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Simultaneous Measurement of Reticulocyte and Red Blood Cell Indices in
Healthy Subjects and Patients With Microcytic and Macrocytic Anemia
By Giuseppe d’onofrio, Rosario Chirillo, Gina Zini, Gianfranco Caenaro, Maria Tommasi, and Giulia Micciulli
Using the newBayer H*3 hematology analyzer (Leverkusen,
Germany), we have determined red blood cell and reticulocyte indices in 64 healthy subjects, in patients with microcytosis dueto iron deficiency (58 patients) and heterozygous
P-thalassemia (40 patients), and in patients with macrocytosis (28 patients). We
found in all cases that reticulocytes
were larger than mature red cells by 24% to 35%, with a
hemoglobin concentration 16% to 25% lower and a similar
hemoglobin content. The correlation between red cell and
reticulocyte indices was strikingly tight ( r = .S28 forvolume,
r = .g29 for hemoglobin concentration, r = .S72 forhemoglobin content) in all four groups, regardless of red blood cell
size. The ratio of reticulocyte to red blood cell mean corpus-
colar volume (MCV ratio) wasconstantly above 1. Inversion
of the MCV ratio was observed only in four patients. It was
always abrupt and transitory and was associated with erythropoietic changes leading to the production of red blood
cells of a different volume (treatment of megaloblastic anemia, functional iron deficiency, bone marrow transplantation). In two cases of marrow transplantation, reticulocyte
volume fell during the aplastic phase after conditioning chemotherapy and then rapidly increased up to values higher
than before; this production of macroreticulocytes was the
earliest sign of engraftment.
0 1995 by The American Society of Hematology.
T
counting using an opticalmethod based onthemeasurement
of
scatter and absorption of helium-neon laser light, associated
with
automatedperoxidasecytochemicalwhitebloodcelldifferential
counting.y The H*3 red blood cell counting method, in particular,
is identical to that of the previous H*l and H*2 systems:“”” after
isovolumetric red blood cell sphering, measurements of monochromatic light scattered at two different angular intervals are electronically processed to derive their volume and refractive index,which is
a linear functionof hemoglobin concentration. Thus, besides directly
measuring mean corpuscolar volume (MCV) and calculating mean
corpuscolar hemoglobin concentration (MCHC) and content (MCH),
the Bayer H series instruments also provide a direct measure of cell
hemoglobinconcentration mean (CHCM), which is compared in
each analyzed sample with MCHC for quality control purposes and
for detection of red blood cell abnormalities. The instruments also
provide the percentage of red blood cells with volume less than 60
fL (microcytes) and with hemoglobin concentration lower than 28
g/dL (hypochromic red blood cells).
The H*3 reticulocyte method. Reticulocytecountingrequiresa
preliminary manual mixing of 3 pL of whole blood with 3 mL of
reticulocyte reagent, containing a surfactant, which spheres RBCs
andreticulocytes,and
the nucleicacid-bindingdyeoxazine
750,
which selectivelystainsreticulocytes
by complexion with cytoplasmic RNA. After a IS-minute incubation, the prepared sample is
aspirated through the H*3red blood cell flowcell, where three detectorsmeasure laser light scatter, at low angle (2”to 3”) andhigh
angle (5” t o IS’), and absorption. On a two-dimensional cytogram
of absorption versus low-angle scatter, the stained reticulocytes are
separated from unstained erythrocytes, platelets, and leukocytes by
appropriate thresholds. Moreover. because the amount
of light absorbed by reticulocytes is proportional to the intensity of staining
and RNA content, reticulocytes are subdivided into three populations
with low, medium, and high RNA content. From the amount of the
light scattered at two different angles, the H*3 is capable of separately measuring reticulocyte mean corpuscolar volume (MCVr) in
femtoliters
and
corpuscolar
hemoglobin
concentration
mean
(CHCMI) in grams per deciliter, together with MCV and CHCM of
matureerythrocytes.
Mean hemoglobincontent
of reticulocytes
(CHr) and red blood cella (CH) is calculated from the product of
volume times hemoglobin concentration of single cells.
Study .\amples. K3EDTA-anticoagulated peripheral bloodsamples were analyzed with the H*3 within 2 to 36 hours from phlebotomy. They were refrigerated at 4°C if the analysis had to be delayed
more than 4 hours. A small number of the study sampleswere mailed
in refrigerated bags from Rome to Treviso using an express daily
courier. These procedures were defined after a stability study,
which
HE INTRODUCTION of flow cytometric methods,
based on the measurement of fluorescence after staining with RNA-binding fluorochromes, has greatly improved
the precision and accuracy of reticulocyte counting.’.’ The
increased sensitivity in the low reticulocyte range and the
availability of reticulocyte maturation indices based on the
measurement of RNA content haverecently extended the
diagnostic applicationsof reticulocyte counting to new clinical settings,such as monitoring of erythroidregeneration
or bone
marrow
transplantation
after
chemotherapy”‘
(BMT)5” and of erythropoietic response to erythropoietin
administration.’
The latest achievement in automatic reticulocyte counting
is the simultaneous flow-cytometric measurement of volume
and hemoglobin concentration on red blood cells andreticulocytes by the Bayer H*3 hematology analyzer(Leverkusen,
Germany). We report the results of a two-institution study
of the H*3 reticulocyte method, including the definition of
the distributionranges of reticulocyteand red bloodcell
indices in healthy subjects and in anemic patients with red
cells of abnormal size, as well as our first observations on
the clinicalutility of such indices in the monitoring of erythropoietic function.
MATERIALSANDMETHODS
The H*-? hemutology analyzer. The Bayer H*3 is an automated
blood cellcounterthatperformscomplete
blood andreticulocyte
Front the Reseurch Centerforthe Development und Clinical Evuluation of Automated Methods in Hemutology, Hemuto1og.y Service,
Universitu Cattvlica del Sacro Cuore, Rome; und the Laborcltop
oj Clinical Pathology, Presidio Multizonale Ospedaliero, Treviso,
Italy.
Submitted Februuy 23, 1994; accepted September 30, 1994.
Address reprint requests to Giuseppr d’onofrio, MD, Research
Center for the Development and Clinical Evaluation of Automated
Methods in Hematology, Servizio di Emutologiu, Universitri Cuttolicu del Sacro Cuore, Largo Francesco Vito l , 1-00168 Rome, Italy.
The publication costs of this urticle were defruyed in purt by pug‘
charge puymml.Thisarticle
must therefore he hereby tnurked
“advertisement” in uccordunce with 18 U.S.C. section 1734 solely to
indicute this juct.
0 1995 by The Americun Sociey of he mu to log.^.
0006-497//9S/8503-0005$3.00/0
818
Blood, Vol 85, No 3 (February 1). 1995: pp 818-823
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RETICULOCYTE AND RED CELL INDICES
819
Table 1. Mean Values and Reference Rangesof Red BloodCell and Erythrocyte Parameters MeasuredWith the H*3 on 64 Healthy Subjects
Males (n = 32)
All Subjects (n = 64)
Parameter
Mean
Reference Range
Mean
Reference Range
Hemoglobin (g/dL)
Red blood cells (x10’2/L)
14.0
4.66
89.9
32.6
31.6
30.3
27.7
0.9
0.9
41.0
111.7
26.3
28.5
12.9-15.7
4.22-5.26
14.5
4.86
89.3
32.7
31.7
30.1
27.6
0.9
41.5
112.2
26.3
28.6
13.5-15.7
4.44-5.35
83.4-97.0
31.5-34.0
30.9-33.2
27.9-31.9
25.6-29.5
MCV (fL)
MCHC (g/dL)
CHCM (g/dL)
MCH (pg)
CH (pg)
83.4-97.0
27.8-32.0
Reticulocytes (96)
Reticulocytes (x109/L)
MCVr (fL)
CHCMr (g/dL)
CHr (pg)
31.1-34.0
30.1-33.2
25.6-29.6
0.5-1.3
0.5-1.4
22.7-66.9
103.2-126.3
23.5-28.7
25.9-30.6
24.4-65.8
104.0-123.6
24.0-28.7
26.3-30.2
Females (n = 32)
Mean
13.5
4.46
90.5
32.7
31.5
30.4
27.8
40.6
111.2
26.4
28.5
Reference Range
P Value of
t-Test (m/f)
12.9-14.3
4.20-4.78
84.5-95.0
31.1-33.6
30.1-32.9
27.9-31.4
26.8-30.5
0.6-1.4
25.4-66.6
103.2-125.8
23.5-28.2
25.8-29.4
1.01
<.Ol
.257
.l36
,396
.382
,503
,446
,783
.553
,837
,786
Hemoglobin, red blood cells, MCHC, and MCH were obtained as a part of the H13 complete blood count. All remaining red blood cell and
reticulocyte parameters were generated in the H*3 reticulocyte method.
had shown that K3EDTA specimens do not show significant changes
in reticulocyte percentage, absolute values, or indices after 8 hours
at room temperature and 72 hours at 4°C.or after 12 hours in
refrigerated bags. The study of H*3 stability was performed as a
part of a complete instrument performance evaluation, which proved
that all H*3 measurements are highly comparable with the reference
methods and have precision, linearity, and carry-over features equal
to or better than the manufacturer’s specifications (unpublished data,
May 1993).
Reference ranges. The reference ranges for the H*3 hemoglobin,
red blood cell, and reticulocyte measurements and indices were obtained from 32 male and 32 female healthy adult subjects, with no
clinical symptoms and normal results at a screening blood test including complete blood count, differential count, and serum femtin
level. Reference ranges were calculated as the 95 central percentiles
of the distribution.
Patient study. We also investigated the reticulocyte and red
blood cell indices in anemic patients with microcytosis or macrocytosis. The microcytic anemia group included 58 patients with overt
iron deficiency anemia (hemoglobin level, 5.4 to 10.7 g/dL; serum
femtin level, below 15 ng/mL”) before iron treatment and 40 with
heterozygous @-thalassemia and no iron deficiency (hemoglobin
level, 8.9 to 12.4 g/&; AZ hemoglobin level, above 5%; serum
femtin level, above 15 ng/mL). The macrocytic anemia group included 28 patients with anemia of different etiology (hemoglobin
myelodysplastic synlevel, 4.5 to 11.2 g/dL; MCV, above 100 a):
dromes, eight patients; chronic liver diseases, seven; AIDS treated
with U T ,nine; folate deficiency, two; and vitamin B12 deficiency,
two. Moreover, during our H*3 evaluation, we were able to follow
with daily H*3 analysis a small number of hematologic patients,
including two patients undergoing bone marrow transplantation.
RESULTS
Reference ranges. Table 1 shows the mean values and
95 central percentile distributions of red blood cell and reticulocyte parameters provided by the H*3 in 64 healthy subjects. Results of hemoglobin measurement and red blood
cell count showed a lognormal distribution, while all other
parameters, including reticulocyte percentage, absolute number, and indices, were normally distributed. With the exception of hemoglobin and red blood cell count, we did not find
any statistically significant difference between sexes. The
calculated red cell indices MCHC and MCH were somewhat
higher than the corresponding directly measured parameters
CHCM and CH, but the correlation between them was as
good as expected ( r = .768 for MCHC v CHCM; r = .881
for MCH v CH).
Reticulocyte and red blood cell indices. Table 2 shows
the mean values and standard deviations of the red blood cell
and reticulocyte indices generated in the H*3 reticulocyte
method. It also shows the mean percentage differences and
ratios between the value of red blood cell MCV, CHCM,
and CH and the corresponding reticulocyte indices MCVr,
CHCMr, and CHr, both in healthy subjects and in patients
with microcytic and macrocytic anemia. The MCVr of reticulocytes is consistently higher than red blood cell MCV in
the four groups we have studied, with a mean difference of
about 24% for normocytic and macrocytic subjects and about
34% for patients with microcytic anemia. The frequency
histograms of MCVr and MCV in healthy subjects show a
Table 2. Average Values (standard deviation), Percentage
Differences, and Ratiosof Red Blood Celland Reticulocyte Indices
Obtained With the H*3 Reticulocyte Method in Normal and
Abnormal Subjects
Healthy
Subjects
Index
(n = 64)
Iron
Deficiency
(n = 58)
MCV (fL)
89.9 14.03)
74.3 (4.89)
MCVr (fL)
111.7 (6.77) 100.1 (8.78)
MCVr - MCV 1%)
24.3 (5.81)
34.9 (8.31)
MCVr:MCV ratio
1.24 (0.06)
1.35 (0.08)
CHCM (g/dL)
31.6 (1.021 27.3 (1.48)
CHCMr (gldL)
26.3 (1.50)
20.4 (2.09)
CHCMr - CHCM (%) -16.7 (3.80) -25.4 (4.89)
CHCMr:CHCM ratio
0.83 10.04)
0.75 (0.05)
CH (pg)
27.7 (1.42)
19.7 12.05)
CHr (pg)
28.6 (1.60)
19.6 (2.46)
CHr - CH (46)
3.04 13.59)
-0.2 (6.94)
CHr:CH ratio
1.03 10.04)
1.00 (0.07)
8-Thalassemia Macrocytosis
Trait (n = 40)
In = 27)
68.6 (3.56)
92.3 (6.87)
34.5 (8.01)
1.35 (0.08)
28.5 (1.28)
111.5 (5.55)
139.0 (11.00)
24.6 (6.25)
1.25 10.06)
31.0 (1.12)
25.7 (1.19)
22.3 (1.74)
-21.9 (3.70) -17.0 (4.11)
0.78 (0.04)
0.83 10.04)
33.7 (2.12)
18.9 (1.64)
34.9 (3.87)
19.9 (2.04)
5.7 14.40)
3.4 (7.70)
1.06 (0.04)
1.03 (0.08)
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D'ONOFRIO ET AL
820
130
85
90
95
l00
105
110
from 0.83 to 0.75 in the four groups. The
hemoglobin content
per cell, on the other hand, wasvery similar in reticulocytes
and matureerythrocytes inall the subjects thatwehave
studied, with small differences ranging from -0.2% in iron
deficiency to+5.7% in@-thalassemia, so that frequency
histograms of CHr and CH were widely overlapped (Fig I),
and the CHr:CH ratio was very close to 1.
These results indicate that,regardless of the final redblood
cell size, reticulocytes are consistently larger than the mature
erythrocytes that originate from them, whereas their hemoglobin concentration is consistently lower: as a consequence
of these changes, hemoglobin content is almost the same.
The constancy of this general pattern of reticulocyte maturation is clearly demonstrated by the comparison of red cell
and reticulocyteindices
usinglinear regression(Fig2),
which shows very high coefficients of correlations (Y range
from .928 to .972) with tight distribution of measurements
around the regression line. The regression lines have slopes
very close to 1 for all three indices, whereas the Y intercept
115
120
125
130
fL
25.
20
15
L
W
n
g 10
z
5
0
2i
.
2'4
26
28
30
32
160. y = 1.047~+ 20.314. r: 0.928
34
1 50.
g/dL
25
140.
. .._
130.
t
C i .
.
70
60
.
,
70
.
,
.
,
,
80130 120
90 110 100
.
.
,
.
,
MCV
304
28.
t
y
=
1.301~
-
14.869, r: 0.929
26.
24.
L41
Fig 1. Frequency distributions of reticulocyte and red blood cell
indices obtained with theH*3 reticulocyte method in 64 healthy
subjects.
L
H
V
I
22.
20.
V
....
18,
normaldistribution andare wellseparated onefromthe
other,with only34
minimaloverlap
32
30(Fig l).28An MCV
26ratio
24
34
32
was calculated as the ratio of MCVr to MCV generated in
theH*3reticulocytemethod;
this simple index describes
and quantifies the size difference between mature red blood
cells andreticulocytes. In all subjects, regardless of red blood
cell size, the MCV ratio was higher than 1. The behavior of
H*3 CHCM and CHCMr all
in our groups was almost specular to that of thevolume. In healthysubjects we found a
mean CHCMr of 26.3g/dL,compared witha CHCM of
31.6 g/dL, with a mean negative difference of 16.7%. The
frequency histograms of CHCM and CHCMr (Fig 1) show
normal distribution and no overlap, similarly to those of the
volume, although their position along the X axis is inverted.
Similar differences were found in patients with microcytic
or macrocyticanemia.Themean
values of the ratio of
CHCMr to CHCM were consistently lower than 1 , ranging
14
22
30
101
28
,
,
.
12.5
17.5
1522.5
2027.5
2532.5
3037.5
35
,
26
.
,
.
,
,
,
.
,
.
,
,
,
,
,
.
,
. 1
40
CH
Fig 2. Linear regression of reticulocyte and red blood cell indices
obtained with the H*3 reticulocyte method in 64 healthysubjects.
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RETICULOCYTE AND RED CELLINDICES
has a positive value for MCV:MCVr and a negative value
for CHCM:CHCMr and is close to 0 for CH:CHr.
Inversion of the MCV ratio. During the 8 weeks of our
study, we were able to discover only four cases of abrupt
and transitory inversion of the MCV ratio, that is, of subjects
having reticulocytes smaller than mature erythrocytes. Case
1 was a 51-year-old male patient who was being treated for
megaloblastic anemia developed 7 years after total gastrectomy for peptic ulcer. At the time of clinical diagnosis, his
hemoglobin concentration was 4.5 g
/
&
and his MCV was
140 fL.He was treated with daily intramuscular vitamin B 12
administration for the first week and then once a week. We
did not have the H*3 system during the first treatment period,
but we observed a rapid reticulocyte response (up to 13.4%)
using the fluorescence reticulocyte counter Sysmex-Toa R1000 (Kobe, Japan). When we analyzed his blood with the
H*3 after 17 days of treatment, his hemoglobin concentration
had risen to 8.5 g/dL, and his MCV was lowered to 109.8
f L , while his MCVr was 108.8 fL, so that the MCV ratio
was 0.95. Eleven days later, hemoglobin concentration was
10.2 g/&, MCV was 98.9 fL, and MCVr was 116.7 fL, with
an MCV ratio of 1.18, within the normal range. It is probable
that in this case the inversion of the MCV ratio was consequent to the delivery from bone marrow of new normocytic
reticulocytes, produced in the presence of restored vitamin
B 12 availability. They were smaller than the circulating, still
macrocytic population of mature red blood cells, most of
which had been formed before the vitamin administration.
Case 2 was a 45-year-old male patient with acquired immunohemolytic anemia of the warm antibody type, which
was monitored with the H*3 for 20 days while he was being
treated with intermediate-dose prednisone and not transfused. He had an almost constant hemoglobin concentration
ranging from 8.1 to 9.7 g/dL, whereas the reticulocyte percentage, which had been above 8% for the first 15 days,
showed a progressive decline to less than l%, preceded by
2 days by a gradual rapid decrease of MCVr, from 130 to
91.5 f L , and the MCV was 93.2. The MCV ratio was 0.98
and stayed close to 1 for the 2 successive days, after which
we had to stop the follow-up for the end of the H*3 evaluation. Similarly, the CHr decreased from a normal value of
29.0 pg at the time of reticulocytosis down to 19.8 pg, a
value typically found in iron deficiency,13while red blood
cell CH was still normal (27.8 pp). Concomitantly with the
decrease in reticulocyte percentage, MCVr, and CHr, the
H*3 showed a progressive increase of the hypochromic red
blood cells up to 23.0%, without any increase in the percentage of microcytes. Bone marrow iron stores of the patient
were normal, but his serum iron was 10 pg/dL and his ironbinding capacity was 66 pg/dL, with a transferrin saturation
of 15%. Thus, in this case the decrease in reticulocyte number and size was probably related to the acute onset of severe
functional iron deficiency, similar to that recently reported
in patients treated with erythropoietin for anemia of chronic
renal f a i l ~ r e ' ~or
. ' ~autologous blood donation.I6
Case 3 was a 22-year-old female patient in complete remission from Phl-positive acute lymphoblastic leukemia
who underwent allogeneic bone marrowtransplantation from
MCVr
MCV
ANC
120
-
100
-
(K) I
1.0-
0.81
80 1,
0.6-
60-
0.4-
40-
0.2:
20 :
0-
0-
-5
0
5
10
15
20
Days
Fig 3. Follow-up of a case of allogeneic bonemarrow transplantation:changes of H*3 mean corpuscolarvolume of reticulocytes
(MCVrl and red blood cells (MCV), R-1000-determined hyperfluorescent young reticulocytes (HFR), and absolute
neutrophilcount (ANCI.
PRBC, packed red blood cells.
her ABO-incompatible sister. Conditioning treatment with
busulphan and cyclophosphamide was started 7 days before
infusion of donor bone marrow cells (3 X lO*/kg). Figure 3
shows the observed changes in absolute neutrophil count
(ANC), hyperfluorescent reticulocytes (HFR) measured with
the R-1000, and H*3-measured MCV and MCVr. From the
time of conditioning treatment, the MCV showed a slight
progressive decrease, whereas the MCVr rapidly decreased
from 120 fL to 68 f
L during the period of aplasia, with
inversion of the MCV ratio from the day of infusion until
day +7. At this time an abrupt and transitory increase of
MCVr occurred after a transfusion of packed red bloodcells,
then a second, sustained increase in MCVr started on day
+12, heralding bone marrow engraftment, which was confirmed 2 days later by the increase in HFR above 4%69 and
10 days later by recovery of ANC above 0.5 X 1 0 9 L
Case 4 was a57-year-old male patient with non-Hodgkin's
lymphoma, immunoblastic subtype, in first complete remission. After conditioning treatment with busulphan and cyclophosphamide, he underwent transplantation of autologous
peripheral blood stem cell collected by leukapheresis (6.7 X
108/kg). During the follow-up, his MCV did not show evident changes, whereas his MCVr decreased after chemotherapy from 120 fL to 92 fL, with inversion of the MCV ratio
on day +4. MCVr increased again on day +9, reaching a
value of 148 fL on day +13. Bone marrow recovery was
confirmed by HFR above 4% on day +9 and ANC above
0.5 X 109Lon day +14.
DISCUSSION
We have determined with the H*3 hematologic analyzer
the mean values and distribution ranges of reticulocyte and
red blood cell indices in healthy subjects and in patients with
abnormal red blood cells. In healthy subjects we have found
that, both in menand in women, reticulocytes are on average
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
822
24.3%largerthan
mature erythrocytes,theirhemoglobin
contentis on average 16.7% lower, and theirhemoglobin
content is almost the same, witha mean difference of 3.0%.
These results confirm the classical concept
that maturation
of the reticulocyte is associateswith loss of size and increase
in density,I7but contrastwith previous reports of hemoglobin
production'Ror loss" during reticulocytematuration. Our
dataare very similartothose
obtained by Clarkson and
Moore'" usingaplanimetricmethod
on microphotographs
of wet preparations stained with brilliant cresyl blue: they
calculated, in fact, a mean reticulocyte volume of 106 fL in
17 normal subjects, compared with our MCVr of 1 1 1.7 fL,
and a difference between reticulocyteand redblood cell
volume of +2O%. Colella et ai,'9 using a laser scatter flowcytometer, obtained in 33 normal blood samples an MCVr
15% higher than that of mature red blood cells, whereas the
CHCMr was 5% lower and the CHr 9% higher.
The study of patients with iron deficiency, &thalassemia
trait, and macrocytic anemia of various origin showed that
changes associated with reticulocyte maturation in abnormal
erythropoiesis are similar tothose of normalsubjects. In
patients with macrocytosis, the mean MCVr was 139 fL, a
value identical to that found by Clarkson and Moore.''In
contrast with the wide range of red cell size in our groups
of patients, we found thata strikingly tight relationship does
exist between reticulocyte indices and the correspondingred
cell indices (Fig 2 ) . We chose the ratio of MCVr to MCV
(MCV ratio) as the simplest index that quantifies the
size
differences between reticulocytes and mature erythrocytes.
1.0 was auniversalfindingin
our
An MCVratioabove
experience: in men and women,normal and abnormal erythropoiesis, and normocytic, microcytic, and macrocytic red
blood cells.Its meanvalues rangedfrom 1.24 in healthy
subjects and in patients with macrocytosis to 1.35 in microcytic patients with untreated iron deficiency or heterozygous
&thalassemia.
We observed rare exceptions to this general rule of the
constancy of the MCV ratio, such as in a patient receiving
vitamin B 12 treatment for megaloblastic anemia, whosenew
normal-sized reticulocytes were smaller than the preexisting
macrocyticerythrocytes, and ina case of functional iron
deficiency developed during the clinical course of autoimmune hemolytic anemia. In this patient, a relative insufficiency of iron supply to erythroblasts caused by prolonged
erythropoietic stimulation was shown by low transferrin saturation in the presence of normal iron s t o r e ~and
' ~ increased
percentage of hypochromic erythrocyte^.'^.'^ It caused a dramatic decrease in both reticulocyte percentage and MCVr,
with inversion ofthe MCVratio, as well as a sudden decrease
of CHr, which hasrecently been reported as an earlyindicator of iron deficiency." During the follow-up of two bone
marrow transplantation patients, the succession of suppression and regeneration of erythropoiesis was associated with
important changes in MCVr and MCV ratio. The decrease
in reticulocyte percentage that occurred after conditioning
chemotherapy, in fact, was associated with progressive decrease of MCVr and transitory inversion of the MCV ratio.
In both caseserythroid regeneration was heralded by an
D'ONOFRIO ET AL
abrupt increase of MCVr, which in a fewdays reached values
above normal. The first transitory increase in MCVr of our
first bone marrow transplant patient immediately after transfusion was probably caused by the presence of normal-sized
reticulocytes in refrigerated blood (unpublished observation,
May1993);alternatively,
it could represent the first sign
of erythroid regeneration and its suppression caused by the
transfusion.
Our results confirm that, in steady conditions, intravascular reticulocyte maturation is characterized by a decrease in
size from reticulocytes to mature erythrocytes, expressed by
an MCV ratio higherthan 1.0. However, whenasudden
change in erythropoiesis takes place and leads to producthe
tion of reticulocytes of different size, a change of MCVr
occurs much earlier than the MCV change, due to the long
red blood cell survival in peripheral blood. A rapid MCVr
Increase, for instance, caused by the production of macroreticulocytes
with
characterizes
RNA
high
the
brisk erythropoietic stimulation that occurs after chemotherapy, bone marrowtransplantation, or erythropoietintreatment. On the other hand, the production of microreticulocytes,leading to rapid MCVrdecrease withinversion of
the MCV ratio, rapidly follows the development of true or
functionalirondeficiency, as it was shown during experimental induction of iron deficiency in humans by repeated
phlebotomies.'" On these bases, simultaneous measurement
of reticulocyte and redblood cell indices,whichis
now
available on automated routine hematology systems, seems
to be a much more sensitive indicator of sudden erythropoieticchanges than conventionalMCV, thusrepresentinga
powerful tool for the real-time monitoring of erythropoiesis.
REFERENCES
1 . Van Hove L, Goossens W, VanDuppen V, Venvilghen R:
Reticulocyte count using thiazole orange. A flow cytometric method.
Clin LabHaematol 12:287, 1990
2. Tichelli A, Gratwohl A, Driessen A, Mathys S, Pfefferkorn E,
Regenass A, Schumacher P, Stebler C, Wernli M, Nissen C, Speck
B: Evaluation of the SysmexR-1000. Anautomated reticulocyte
analyzer. Am J Clin Pathol 93:70, 1990
3. TankeHJ,vanVianen
PH, Emiliani FMF, Neuteboom I, de
Vogel N. Tates AD, de Bruijn EA, van Oosterom AT: Changes in
erythropoiesis due to radiation or chemotherapy as studied by flow
cytometric determination of peripheral blood reticulocytes. Histochemistry 84544, 1986
4 . Kuse R: The appearance of reticulocytes with medium or high
RNA content is a sensitive indicator of beginning granulocyte recovery after aplasiogenic cytostatic drug therapy in patients with AML.
AnnHematol 66:213, 1993
5. Davis BH,Bigelow N. Ball ED, Mills E, Comwell CG 111:
Utility of flow cytometric reticulocyte quantification as a predictor
of engraftmentin autologous bone marrowtransplantation.Am J
Hematol 32:81, 1989
6. Davies SV, Cavil1 I, Beltley N, Fegan CD, Poynton CH, Whittaker JA: Evaluation of erythropoiesis after bone marrowtransplantation: Quantitative reticulocyte counting. Br J Haematol 81: 12,
1992
7. Kanold J, Bezou MJ, Coulet M, Quainon F, Malpuech G,
Travade Ph, Demkocq F: Evaluation of erythropoietichematopoietic
reconstitution after BMT by highly fluorescent reticulocyte counts
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
RETICULOCYTE AND REDCELL INDICES
compares favorably with traditional peripheral blood cell counting.
Bone Marrow Transplant 11:313, 1993
8. Tatsumi N, Kojima K, Tsuda I, Yamagami S, Itoh Y,Tanaka
H: Reticulocyte count used to assess recombinant human erythropoietin sensitivity in hemodialysis patients. Contrib Nephrol82:41, 1990
9. Ross DW, Bentley SA: Evaluation of an automated hematology
system (Technicon H-l). Arch Pathol Lab Med 110803, 1986
10. Tycko DH, Metz MG, Epstein EA, Grinbaum A: Flow cytometric light-scattering measurement of red cell volume and hemoglobin concentration. J Appl Optics 24:1355, 1985
11. Mohandas N. Kim YR, Tycko DH, Orlik J, Wyatt J, Groner
W: Accurate and independent measurement of volume and hemoglobin concentration of individual red cells by laser light scattering.
Blood 68:506, 1986
12. Dacie JV, Lewis SM: Practical Hematology (7th ed). Edinburgh, Scotland, Churchill Livingston, 1991
13. Brugnara C, Laufer MR, Friedman AJ, Bridges K, Platt 0:
Reticulocyte hemoglobin content (CHr): Early indicator of iron deficiency and response to therapy. Blood 83:3100, 1994 (letter)
14. Macdougall IC, Hutton RD, Cavill I, Coles GA, Williams
823
JD: Poor response to treatment of renal anaemia with erythropoietin
corrected by iron given intravenously. Br Med J 299:157,
1989
15. Macdougall IC, Cavill I, Hulme B, Bain B, McGregor E,
McKay P, Sanders E, Coles GA, Williams JD: Detection of functional iron deficiency during erythropoietin treatment: A new approach. Br Med J 304:225, 1992
16. Brugnara C, Chambers LA, Malynn E, Goldberg MA,
Kruskall MS: Red blood cell regeneration induced by subcutaneous
recombinant erythropoietin: Iron-deficient erythropoiesis in iron replete subjects. Blood 81:956, 1993
17. Lowenstein LM: The mammalian reticulocyte. Int Rev Cytol
8:135, 1959
18. Papayannopoulou T, Finch C: Radioiron measurements of red
cell maturation. Blood Cells 1535, 1975
19. Colella GM, Fan S , Mohandas N: Changes in cell volume,
hemoglobin content, and hemoglobin concentration during maturation of normal human reticulocytes. Blood 78:407a, 1991 (abstr)
20. Clarkson DR, Moore EM: Reticulocyte size in nutritional
anemias. Blood 48:669, 1976
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1995 85: 818-823
Simultaneous measurement of reticulocyte and red blood cell indices
in healthy subjects and patients with microcytic and macrocytic
anemia
G d'Onofrio, R Chirillo, G Zini, G Caenaro, M Tommasi and G Micciulli
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