic activity in anemia. Interpretation by integrating IRF and w o r d s : I m m a t u r e reticulocyte fraction; Absolute reticulocyte
reticulocyte enumeration (ARC and RPI) provided useful infor- c o u n t ; R e t i c u l o c y t e p r o d u c t i o n i n d e x ) Am J C l i n P a t h o l
mation for further subclassification of anemia. Increased IRF 1997;108:69-73.

Besides the actual reticulocyte count, assessment of enumeration. 1 5 - 1 8 Furthermore, some counters, like
reticulocyte maturation is important for evaluating the the Sysmex R-3000 (TOA Medical Electronics, Kobe,
degree of effective erythropoiesis, for understanding Japan), can evaluate the maturation of reticulocytes
the pathophysiologic changes of anemia, and for the by quantitating the fraction of reticulocytes within
differential diagnosis of anemia. 1-13 Historically, reticu- low (LFR)- , middle (MFR)-, and high-fluorescence
locytes have been classified into different maturational (HFR) intensity regions. The HFR represents the
stages by morphology. 1-4 Various reticulocyte indexes most immature reticulocytes; MFR represents imma-
have been used to estimate these maturational stages, ture reticulocytes, and LFR represents mature reticu-
but they are of limited clinical applicability because of locytes. However, to date, the clinical relevance of
variability in manual microscopic methods. 1 " 4,14 these distinctions is not completely clear.
Automated flow cytometric reticulocyte coun- In the p r e s e n t study, we o u t l i n e d t w o goals.
ters have increased the precision of reticulocyte Initially, we defined the immature reticulocyte frac-
tion (IRF) as the sum of the fraction of HFR plus the
fraction of MFR, as proposed by Davis. 19 First, we
asked whether the IRF was clinically informative
From the Department of Pathology, MetroHealth Medical Center and about the bone marrow response to anemia, by com-
Case Western Reserve University School of Medicine, Cleveland, Ohio. paring the IRF to the absolute reticulocyte count
M a n u s c r i p t received A u g u s t 16, 1996; revision accepted (ARC) and to the reticulocyte production index (RPI).
December 3,1996. The RPI is a mathematical expression of the erythro-
Address reprint requests to Dr Kass: Department of Pathology, poietic capacity of bone marrow. 2 ' 3 According to
MetroHealth Medical Center, Case Western Reserve University
School of Medicine, 2500 MetroHealth Drive, Cleveland, OH Hillman and Finch, 3 this numerical index can be
44109-1998. obtained as follows:

69
70 HEMATOPATHOLOGY
Original Article

Patient hematocrit Patient reticulocyte % by Davis19 who stated that the normal range for the IRF
Ideal hematocrit Reticulocyte maturation (in days) value was 0.05 to 0.22 in the American population. In this
group of healthy persons without anemia, the normal
With the use of the reticulocyte maturation time, a reference value for ARC was 30 to 82 x 10 9 /L. This value
compensation can be made for premature release of agrees with published normal values for ARC.20,21
reticulocytes (so-called shift reticulocytes) from the By using simple regression analysis, our results
bone marrow under the influence of erythropoietin in showed that ARC had a weak but significant positive
patients with anemia. Clinically, an RPI less than 2 has correlation with IRF for samples from healthy persons
been widely accepted as an indicator of inadequate without anemia (Fig 1, A, R2 = 0.27, P=.018). A similar
bone marrow response to anemia. 2,3 Second, we asked positive correlation also existed for samples from

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whether the IRF representing immature reticulocytes
used along with the ARC or with the RPI was helpful
in the differential diagnosis of anemia.

MATERIALS AND METHODS

Between December 1995, and March 1996, reticulo-

cyte counts were performed on 132 specimens of
peripheral venous blood anticoagulated with EDTA
from 102 patients undergoing evaluation of anemia.
There were 71 females and 31 males, ages 12 to 92
(mean, 52.6 years). As normal controls, specimens
from 20 healthy persons (10 women and 10 men, ages
18 to 81 years; mean, 43.6 years) who had normal val-
ues for hemoglobin, hematocrit, mean corpuscular
volume, mean corpuscular hemoglobin, mean corpus-
cular hemoglobin concentration, leukocyte count,
platelet count, and leukocyte differential count were
analyzed in the same way as the patient specimens.
Specimens were prepared according to manufac-
turer's guidelines 20 and were analyzed within 4 hours
of specimen collection. The reticulocyte percentage, 1.4
B
ARC, and fractions of HFR, MFR, and LFR in the retic- B

ulocyte population were determined by the Sysmex R- y = - 0.36463 + 2.5220X R2 = 0.25

3000, and hematocrits were determined by the Coulter
STKS (Coulter, Hialeah, Ha). Reticulocytes were iden- B

tified in the Sysmex R-3000 by fluorescence based on B

binding of auramine O to RNA. Depending on the flu- B

orescence intensity, the reticulocytes were further dif- a. / ^ B B
DC
ferentiated into HFR, MFR, and LFR. 20 With these ^ ^
data, the IRF and RPI were calculated. 0.6
B " V ^ "
B
B
Finally, completed charts were available for 90 of the
B
102 patients. We reviewed these charts and recorded ^ B

pertinent clinical information. Simple regression analy-

sis and the x2 were applied for statistical analysis.
—, a , r-
0.0 0.1
RESULTS IRF

FIG 1. A, Correlation between the absolute reticulocyte count

The mean value and SD for healthy persons without
(ARC) and the immature reticulocyte fraction (IRF) for samples
anemia in this study for IRF were 0.136 and 0.046, respec- from healthy persons without anemia. B, Correlation between the
tively (normal reference value is 0.044-0.228). These reticulocyte production index (RPI) and the IRF for samples from
results are consistent with the results reported recently healthy persons without anemia.

AJCP • July 1997

 CHANG AND KASS 71
Significance of Immature Reticulocyte Fraction

patients (Fig 2, A, R2 = 0.18, P=.0001). In healthy persons

B El
without anemia and patients with anemia, the correla-
tion between the RPI and IRF was similar to but slightly
y = - 17.959 + 515.29x R2 = 0.18
weaker than that between the ARC and IRF (Fig 1, B, R2
= 0.25, P=.02 for healthy persons without anemia; Fig 2, 500-
B, R2 = 0.15, P-0001 for patients with anemia).
As shown in sections a and b of Figure 2, A, 45 of the
132 specimens had an increased ARC (>82 x 10 9 /L). As o
depicted (see Fig 2, A, section a), 26 (58%) of these 45 a.
<
specimens had an IRF of 0.23 or more (the upper limit of
normal range for IRF). Nine of the 27 specimens showed

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an RPI greater than 2. The majority of these specimens
were from patients with sickle disease with crises. Of the
45 specimens, 19 (42%) had an IRF of less than 0.23 (see
Fig 2, A, section b). All but three of the 19 specimens
showed an RPI of 2 or less. These specimens were
mainly from patients with hypothyroidism, sarcoidosis,
eating disorder with malnutrition, and neurosyphilis.
The remaining 87 specimens had a normal or sub-
normal ARC (<82 x 10 9 /L). All of these specimens
showed an RPI of 2 or less. Of the 87 specimens, 59
y = -9.8161e-3 + 4.0089x R2 = 0.15 B
(68%) showed an IRF of less than 0.23 (see Fig 2, A, sec- 6.0-

tions c through e). Thus, the specimens with a normal B

or subnormal ARC were more likely to have an IRF of B

5.0 - a
less than 0.23 compared with specimens with an
increased ARC (68% vs 42%; P=.008, %2)- This trend is 4.0 -
B

consistent with the results of simple regression analysis. Q. B

However, there were 28 other specimens that also had B
3.0-
B
a normal or subnormal ARC but had an IRF of 0.23 or B
B
higher (see Fig 2, A, sections f and g). A review of the B
2.0- B E) B
patient charts revealed that specimens with a normal or B
B
B °G> B ^_^,- - " ' ^ B
subnormal ARC were from two different groups of #3 B B | ^
1.0-
S B
patients corresponding with the value of IRF. Specimens B a
with a normal or subnormal ARC and an IRF of less than B e
0.0 -
0.23 were from patients with anemia and underlying dis-
eases known to lead to decreased erythropoietic activity, IRF
such as chronic renal insufficiency and eating disorder,
predominantly chronic renal insufficiency (see Fig 2, A,
sections c through e). Conversely, specimens with a nor- FlG 2. A, Correlation between the absolute reticulocyte count (ARC)
and the immature reticulocyte fraction (IRF) for samples from
mal or subnormal ARC but an IRF of 0.23 or higher were patients. Section a, sickle disease with crisis (n = 8), physiologic ane-
from patients with anemia associated with various under- mia secondary to respiratory insufficiency (n = 1)*, pregnancy (n =
lying conditions, including acute infection, iron deficiency 2), acute infection (n = 3), lead poisoning (n = 1); section b, sickle dis-
anemia, human immunodeficiency virus infection, sickle ease with crisis (n = 1), physiologic anemia secondary to respiratory
insufficiency (n= 1)*, hypothyroidism (n = 4), sarcoidosis (n = 2),
disease with crisis, pregnancy, and myelodysplastic syn-
neurosyphilis (n = 1), eating disorder with malnutrition (n= 2); sec-
drome (see Fig 2, A, sections f and g). tion c, chronic renal failure (n = 30), eating disorder with malnutri-
tion (n = 3); section d, chronic renal failure (n = 1); section e, chronic
renal failure (n = 8); section f, iron deficiency anemia (n = 3), human
DISCUSSION immunodeficiency virus (HIV) infection (n = 3), acute infection (n =
2); section g, iron deficiency anemia (n = 2), HIV infection (n = 2),
Automated reticulocyte counting has helped labora- pregnancy (n = 1), acute infection (n = 5), sickle disease with crisis (n
tories not only to improve precision in reticulocyte = 4), myelodysplastic syndrome (n = 1). 'Different specimens from
enumeration, but also to stimulate interest in the clini- the same patient. B, Correlation between the reticulocyte production
index (RPI) and the IRF for samples from patients.
cal utility of quantitating the immaturity of reticulocyte

Vol. 108 • No. 1

72 HEMATOPATHOLOGY
Original Article

populations. Limited pilot studies indicated that the itself through the enhanced (increased or accelerated)
level of immaturity of reticulocyte populations could release of immature reticulocytes from bone marrow.
have clinical utility in the assessment of erythropoietic This effect has been suggested to be erythropoietin-
activity in cases of bone marrow transplantation 4-8 and related, at least partially. 26 ' 27
anemia. 10 - 11 However, the clinical significance of the It is noteworthy that all but three specimens with an
level of immaturity of reticulocyte populations is still IRF of less than 0.23 showed an RPI of 2 or less (see Fig
unclear. Furthermore, to date, no international consen- 2, A, sections b through e). The RPI was defined before
sus on the definitions of terms and the methods for the advent of the automated reticulocyte counters and
measuring reticulocyte immaturity exists. 4 ' 9 ' 10 ' 16,22-25 has been widely accepted as an indicator of bone mar-
In our study, the term immature reticulocyte fraction rep- row response to anemia. 2 ' 3 Clinically, it has been widely

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resents the immature reticulocyte population. The advan- accepted that an RPI of 2 or less indicates a bone mar-
tages of our definition are as follows. First, HFR has been row that is nonresponsive or hyporesponsive to anemia.
shown to have a much higher coefficient of variation than Accordingly, an IRF less than 0.23 is an indicator,
MFR.16 Thus, the use of IRF should significantly decrease corresponding with an RPI of 2 or less, for patients
the coefficient of variation compared with using the frac- with anemia and bone marrow that is nonresponsive
tion of HFR alone, a value that has been used by some or underresponsive to anemia. This finding is further
investigators as the sole indicator of reticulocyte immatu- supported by the observation that these specimens
rity.23'24 Second, IRF gave a pathophysiologic meaning for were from patients with chronic diseases known to
representing total immature reticulocytes and showed a lead to decreased erythropoietic activity, such as
significant positive correlation with the ARC and with the chronic renal failure, eating disorder with malnutri-
RPI in our study. The associations between the fraction of tion, hypothyroidism, sarcoidosis, neurosyphilis, and,
HFR and the ARC and between the fraction of HFR and particularly, chronic renal failure. Our results are con-
the RPI (R2 = 0.064, for ARC; R2 = 0.042 for RPI) were sistent with those of Davis et al. 10 ' 26 By using a differ-
much less significant than those between the IRF and ent automated reticulocyte counter, they suggested
ARC and between the DRF and RPI. Recently, Davis19 sug- that only the patients with anemia with low reticulo-
gested that the determination of IRF may be the best way cyte counts and low mean fluorescence intensity of the
to detect changes in erythropoietic activity. Our studies reticulocyte population have true hypoproliferation.
support his suggestion. The significance of the three specimens among those
As shown by regression analysis in the present with an IRF of less than 0.23 with an RPI greater than 2 is
study, a weak but statistically significant positive cor- uncertain. It has been suggested that in cases of reduced
relation between the ARC and IRF and between the erythropoiesis, application of an RPI correction may con-
RPI and IRF, agreed with the findings by Davis et al. 26 ceal a failure of the bone marrow response, ie, a falsely
By using a different flow cytometry-based method elevated RPI, because the shift does not occur fully or at
with thiazole orange, their results also showed weak all. These three specimens could reflect this situation.
significant positive correlation between the IRF and Thus, in cases of an RPI greater than 2 but a low IRF, this
ARC. Our study further confirmed that these correla- possibility must be considered in the differential diagno-
tions are independent of the fluorochrome and the sis of anemia. Our findings support the concept that the
instrumentation. In their study, a correlation between ERF may be better than, or at least as good as, the RPI for
the RPI and IRF was not specifically addressed. evaluating hyporesponsive marrow. Furthermore, the ERF
The weak but significant correlations between the had the advantage of being directly obtained from the
ARC and ERF and between the RPI and IRF indicated that automated reticulocyte counter over an off-line or labora-
the IRF is also a useful parameter to evaluate erythropoi- tory information system-generated RPI calculation.
etic activity in anemia. Further analysis by interpreting Another set of parameters, a normal or subnormal
the integration of IRF and reticulocyte enumeration (ARC ARC but an ERF of 0.23 or higher, defines an additional
and RPI) is necessary and may help to further understand group of patients with various underlying conditions (see
erythropoiesis in anemias and to subclassify them. Fig 2, A, sections f and g). This finding may be clinically
In our study, the majority of specimens with an important because the majority of the patients need fur-
increased ARC (>82 x 10 9 /L) showed an IRF of 0.23 or ther immediate examination for proper diagnosis and
higher. This finding indicated that an increased level treatment. This finding further highlighted the value of
of erythropoietic bone marrow activity could result in the ERF in the differential diagnosis of anemia. Without
an increased IRF. The increased IRF suggested that an the information provided by the ERF, these patients would
erythropoietic bone marrow response could manifest be believed to have the same pathophysiologic causes as

AJCP • July 1997

 CHANG AND KASS 73
Significance oflmmm Reticulocyte Fraction

those with a normal or subnormal ARC (with a corre- 8. Batjer JD, Riddell K, Fritsma GA. Predicting bone marrow trans-
plant engraftment by automated flow cytometric reticulocyte
sponding RPI <2) and an IRF of less than 0.23. analysis. Lab Med. 1994;25:22-26.
However, the pathophysiologic changes of this set of 9. Kuse R. The appearance of reticulocytes with medium or high
parameters remains unclear. Tatsumi and Izumi 13 and RNA content is a sensitive indicator of beginning granulo-
cyte recovery after aplasiogenic cytostatic drug therapy in
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or megaloblastic anemia may result from a qualitative tion using thiazole orange provides clinically useful reticulo-
cyte maturity index. Arch Pathol Lab Med. 1989;113:684-689.
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