Vol. 45 No.

1 SCIENCE IN CHINA (Series C) February 2002

Microrheological characteristics of reticulocyte in vivo

XIE Lide (
), SUN Dagong (), YAO Weijuan ( )

& WEN Zongyao (
)

Hemorheology Center, Department of Biophysics, School of Basic Medical Sciences, Peking University, Beijing
100083, China
Correspondence should be addressed to Wen Zongyao (email: rheol@mail.bjmu.edu.cn)

Received September 29, 2001

Abstract Using the method of phenylhydrazine-induced anemia in rabbits, a mass of new reticu-
locytes, which synchronously grow, were got in vivo. The measurements of deformation index,
orientation index, electrophoresis mobility etc. were performed for more than 72 h in the process of
reticulocytes turning into red blood cells in vivo. There were obvious changes in the micro-
rheological characteristics of reticulocytes in the course of turning into erythrocytes. The present
study is significant in clinic for studying erythrocytes microrheological characteristics when there
are a lot of reticulocytes in blood, and also important in basic theorem for studying reticulocytes
microrheological characteristics. It makes up a deficiency in the study on microrheological charac-
teristics of reticulocytes turning into new RBCs from reticulocytes during reticulocytes life span.

Keywords: phenylhydrazine, reticulocyte, microrheological characteristics, deformation indexes, orientation indexes, new
ektacytometry.

Reticulocytes exist in the course of acidophilic erythroblasts turning into erythrocytes. About
one percent of reticulocytes are in normal blood, and their influence on the blood flow is small.
But the influence should not be neglected when a mass of reticulocytes exist in the blood of some
patients with anemia. So it is significant in clinic to study the microrheological characteristics of
reticulocytes, as well as to find out how the reticulocytes influence the microcirculation.
The changes in ion transport during aging of rabbit RBCs were studied by Brugnara[1] with
the phenylhydrazine-induced anemia model. Waugh et al.[2] also investigated the alterations in
surface/volume of aged RBCs marked with biotin in the same animal model. Many studies focus
on the rheological characteristics of erythrocytes, and the characteristics of reticulocytes, espe-
cially its mutational change during the tens of hours after they are released from the bone marrow
and turn into mature erythrocytes have been rarely studied. Using the method of phenylhydra-
zine-induced erythrocytosis in rabbits, a mass of new reticulocytes were got in vivo. In this paper,
the measurements of deformation index, orientation index, electrophoretic mobility etc. were per-
formed to obtain reticulocytes rheological characteristics and the changing rules in the process of
their turning into red blood cells in vivo.
 No. 1 MICRORHEOLOGICAL CHARACTERISTICS OF RETICULOCYTE IN VIVO 51

1 Materials and methods

1.1 Materials
(i) LBY-BX2 Ektacytometer, LIANG-100 Cell Electrophoresis Meter.
(ii) Five Beijing rabbits (2.53 kg each, about 8 months) were obtained from Experimental
Animal Department of Peking University.
(iii) Phenylhydrazine, Heparin (Sigma Company), PBS (0.13 mmol/L NaCl, 0.2 mmol/L
Na2HPO4, 0.5 mmol/L KH2PO4, pH 7.4, Osmotic pressure: 300 mOsm/kg).
(iv) PVP suspending medium 1 (3% PVP, MW 30000, osmotic pressure 300 mOsm/kg, pH
7.4, viscosity 1.2 mPags), PVP suspending medium 2 (15% PVP, MW 30000, osmotic pressure:
300 mOsm/kg, pH 7.4, viscosity 8 mPags) for ektacutometry.

1.2 Methods
0.1-milliliter phenylhydrazine was injected into the ear vein of five rabbits respectively. On
the second day after injection, 1 mL of fresh heparinized blood was taken from ear vein. Hema-
tocrit of blood samples was determined by centrifugation at 3000hg for 5 min in a micro-
hematocrit tube. The alteration of hematocrit of blood was monitored every day. The percentage of
reticulocytes was determined by the brilliant cresyl blue coloration technique[3]. After the per-
centage of reticulocytes rose to about 35% on the third day, 2.5-mL heparinized blood was taken
from the rabbit ear veins every 6 hours and the following measurements were completed.
(i) 0.5-mL fresh heparinized blood was taken for determination of the percentage of reticu-
locytes and electrophoretic mobility of RBCs and the measurements for ektacytometry.
(ii) The reticulocytes were separated from the other 2.0-mL fresh heparinized blood by den-
sity gradient separation technique.
(iii) Electrophoretic mobility of RBCs and reticulocytes was measured with LIANG-100 Cell
Electrophoresis Meter.
(iv) Deformation index DI (501000 s1) and orientation indexes (50k140 s1) of RBCs
were measured with LBY-BX2 Ektacytometer[4].
(v) Deformation index DI (501000 s1) and orientation indexes (50k140 s1) of reticulo-
cytes were measured with LBY-BX2 Ektacytometer.
From the time when the percentage of reticulocytes reached its maximum on, the heparinized
blood was taken every 6 hours and the above measurements were performed for more than 72 h.
Each blood sample was measured three times before the average value was taken. The data were
statistically treated with SAS software.

2 Results

Fig. 1 shows that reticulocytes in the blood decreased gradually and reached its minimum at
about 72 h. The percentage at different time points between 30 h and 72 h was significantly dif-
 52 SCIENCE IN CHINA (Series C) Vol. 45

ferent from that at the start point (0 h). This means that the reticulocytes turned into mature eryth-
rocytes in about 48 h.

Fig. 1. The percentages of reticulocytes in blood. **, p<0.05.

The average value of electrophoretic mobility of RBCs and reticulocytes was measured with
a LIANG-100 Cell Electrophoresis Meter (fig. 2). In the process of turning into erythrocytes, the
decreased electrophoretic mobility indicates the reduced density of surface charge on the reticylo-
cytes membrane. With the same time span at the early stage of transition, that reticulocytes have a
higher electrophoretic mobility than RBCs in blood shows that the younger cells possess more
surface charge than mature ones.

Fig. 2. Average value of electrophoretic mobility of RBCs and reticulocytes. *, p<0.1; **, p<0.05.

The deformation index DI (1000 s1) of RBCs and that of reticulocytes were measured with
LBY-BX2 Ektacytometer (fig. 3). It can be seen that reticulocytes had lower deformability than
mature erythrocytes. During the about 48 h of transition, the deformability of reticulocytes became
higher. That consisted with the change in the structure of reticulocytes.
 No. 1 MICRORHEOLOGICAL CHARACTERISTICS OF RETICULOCYTE IN VIVO 53

Fig. 3. The deformation index DI (1000 s1) of RBCs and reticulocytes. **, p<0.05.

The integral deformation index IDI (1000 s1) of RBCs and that of reticulocytes measured
with LBY-BX2 Ektacytometer are shown in fig. 4. IDI is the integral of deformation index (DI). It
reflects the deformability of the RBC membrane. The higher IDI indicates the higher deform-
ability of RBC membrane. The IDI of reticulocytes increased significantly in the late stage of
transition (p<0.05) but its value was still lower than the average value of RBCs.

Fig. 4. The deformation index IDI (1000 s1) of RBCs and reticulocytes. **, p<0.05.

The orientation indexes (140 s1) of RBCs and that of reticulocytes measured with LBY-BX2
Ektacytometer are shown in fig. 5. The orientation index reflects the alignment of cell in the flow
field and indicates the morphological regularity and integrity of the cells. The increased value of
orientation index at the end of the transition shows that shape of the reticulocytes became close to
biconcave.
 54 SCIENCE IN CHINA (Series C) Vol. 45

Fig. 5. The orientation indexes (140 s1) of RBCs and reticulocytes. **, p<0.05.

3 Discussion

(i) The reticulocyte count increased gradually from 1 to 3 days after injection of phenylhy-
drazine and reached its maximum on the third day (about 35%), because the phenylhydrazine has
destructive effect on RBC membrane and results in fragility of the membrane and RBCs hemoly-
sis due to action of phenylhydrazine with RBC membrane protein[4]. The degree of RBCs hemoly-
sis depends on the dose of phenylhydrazine. The higher dose makes it difficult for the rabbits to
survive because of the toxicity of phenylhydrazine. From 0 to 3 days, the hematocrit decreased
rapidly and reached its least value on the third day (about 12%). As the RBCs of hemolysis were
removed from the circulation, they were replaced by reticulocytes produced from the bone mar-
row in response to the anemic stress. The reticulocyte count increased rapidly[5]. In the process of
about 48 hours for the reticulocytes turning into new RBCs continuously and rapidly, it can be
seen in the following discussion that the microrheological characteristics changed obviously. The
phase is about 48 hours for reticulocytes turning into RBCs completely and this result consists
with ref. [6]. The hematocrit increased continuously with the increasing new produced RBCs, and
the reticulocyte count decreased to below 3 % after 72 hours. From then on, the elimination rate of
RBCs keeps in balance with the production rate of RBCs, so the percent of reticulocytes in blood
keeps stable.
(ii) As shown in fig. 2, the electrophoretic rate vs. hours curve for reticulocytes rapidly de-
creased during 072 hours. The cause is that the surface area of reticulocytes is larger than that of
RBCs, so they carry more negative charge than RBCs in the surface and are affected by bigger
force. On the other hand, the density of reticulocytes is smaller than RBCs[7], so the whole weight
is lighter than RBCs. That results in faster electrophoretic mobility. With the time going by, the
reticulocytes turned into mature RBCs continuously. In the course of turning, the surface area of
reticulocytes becomes smaller, and the surface charge of reticulocytes also decreases. On the other
 No. 1 MICRORHEOLOGICAL CHARACTERISTICS OF RETICULOCYTE IN VIVO 55

hand, the density of reticulocytes is getting larger with time, so the electrophoretic rate decreased
obviously. About 42 hours later, the electrophoretic rate of reticulocytes was significantly different
from that at the 0th hour (p<0.05). A lot of reticulocytes existed in the blood, so the electropho-
retic rate of RBCs was changing similar to the reticulocytes but their value was smaller than that
of reticulocytes. As the proportion of reticulocytes in blood became small, the electrophoretic rate
decreased to the normal value of new RBCs. But there still was more surface charge on new RBCs
as can be seen in the model of antibody-induced spherical anemia put forward in ref. [8].
(iii) As shown in fig. 3, deformation indexes DI[9] of reticulocytes increased quickly during 0
72 hours. That is to say the deformability of reticulocytes is getting higher in the course of
turning into new RBCs. The cause is that lots of chromatin are still in its cytoplasm so that de-
formability of reticulocytes was lower than of new RBCs[10]. Therefore, obviously there is differ-
ence between the beginning and the end of the experiments (p<0.05). With the time going by, the
majority of chromatin was excluded from reticulocytes continuously, and the cytoplasm in them
increased. So the deformability of the reticulocytes became higher than before, and the deforma-
tion indexes DI increased[11]. About 42 hours later, DI was obviously different from that of
reticulocytes at the 0th hour in their life space (p<0.05). Because a lot of reticulocytes
synchronously grew in the blood, the change of deformation indexes DI actually reflected that of
deformability for reticulocytes in their life space. As the time went by, the proportion of
reticulocytes in blood was gradually decreased and then the deformation indexes DI also gradually
increased to the normal value of new RBCs with time in about 48 hours. The deformation index of
the new RBCs was greater than that of RBCs in later half time of life, as can be seen in the model
of antibody-induced spherical anemia put forward in ref. [12]. For the same reason, the
deformation indexes IDI reflecting the membrane structure were changing similar to DI, as shown
in fig. 4.
(iv) As shown in fig. 5, the orientation indexes (DI)or[13] (cf. ref. [14]) of reticulocytes in-
creased quickly during 072 hours. This means that the orientational ability of reticulocytes was
getting higher in the course of turning into new RBCs. The orientation indexes (DI)or are corre-
lated with the deformation indexes DI and the shape of cells[9]. The larger the deformation indexes
DI, the larger the orientation indexes (DI)or. The better the shape of cells, the larger the orientation
indexes (DI)or[11]. In the course of turning into new red cells, the new reticulocytes were getting
thinner and thinner and their shape was turning into biconcave disk continuously. So the ratio of
surface area to volume increased and the orientation index (DI)or was getting larger. At the same
time, with the time going by, chromatin was excluded from reticulocytes continuously, and the
cytoplasm increased, so the orientation ability of reticulocytes was getting higher than before, and
the orientation indexes (DI)or increased[9]. About 42 hours later, the orientation indexes were ob-
viously different from that of the 0th hour (p<0.05). A mass of reticulocytes in the blood at the 0th
hour made the orientation indexes (DI)or of RBCs keep at a similar changing trend to those of re-
ticulocytes after the 0th hour, but the value for the latter was bigger than reticulocytes at the 0th
 56 SCIENCE IN CHINA (Series C) Vol. 45

hour. With the time going by, the proportion of reticulocytes in blood was getting smaller and the
orientation indexes (DI)or increased to the value for new RBCs.
From the above discussion, it can be seen that the quick change in microrheological charac-
teristics of reticulocytes is one of the two phases of the change in the whole life. There are two
quick changes in microrheological characteristics of RBCs in the whole life. (i) The rebirth salta-
tion changes for microrheological characteristics of reticulocytes happened in the process of turn-
ing into new RBCs from reticulocytes within 48 hours. The density of surface charge, ability of
deformation and orientation of reticulocytes increase quickly, and reach the best value when they
become new RBCs till the former half time of life. (ii) It is known from previous study[8, 9] that the
microrheological characteristics of RBCs have similar changes to reticulocytes in the latter half
time of life, but this is a disintegration of RBCs in their life space. There is obvious difference
between the former and the latter of the turning from reticulocytes into RBCs. The discovery of
this regulation is significant in understanding microrheological characteristics of RBCs, aging of
RBCs and diagnosis of rheology index in clinical practice.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No.
39770203) and Doctoral Foundation of China (Grant No. 9729).

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