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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3986-3991
By
From the Departments of Cardiothoracic Surgery and of Hematology,
Umeå University Hospital, Umeå, Sweden.
Hydroxyurea (HU) is used in suppressing the bone marrow and
producing fetal-like red blood cells (RBCs). These RBCs are large in
size and may theoretically disturb the microcirculation. In five
patients with myeloproliferative disorders (MPD), the RBC geometry and
deformability were analyzed before and after 6 to 8 months of HU
treatment. In untreated MPD, the RBC geometry and filterability was
normal. After HU, the RBC membrane area increased 24% and the cell
volume increased 39% (P < .005). This change resulted in a
12% increase in the minimum cylindrical diameter (MCD). From a static
bending model of initial deformation, the RBC diametrical cross-section
had a significantly increased section modulus. However, this increase
in profile stiffness was compensated for by its larger projected cell
area and, thus, pressure load on the RBC corpuscle. The resulting
resistance to initial deformation therefore remained unchanged after
HU. These findings were tested experimentally; with 3-µm filter
membranes, HU treatment caused a significant increase in flow
resistance (P < .02), in accordance with MCD. However, with
5-µm pores, no difference was seen, again in consonance with the
theoretical findings of initial deformation. Because most capillaries
are larger than 3 µm, we suggest that HU is acceptable from a
perspective of cellular microrheology.
THE CIRCULATING red blood cells (RBCs)
reflect the bone marrow, both cell geometrically and functionally, such
as with growth hormone stimulation1 and with neonatal
RBCs.2 Diseases of the bone marrow may also cause abnormal
geometry of the circulating RBCs, an example being sickle cell anemia,
in which the hemoglobin (Hb) is abnormal with a change in a terminal
amino acid and structural defect of the protein configuration and
solubility. Myeloproliferative disorders (MPD) are another group of
bone marrow diseases, including polycythemia vera (PV), essential
thrombocythemia (ET), and myelofibrosis (MF), with various degrees of
changes in the myelopoiesis and circulating number of erythrocytes and
platelets.3 When there is a need for myelosuppression,
hydroxyurea (HU), a DNA synthesis inhibitor, has been increasingly
used. In both sickle cell anemia4,5 and MPD,6,7
HU has been found useful to modify the gene expression to produce fetal
Hb (HbF).6 HU reduces the production of sickel Hb and
retards the myeloproliferation, respectively. However, a side effect of
HU treatment is megaloblastic change of the RBCs.8
Although the megaloblastic effects of HU has been known for many
years,8 the consequences of this RBC change on the
microcirculation is still under debate. Most of this research is from
sickle cell anemia,9 during which the HU changes per se are
affected and partly masked by the sickle cell disease. Not much is
known about the blood cell rheology in MPD and the possible effects of
HU treatment on relatively normal RBCs. We addressed this problem by
analyzing the geometry and filterability of RBCs on previously untreated patients with MPD before and after 6 to 8 months of HU
treatment. The RBC geometry was analyzed using cell curvature profile
generation with the possibility of calculating and estimating the
theoretical consequences of the HU-induced RBC change.
These theoretical interpretations were also tested experimentally by RBC filterability measurements using both 5-µm and 3-µm Nucleopore membranes. The 5-µm pores measure the effects on initial bending deformation, whereas the 3-µm pores are sensitive to maximum
deformation close to the critical minimum cylindrical diameter (MCD)
limit.
Patients and HU treatment.
Five patients with newly diagnosed MPD were selected. Four of the
patients had ET, whereas one was subgrouped to have MF with thrombocytosis. All five patients were about to begin receiving chemotherapy treatment because of thrombocytosis, symptoms,
and/or thrombotic/hemorrhagic complications. The inclusion in
the study had no influence on the clinical routine and treatment. The
patients were analyzed immediately before HU and were then observed 6 to 8 months later during ongoing HU treatment. HU was administered daily, using an oral dose of 0.5 to 1 g/d. Normal controls were healthy
age-matched hospital personal, who were studied in parallel. These
controls were not the same individuals before HU and at the follow-up,
thus producing 5 + 5 control observations.
Blood sampling and media.
Blood samples were from venopuncture using standard heparinized 10-mL
test tubes. The blood was centrifuged at 1,500g for 10 minutes
with buffy coat removal. This was repeated three times to wash the
RBCs, each time by topping up the suspension to 10 mL. Using this
standard routine, most of the plasma, leukocytes, and platelets were
removed. The final resuspension hematocrit for filtration analyses was
set to 5%. The resuspending medium consisted of a Krebs-Ringer
solution (135 mmol/L NaCl, 4.7 mmol/L KCl, 1.2 mmol/L
KH2PO4, 1.2 mmol/L MgSO4) buffered
with 20 mmol/L HEPES and supplemented with 2.56 mmol/L
CaCl2, 5 mmol/L D-glucose, and 0.2 g/L human serum albumin
(Fraction V Albumin; Sigma Chemical Co, St Louis, MO). The pH was set
to 7.40 and the final medium had an osmolality of 299 ± 1 mOsm.
Medium osmolality was measured by freezing-point depression (Osmette
Osmometer; Precision Systems, Inc, Natick, MA). The medium was also
prefiltered through a 0.45-µm Millipore filter (Millipore Corp,
Molsheim, France).
RBC geometry.
About 5 µL of the whole blood was diluted in 1 mL of RBC suspension
medium and incubated for 30 minutes before analysis. A chamber for RBC
geometry measurement was made from coverslips on a microscope slide to
make a rectangular compartment (18 × 10 × 0.17 mm). A drop of suspended RBCs was introduced into the slit entrance of
the chamber, and the openings were sealed with immersion oil. RBCs were
allowed to sediment with the chamber held upside down. After 2 minutes,
the chamber was turned right side up and mounted in the microscope. A
large proportion of the previously settled RBCs were thus hanging
vertically from the upper glass surface. On these vertical RBCs, the
diametrical cross-sections were focused on using bright field
illumination (Carl Zeiss ×100/1.25 oil objective lens; Carl
Zeiss, Oberkochen, Germany). A digitizing table with light-spot cursor
(MOP-Videoplan; Kontron Bildanalyse GmbH, Munich, Germany) was
optically superimposed on the microscopic image. The RBC diameter,
maximum thoroidal thickness, and central thickness were measured. A
thin diffraction band surrounded the RBC cross-sections due to
differences in refractive indices between the RBC and the suspending
medium to which the uniform shell rule was applied during
measurements.10 The method used here is consistent with
that described in previous publications, to which readers are referred
for further details.1,2,11,12 RBCs (50 cells per sample)
were focused on one by one and measured at their diametrical cross-section, a procedure that required about 15 minutes of microscopy per blood sample.
RBC profile calculations.
The above measurements were loaded into equation 1 expressing the RBC
profile curvature, originally derived from Evans and Fung13
and later remodelled11,12:
RBC folding resistance.
It is possible to mathematically calculate the mechanical resistance to
initial bending deformation of the RBC corpuscle in terms of a section
modulus value. The section modulus only considers the RBC shape and
size and not the membrane mechanical properties as such, eg, elastic
shear, dilation, or surface bending moduli or their interrelations. The
section modulus is calculated for the RBC corpuscle, consisting of a
0.01-µm-thick membrane shell model12 with linear elastic
behavior.15 The unit becomes µm3.
The static bending model.
The corpuscle section modulus value is evaluated using a static bending
model in which the RBC is stressed due to a pressure difference between
the two sides of the cell (here set to a fictitious pressure of 1 kPa).
The pressure generates a bending moment and hence a membrane tension,
both having a maximum at the diametrical cross-section of the
RBC12; the higher the tension, the more deformable the RBC.
The tension is mathematically given by the quotient of bending
moment-to-section modulus, with the unit
nanoNewton/µm2 (nN/µm2). This method of
theoretical analysis is useful to evaluate the influence of RBC shape
and size only on corpuscle deformability.11,12
RBC filterability.
The capillary function of RBC was studied by a filterability model on
resuspended cells. This was studied by means of a specially developed
filtration system.1,2,16 This system uses a digital balance
(Mettler PM480; Mettler Toledo AG, Greifensee, Switzerland) in
connection with a computer (IBM PS/2 Model 50; IBM UK Ltd, Greenock,
Scotland) for high precision monitoring of the filtration flow. The
accumulated weight is transmitted by a frequency of 7.5 Hz, with
on-line calculation of flow rate versus time, and is not limited in
volume. This sampling rate gives a sufficient resolution
in time in relation to the obtained flow rate and the resolution of the
balance, corresponding to 1 µL/0.13 seconds. All analyses were made
at room temperature (22°C to 24°C).
Statistics.
Data are presented as the mean values ± SEM. For statistical
differences, both paired and unpaired t-tests were used for
matched analyses with respect to differences induced by HU and for
analysis versus control individuals, respectively. With unpaired
testing, correction was made for unequal variance and different number of observations between groups. The different numbers of observation are indicated in the tables (n). Five levels of significance were tested; P < .051, P < .022,
P < .013, P < .0054, and
P < .0015.
Clinical response.
The patients were diagnosed according to required criteria for MPD. The
Hb concentration varied with the MPD spectrum. One patient had
supranormal Hb and an MPD profile towards PV, but with normal RBC mass
and classified as ET; the Hb was reduced after HU. In another patient,
the Hb was subnormal (MF) and increased during HU treatment. In the
remaining three patients, an isolated ET was observed in which the Hb
remained largely unaffected by HU (Table
1). In all included patients, the platelet counts were increased and
became successfully reduced after HU (P < .05).
Effects of HU on the RBC geometry.
The circulating RBCs of patients with MPD were normal in cell geometry,
with close to identical cross-section profiles to those of matched
normal control individuals (Table 2). After HU treatment, the RBC geometry was increased dramatically (Table 2).
This was seen in both diameter and thoroidal thickness by 10.0% ± 2.0% and 15.1% ± 2.6%, respectively. The central thickness remained unchanged. The resulting cell profile was generated by computer iteration to produce a larger RBC with marked increase in both
membrane area and cell volume by 24.0% ± 4.5% and 39.0% ± 6.3%, respectively. The area-to-volume ratio decreased, whereas the
surface area index remained unchanged.
Effects of HU on calculated RBC deformability.
The patients with MPD had normal RBC geometry and, thus, no difference
in calculated deformability indices (Table 2). However, the HU-induced
change in RBC size produced highly significant alterations in these
calculated RBC deformability indices. These changes were found in terms
of both maximum deformation, represented by MCD, and initial bending
rigidity, as judged from the static bending model. The MCD increased
significantly by 11.7% ± 0.9%; furthermore, the percentage of RBC
having an MCD greater than 3.5 µm increased by 263% ± 39%
(Table 2). This dramatic percentage increase suggests a larger
resistance to pass a narrow capillary and with more capillary blockage.
Effects of HU on the measured RBC filterability.
The RBC filterability in patients with untreated MPD was not
significantly different from those of normal controls. This was seen
using both 3-µm and 5-µm pore filters, although there was a small
tendency for a decreased filterability in MPD patients using the 5-µm
filtration model (interpreted as a small increase in both
RBCiv and RBCcr;
Table 3). After HU treatment, a significant reduction in filterability was recorded with 3-µm filters as the RBCiv increased by 221% ± 63% (P < .02).
There was also a 23% ± 13% increase in the the corresponding
RBCcr, but without becoming significant. No significant
changes were seen using the 5-µm pore size (Table 3).
The oxygen delivery from the blood to the surrounding tissue is
promoted by the narrow dimensions of the capillaries. The RBC are
folded in the capillaries because these are narrower than the cell
diameter, of the order of 4 to 5 µm.18 The RBC therefore relies on its ability to deform (ie, cellular deformability), which in
large part is due to the relative excess of membrane area to cell
volume. RBCs of unfavorable shape and size may impair the blood flow in
the macrocirculation by viscosity increase, but more obviously in the
microcirculation with retarded cell passage through the capillary
network. Impaired RBC deformability is thought to
contribute to the sequestration of senescent cells.19
Submitted August 12, 1997;
accepted January 13, 1998.
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Abstract
Introduction
Abstract
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