Volumetric Erythrocyte Macrocytosis Induced by Hydroxyurea
EDWARD R. BURNS, M.D., L. JUDEN REED, M.D., AND BARRY WENZ, M.D., F.A.C.P.
An atypical form of macrocytosis termed volumetric macrocytosis
is described. In contrast to the macrocyte associated with megaloblastic anemia and the pseudomacrocyte caused by viscoelastic
defects, the volumetric macrocyte is characterized by an increased
mean corpuscular volume and a normal cell diameter. The volumetric macrocyte proves to be thicker than the normocytic red
blood cell. This large erythrocyte is overhydrated and contains
an increased quantity of hemoglobin. The cell has many characteristics in common with the red blood cells of neonates. Volumetric macrocytosis accompanies sustained hydroxyurea therapy and may represent a drug-induced dyserythropoiesis. (Key
words: Macrocytosis; Hydroxyurea; Ektacytometer; MCV;
NMR) Am J Clin Pathol 1986; 85: 337-341
LARGE RED BLOOD CELLS (RBCs) are traditionally
classified as either true macrocytes or pseudomacrocytes. 1316 The true macrocyte has an increased cell diameter, mean corpuscular volume (MCV), and mean
corpuscular hemoglobin (MCH).22 True macrocytes are
characteristically found in patients with megaloblastic
anemia, brisk reticulocytosis, and as a physiologic variant
in the neonate. The average reticulocyte is 20% larger than
the mature RBC and can effectively elevate the MCV of
a mixed-cell cohort." The MCV of a neonate's red blood
cells is greater than 100 fL for the first two months of
life.15-18
The pseudomacrocyte has an increased diameter, but
unlike the true macrocyte, this RBC only appears to be
large; its MCV and MCH are normal. This apparent paradox is caused by a membrane abnormality that allows
the cell to expand along the plane of rigid surfaces such
as a microscope slide. While the diameter of the pseudomacrocyte appears large to the microscopist, its volume
remains constant. Pseudomacrocytes are found in splenectomized patients and in those with liver disease.
The current study describes a third type of macrocyte,
the "volumetric macrocyte." When examined microscopically, this cell appears to be normocytic; however, it
is characterized by increased MCV and MCH. This atypical form of macrocytosis is associated with sustained hydroxyurea therapy.
Received April 16, 1985; received revised manuscript and accepted
for publication August 5, 1985.
Supported by a grant from the New York Community Trust.
Address reprint requests to Dr. Burns: Bronx Municipal Hospital
Center, Room 6N29, Pelham Parkway and Eastchester Road, Bronx,
New York 10461.
Department of Laboratory Medicine and Medicine,
Albert Einstein College of Medicine, Bronx, New York
Materials and Methods
Routine Studies
Complete blood counts (CBC) including red blood cell
indices and red blood cell distribution widths (RDW) were
performed by electronic particle sizing using the Coulter
S + IV® blood cell analyzer (Coulter Electronics, Hialeah,
FL). Aberrant findings were confirmed by repeat analyses
using both the laser based ELT-8® (Ortho Diagnostics,
Raritan, NJ) and the H6000® systems (Technicon Instruments Corporation, Tarrytown, NY). The latter instruments measure particle size as a function of light scatter.
Fetal hemoglobin was quantified by the alkali denaturation technic.20 RBC osmotic fragility3 and autohemolysis
tests' 9 were performed according to standard procedures.
Special Studies
Red Blood Cell Aggregation. RBCs obtained from patients and control subjects were washed three times in 50
to 60 volumes of physiologic saline and resuspended to a
3% volume in either Hanks Balanced Salt Solution supplemented with 0.5% albumin or in autologous plasma.
Aliquots (0.4 mL) of these cell suspensions were added
to siliconized glass cuvettes, stirred at 800 rpm in a PAP4® multichannel platelet aggregometer (Bio-Data, Hatboro, PA), and monitored for spontaneous aggregation
over 15-minute intervals.
Red Blood Cell Diameter Measurements. Mean RBC
diameters were determined for RBCs in wet- and drymount preparations. Phase microscopy was used to measure the diameters of RBCs suspended in autologous
plasma, compatible normal plasma, or commercial cell
counting diluent (Isoton III®, Curtis-Matheson, Houston,
TX). RBC to diluent suspensions varied from 1:10 to
1:1,000, (V:V). The latter experiments were performed to
include the possibility that the abnormal RBC indices were
an in-vitro phenomenon induced by an osmotic effect of
the cell-counting diluent solution. The mean RBC diameters were calculated from the measurement of 500
cells obtained with a standard eyepiece micrometer.
337
BURNS, REED, AND WENZ
338
A.J.C.P. • March 1986
Table 1. Erythroocyte Data of Patients with Hydroxyurea-induced Macrocytosis
Patient
Diagnosis
MCV
MCH
MCHC
RDW
HbF
1
2
3
4
5
6
Psoriasis
CLL
CML
CML
CML
SC disease
130
108
114
123
122
123
42.9
35.4
37.5
39.3
39.6
39.1
33
33
33
32
32
32
15.3
22.5
15.7
14.7
15.9
18.9
4.32
1.97
2.11
4.32
2.65
20.0
120
±8
39
3
33
1
17.2
3
2.6
1.6
90
±5
31
2
35
2
15
2
1
0.5
Mean ± SD
Lab Mean ± SD
See text for abbreviations.
The mean cell diameters of fixed RBCs were obtained
from stained peripheral blood smears using the Hematrak
590® analyzer (Geometric Data, Valley Forge, PA). The
instrument was programmed to measure the diameter of
200 cells and construct a Price-Jones histogram from
which it derived the mean cell diameter.
Red Blood Cell Deformability. RBC deformability
measurements were obtained with the Ektacytometer®
(Technicon Instruments). This instrument is a laser defraction viscometer that measures the elongation of RBCs
in suspension at varying levels of shear.9 The elongation
index (EI), a parameter measured by the Ektacytometer®,
is an indirect measurement of viscosity/deformability. The
Ektacytometer® also can be used to construct an "osmograph" that correlates the EI, at a constant level of
shear, as a function of the suspending medium's osmolality. Specific features of the osmograph have been shown
to relate to the cells' deformability characteristics, state
of hydration, and osmotic fragility.2 Measurements of the
O min , defined as the osmolality at which the minimum
EI occurs, were made. The Omin value corresponds to the
50% hemolysis point in the traditional osmotic fragility
curve. The O', defined as the osmolality at which EI is
equal to one-half its total variation, indicates the cells'
relative level of hydration. Measurements of these parameters were performed over a range of 100 to 400 mOsm.
Red Blood Cell Viscosity. RBC viscosity was measured
directly using a Wells-Brookfield Model LTV® microconeplate viscometer (Brookfield Engineering Laboratories,
Brookfield, MA). 10 Measurements were obtained in the
same diluents described previously at 30% RBC concentrations.
Red Blood Cell Density. Density measurements of RBC
cohorts were obtained by centrifuging cells on a continuous, self-forming gradient of colloidial silica coated with
polyvinylpyrrolidone (Percoll, Pharmacia, Inc., Piscataway, NJ) and arabino-galactan polysaccharide (Stractan,
St. Regis Paper Co., West Nyak, NY). The technic is described elsewhere.7
Red Blood Cell Free- Water Lifetime. The lifetime of
free water within the RBC was measured using proton
nuclear magnetic resonance (NMR) relaxation spectroscopy.6 This technic measures the egress of "labeled" molecules from intracellular to extracellular compartments.
The "labeling" is accomplished by orienting protons of
water molecules within the cell in the direction of an applied magnetic field. Measurements are obtained of the
rate at which net magnetization returns to its normal orientation. This measurement, called spin lattice relaxation
time, averages 570 milliseconds (ms). This relaxation time
is then measured relative to the relaxation time of the
extracellular plasma, treated with 2 mM manganese (Mn
II). The latter averages only a few milliseconds.
The plasma-Mn II complex cannot enter the RBC.
Thus, a relaxation time less than 570 ms becomes a function of the rate at which water can pass from the cell into
the plasma. The measured relaxation time is related to
the half-life of free water by a complex series of equations
describing the decay of magnetization in a dual compartmentalized system.6 In turn, the half-life of free water
is related to the permeability of the cell to water.
Scanning Electron Microscopy (SEM). Whole-blood
specimens were collected into 3% buffered gluteraldehyde,
dehydrated in a graded series of ethanol and freon, and
dried by critical point technic. The preparations were
coated with gold and examined with JEOL® lOOcx SEM.
Direct measurements of cell diameter and thickness were
performed at 200 RBC per specimen.
Results
Six patients, all receiving a minimum daily dose of 1
g of hydroxyurea for at least two months were studied.
The patients had a mean MCV of 120 ± 8 fL which ranged
from 108 to 130 fL. Five patients had elevated percentages
of fetal hemoglobin (Table 1). All of the patients' peripheral blood smears were characterized by anisocytosis. No
correlation could be established between the observed
RBC diameters and the measured cell indices. Serum vitamin B12 and folate levels were normal in all subjects.
Hydroxyurea was discontinued in one patient, who was
subsequently monitored over two months. During this
HYDROXYUREA-INDUCED MACROCYTOSIS
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CUBIC MICROMETERS
FIG. 1. RBC size/frequency histogram generated by the Coulter Model
S + IV® cell counter. Note the unimodal distribution of cell sizes on this
hydroxyurea-treated patient with an MCV of 130 fL.
period, her MCV decreased from 130 (Fig. 1) to 96 fL,
and her fetal hemoglobin concentration fell from 4% to
2%. The patient's MCV was consistent with the quotient
of her centrifugally measured hematocrit and her RBC
count obtained with a ZBI® particle counter (Coulter
Electronics). The same MCV values were obtained from
measurements performed on the ELT 8® and H6000®
instruments. Despite these consistently elevated MCV
readings, her RBC diameters were normal when measured
by phase microscopy (patient/control = 8.86/8.73 ji) and
by the Hematrak 590® (7.1/7.8 n) (Fig. 2). The diameter
measurements were not altered significantly by suspending
her cells in the various concentrations of plasma or diluent.
-290
Osmolality (mosm/Kg)
FlG. 3. Osmograph produced by the Technicon Ektacytometer®. The
study patient with an MCV of 130 has a right-shifted curve at high
osmolalities, indicating hyperdeformability.
Aggregation studies on this patient's RBCs proved that
they did not aggregate spontaneously. The cells were
shown to have a negative direct antiglobulin test. Both
phenomena are known to cause spurious elevations of the
MCV as measured by automated particle counters.
Viscoelastic examination of this patient's RBCs dem-
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FIG. 2. Price-Jones cell diameter histogram generated by the Hematrak
590® pattern recognition system. Note the normal mean cell diameter
of 7.1 n obtained from the patient with an MCV of 130.
FlG. 4. Percoll-Stractan density gradients showing the significant population of hypodense cells (at top of gradient) in the hydroxyurea-treated
patient.
340
BURNS, REED, AND WENZ
A.J.C.P. • March 1986
FIG. 5. A (left). Scanning electron micrograph of hydroxyurea-treated patient's RBC thickness (arrow) (X3,000).
FIG. 5. B (right). Scanning electron micrograph of normal RBC chosen to contrast its thickness with that of the cell in 5/1 (X3,000).
onstrated fragility but no autohemolysis. Ektacytometer®
studies of these RBCs revealed that the cells had a normal
EI maximum. However, the patient's osmograph was
shifted to the right (Fig. 3). Both the O min and O' occurred
at higher osmolalities than the control. The direct RBC
viscosity was increased, 4.7 cP at 10 s _1 (control = 3.7).
Gradient separation of the patient's cells revealed a population of hypodense, presumably overhydrated RBCs
(Fig. 4). The approximate density of this cell cohort was
1.070 g/mL, equivalent to a mean corpuscular hemoglobin concentration (MCHC) of 28 g/dL. This MCHC was
lower than the mean value calculated by the Coulter
Counter® (33 g/dL). NMR measrements of the half-life
of free water in the cells was decreased at 8.5 ms (control
= 11.5 ± 1 ms, N = 5). SEM demonstrated that the RBCs
had normal diameters; however, they were abnormally
thick, having a longitudinal value of 3.4 n compared with
a normal control value of 1.2 n (Fig. 5).
Discussion
The findings associated sustained hydroxyurea therapy
with the appearance of a population of true but atypical
macrocytes. These atypical RBCs are characterized by a
normal cell diameter, an increased MCV and MCH, and
an MCHC below the mean of the hospital population.
The cells are overhydrated and hypodense.
Gilbert and colleagues8 reported that the deformability
of RBCs obtained from patients treated with hydroxyurea
is decreased when compared with erythrocytes of similar
size obtained from patients with pernicious anemia. In
contrast, the EI maximum of our patient's RBCs was not
substantially different from that of a normal control's cells.
However, the right-shifted osmograph is consistent with
the cells' independently confirmed state of overhydration
and an increased susceptibility to osmoic lysis. Identical
osmographs have been obtained by Clark and co-workers
for cells that were artificially overhydrated in the presence
of an ionophore.2
The nuclear magnetic resonance (NMR) data demonstrate a decrease in the half-life of intracellular free water
in these cells, which can be explained most readily by an
increase in cell permeability.
Hydroxyurea has been shown to increase levels of fetal
hemoglobin in anemic monkeys. This influence is attributed to a drug-induced alteration of erythrokinetics.14 Al-
Vol. 85 • No. 3
HYDROXYUREA-INDUCED MACROCYTOSIS
ter and Gilbert have also reported elevated fetal hemoglobin levels in 4 of 13 patients on hydroxyurea therapy.1
The effect appears to be dose related; however, no significant correlation was established between the patients' total number of fetal cells and the degree of macrocytosis.
It is plausible to assume that these phenomena are causally
related, but not necessarily dependent functions. The
MCH of fetal cells is no greater than that of the average
RBC.5 It is, therefore, not possible to explain the observed
increase in both cell size and hemoglobin content as a
result of an increase in the fetal cell population. The conclusion is consistent with our patient's RBC histogram
(Fig. 1), which is a right-shifted, unimodal distribution.
A significant subpopulation of F-cells would be expected
to result in either a bimodal distribution pattern or a nonGaussian curve.
Letvin and associates have suggested that S-phase-specific agents, such as hydroxyurea, preferentially arrest the
development of frequently cycling erythroid precursors
and mature erythroid progenitors.14 This selected arrest
in development causes less mature progenitors to undergo
terminal differentiation in an effort to support reticulocyte
production. Accordingly, the peripheral blood is progressively populating with ontogenically younger RBCs. Our
findings are consistent with this hypothesis.
Human neonatal RBCs are large (mean = 105 fL),
contain an increased quantity of hemoglobin (mean = 36
pg), and are somewhat overhydrated (MCHC = 31.7) in
comparison with an adult's RBCs.17 The neonate has a
cell population relatively resistant to osmotic lysis but
also has a subpopulation of RBCs that displays increased
osmotic fragility.21 The properties endow the neonatal
RBC with distinct rheologic and biochemical properties
that are highly analogous to the cells described in our
patient.
Based on these similarities and the known potential of
S-phase-specific drugs to increase the production of fetal
hemoglobin, we propose that the appearance of macrocytic RBCs in patients receiving hydroxyurea represents
selective, drug-induced s-dyserythropoiesis. Hydroxyurea,
as well as other drugs such 5-azacytadine,4 induces a shift
in the source of erythroblasts, which results in the differentiation of immature progenitors. These cells retain features of a younger cell cohort, specifically an increased
MCV and MCH. Although the cell's diameter is not increased, it is markedly thickened. This readily distinguishes it from the normally thin macrocytes found in
pernicious anemia.12 The clinical sequelae of this phenomenon are presently unknown.
Acknowledgments. The authors express their appreciation to Dr.
Maurice Eisenstadt for the NMR studies; Dr. Dhananjaya Kaul for his
341
viscometry; and Mr. Nicholas Kowatch and Mr. William Rooney for
their technical assistance. The thoughtful advice and density gradient
studies of Dr. Mary Fabry also are gratefully acknowledged.
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