Original

Chromatography, Vol.23 No.1 (2002)

Original

Red Blood Cell Lysis at the Single Cell Level
by Using a Mini Electrophoresis Apparatus
Chaiyavat Chaiyasut1, Takao Tsuda1, Udompun Khansuwan2 and Siriwan Ong-chai2
Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso, Showa, Nagoya 466-8555, Japan 1
Department of Biochemistry, Faculty of Medicine, Chiangmai University, Chiangmai, Thailand 2
Received August 8, 2001.

Revised manuscript received November 22, 2001.

Accepted November 26, 2001.

Abstract
Observation of the lysis phenomenon of human erythrocyte at the single cell level under a mini electrophoresis apparatus was studied. The
lysis-inducing reagent (hydrogen peroxide in saline) was introduced to the uptake ferrous red blood cells at the end tip of the rectangular capillary by electroosmotic flow, generating hydroxyl free radical (Fenton reaction). Red blood cell exposed to hydroxyl free radical underwent
progressive lysis, determined by observation through a microscope-CCD camera recording system. The erythrocyte lysis time of several single red blood cells varied between 450 sec and 510 sec. Distinct lysis behavior was delineated by monitoring the apparent light density ofeach single erythrocyte through time. The time dependence apparent light density curve of single red blood cell was used to assess the lysis
phenomenon. It was found that the rate of red blood cell alteration dramatically increased after some initial period leading to eventually complete lysis. The half-lives of the initial step and the second step were estimated as kinetic parameters. The proposed technique stabilized thelysis mechanism by minimizing the depletion effect of the enveloped solvent layer of red blood cell. Moreover, electric field strength provided more homogeneity of the enveloped solvent layer and enabled the erythrocyte to maintain at the observed position.
Keywords: red blood cell, lysis, single cell, electric field, hydroxyl free radical

the lysis phenomenon of the cell was not well observed.

Introduction
Electric field application in separation science has recently

The main purpose of this study was to develop a new method

emerged as a powerful separation technique in applying to single

for the investigation of lysis phenomenon of single cell in detail.

cell studies [1-13]. Electric field was applied as a sample handling

The study was designed in order to observe the lysis phenomenon

technique to manipulate the single cell [2]. Manipulation of a sin-visually from the time the cell is in contact with the lysis induced

gle cell could be performed in a capillary tube under electric fieldreagent, carried through the electroosmotic flow (EOF) into thesuch as the manipulation of rat clonol pleochromocytoma, snail

capillary within the simple homemade electrophoresis apparatus,

neurons or adrenal medullar cells [1, 6-9]. Several researchers have

until it is lysed completely. A new approach to follow the lysis

mentioned the use of capillary electrophoresis as a good tool forprocess was invented. Then we further observed and analyzed thestudying the components in single cell [9-13]. Recently, the neuro-lysis phenomenon into different stages according to the behavior oftransmitters in single neuron cell [9] or the components inside thethe cell lysis. The half-life of each alternation stage was also deter-

single erythrocyte [13] were analyzed and perceived as a progress

mined. In response to these objectives, the red blood cell lysis wasin the single cell studies. Most of the research works on the analy-

selected as a study model. The erythrocyte damage in this experi-

sis of components inside a single cell were performed throughment was induced by the hydroxyl radical formation in the pres-lysing the cell immediately after led into the capillary for further

ence of hydrogen peroxide and ferrous ion (Fenton reaction) [14-separation and determination of the inner components. However,16]. Hydroxyl radical had been shown to cause hemolysis in sev-

Corresponding author: Fax, +81-52-735-5220;
e-mail, takao@ach.nitech.ac.jp―３３―

Chromatography, Vol.23 No.1 (2002)

eral studies [14-16], but the behavior of lysis had not been well es-

power supply (Kikusui Electronics Corp., Kawasaki, Type PMC

tablished. The experiment was designed to obtain the ferrous-500), a microscope (Shimadzu, Kyoto), a CCD camera (Shimadzu,

tagged red blood cells utilizing the simple in vitro method. Red

Kyoto, Type CCD-X2), a video recorder-CRT monitor (Aiwa,

blood cell lysis was studied as a macroscopic parameter for mem-

Type VX-T14G3) and a video timer (minimum time resolution 10

brane damage and red blood cell deformability which are micro-

ms, ForA, Tokyo, Type VTG-33).

scopic parameters. In the current study, the influence of the hy-

The column used for the observation of red blood cell lysis

droxyl radical generating system on erythrocyte lysis observed

was the rectangular column (0.05 mm × 0.5 mm) purchased fromthrough the microscope-CCD camera was monitored in order toWilmad Glass (Buena, NJ, USA).
Ferrous sulfate, sodium chloride and 30% hydrogen peroxide

obtain further insights into the behavior of the lysis process of thissystem.

were purchased from Wako Pure Chemical Industries (Osaka, Ja-

Experimental

pared by dissolving in 0.9% saline.

pan). One percent agarose gel (Nakarai Chemicals Ltd.) was preApparatus, column and materials
Erythrocyte separation

The schematic diagram of an apparatus for the observation of
red blood cell lysis was shown in Figure 1. The apparatus consisted

Blood was obtained from healthy subject and diluted withof a rectangular capillary column (0.05 mm × 0.5 mm, 2 cm long,

0.9% saline. After centrifugation at 500 xg for 5 min at room tem-

500 nl), two polyethylene reservoirs (approximate volume of each:perature, the supernatant, buffy coat, and upper 15% of the packed

250 µl) on a slide glass (Matsunami, Osaka, Type S-1214), two

red blood cell were removed by aspiration. The remaining erythro-platinum wire electrodes inserted to the two reservoirs, a voltage

cytes were washed three times with 0.9% saline at room temperature. Packed erythrocyte 40 µl was then diluted with 0.9% saline
960 µl and stored in refrigerator until used. Incubation of the red
blood cell suspension with ferrous sulfate (0.1 mM final concentration of incubation suspension) for 5 min was freshly prepared before observing the lysis phenomenon.
Procedure
The mini electrophoresis apparatus as shown in Figure 1 wasused. Red blood cells in precubation mixtures of 0.9% saline andferrous sulfate are led in the short rectangular capillary column bycapillarity. Then, both ends were sealed with 1% agarose gel so as
to prevent the pressurized flow in the capillary generated by thedifference of the level of mediums between the two reservoirs. The
capillary was then placed in the homemade mini electrophoresis
apparatus.
The precubation mixture of 0.9% saline and ferrous sulfate
was filled into the polyethylene reservoir at cathodic end side of
the capillary. Then, the 0.03% hydrogen peroxide in saline wasfilled into anodic reservoir. The concentration used in this experiment was adjusted in order to generate the adequate slow process
for the observation of the lysis behavior. After focusing the microscope (magnification of 1,000 at the Cathode Ray Tube screen, 14
inches size) on red blood cells at the anodic end side of the capillary, electroosmotic flow of hydrogen peroxide in saline was intro-

Figure 1. Schematic diagram of apparatus for observation of erythrocyte lysis. (1) dc power supply (2) slide glass (3) short
rectangular capillary (0.05 mm × 0.5 mm, 2 cm long) (4)small polyethylene reservoirs (volume, 250 µl) (5) platinum wire electrode (6) microscope (7) CCD camera (8)video timer and video recorder with CRT monitor.

duced to the column by applying dc 100 V (50 volt/cm) along thecapillary column. The images of the alteration of red cells werefirst recorded on videotape via the microscope-CCD camera video
recording system. The video timer counting, superimposed on thealteration of the red blood cells, was also recorded. Then the im-―３４―

Chromatography, Vol.23 No.1 (2002)

Chaiyavat Chaiyasut , Takao Tsuda , Udompun Khansuwan and Siriwan Ong-chaiages at small different time progress were captured by using a

the starting time of the lysis reaction among all the observed red

FDMCAP software (Photron Ltd.) and printed with a canon BJF

blood cells was less than 1 sec calculated from the electroosmotic

300 printer. The lysis of red blood cell was, therefore, observed at a

mobility (which is 2.0 × 10-4 cm2 V-1 s-1) of the unidentified neutral

single cell level through time. Lysis time of each single red blood

particles toward the cathode with a microscope. The starting time

cell under free radical generating system was finally estimatedof the lysis reaction of each red blood cell was immediately after

through the direct observation method. From the captured photo-

applying voltage.

graphs of red blood cells, the color intensity of each erythrocyte
within a constant limited area was determined by using the Adobe

Observation of erythrocyte lysis at a single cell level

Photoshop software (Adobe Systems Incorporated). Within the

Observation of the visual cell lysis was performed with a mi-

constant limited area of single red blood cell, the average red color

croscope equipped with a CCD camera. Figure 2 shows visual im-

intensity, green color intensity, and blue color intensity of the se-ages of the treated red blood cells in the rectangular capillary at dif-

lected single red blood cell were 168.39, 44.40 and 8.77, respec-

ferent time. Figure 2 a shows the visual images of red blood cells attively. Then the summation of these three main colors (RGB color

the starting of the electric field application. While Figure 2 b showsmode) became 221.56 unit, defined as the color intensity of single

the change in image of red blood cells after the first half observa-

red cell in this experiment. For each red blood cell, the subtraction

tion period. Then in Figure 2 c, shows the image of several redof the color intensity at each time from the maximum color inten-

blood cells completely lysed.

sity obtained from the whole lysis process gave the values intro-

Red blood cell lysis as determined by monitoring under a micro-

duced by the term “apparent light density of red blood cell”.

scope was achieved within 510 sec. The erythrocyte lysis time forseveral observed single red cells varied from 450 to 510 sec in thisResults and Discussion

experiment as shown in Table 1.

In order to clearly observe each red blood cell, the rectangular

For more distinct lysis phenomenon observation, the light ab-capillary column was selected. The transparency property of thesorption property of red blood cell structure and components wasrectangular capillary is the benefaction for the clearer observation

used and determined as the color intensity of red blood cell. Lightof the particles inside the capillary column [17]. Furthermore, inabsorption property of the hemoglobin, which is the main compo-

comparing to the circular type column, the geometrical dimension

nent (33.5%) of the inner compounds of erythrocyte, was used as aof the rectangular column allowed the easier focussing and obser-

detection method in separation of hemoglobin variants in single hu-

vation on each red blood cell.

man erythrocyte [13]. The red blood cell membrane, which is con-

In capillary, since the absolute value of the electrophoretic-4

mobility of the red blood cell (2-1stituted roughly half (49.2%) by protein and half (43.6%) by lipid,-1

4.3 × 10 cm V s [18]) was

can also absorb the light [19]. When the erythrocyte lysis tookgreater than the absolute value of electroosmotic mobility of the sa-

place, remarkably known from the release of hemoglobin from the

-4

2

-1

-1

line medium (2.0 × 10 cm V s ), the red blood cell was able tocell and cell membrane deformability, red blood cell would absorb

maintain around the agarose gel frit without moving to the cathode.

less light leading to higher color intensity of the single cell photo-

Eventhough for the red blood cell incubated with ferrous ion for 5graph. Thus, the color intensity of single red blood cell photographmin in the system without hydrogen peroxide, it moved toward theis correlated to the red blood cell lysis phenomenon. In this report,

anode against the electroosmotic flow once applying the electro-

the inversion of the color intensity was observed as the apparent

voltage. This implied that the net charge of the ferrous uptake red

light density of red blood cell.

blood cell was still negative. This gave the contribution to the sta-

The more apparent light density value the cell has, the more

ble position of red blood cell at the observed chamber, enabling

stable the cell is presumed. The less apparent light density value

precise observation of lysis behavior under the microscope.

the cell possesses, corresponds to the cell that experiences partial
lysis and lesser dense cell existed more lysis action. Lysis wasshown to increase with a decreasing apparent light density value ofStarting time of lysis reaction

cell. The apparent light density of red blood cell - time curve could

It was found that the starting time of the lysis reaction of thebe assumed to correlate with lysis curve.

red blood cell at the end tip of the column next to the agarose gel
frit, was approximate to be the same as the starting time of the ap-

The time dependence of the erythrocyte lysis phenomenon af-

plication of electrovoltage. This was due to that once the electro-

ter applying voltage of six red blood cells was shown as the curves

voltage was applied, electroosmotic flow had carried hydrogen per-in Figures 3 and 4. Each curve represents each single red blood

oxide solution to the capillary and surrounded the observed red

cell. Similar pattern of these curves was observed.
Three different curve patterns were observed from Figure 3

blood cell at the end of the column immediately. The difference of―３５―Chromatography, Vol.23 No.1 (2002)

A

Table 1. Erythrocyte lysis time and half-life of several single red
blood cells obtained from the red blood cell lysis phenomenon by using the mini electrophoresis apparatus.
Lysis time Lysis timeComplete(sec) by(sec) bylysis timevisualapparent(sec) byobservation light density
apparent
methodmonitoring light density
monitoring
methodmethodB

Half-life
Half-life
for thefor theinitial step second step
(sec)
(sec)

450

451

490

172

54

464

467

496

172

62

469

466

505

165

68

453

451

495

165

60

468

467

510

165

68

450

451

490

172

54

C

Figure 3. Relationship between apparent light density of red blood
cell and time for the red blood cell lysis phenomenon observation. Each curve represented each observed single
red blood cell. Other experiment conditions were thesame as in Figure 2.

Figure 2. Visual images of the red blood cell confronting the hydroxyl free radical generating system at different time.
(a) at the starting of the reaction (b) at the first half observation period (c) at the latter observation period. Images captured by FDMCAP software from the recorded
visual images under the microscope-CCD camera system. The lysis phenomenon was performed in the rectangular capillary within homemade mini electrophoresis
apparatus.

expressed as the three steps of cell lysis process : initial step, second step and third or final step. The apparent light density values of
the curves in Figure 3 dramatically changed at the end of each step.
This implied the difference in the lysis phenomena of the threesteps. Distinct observation could be seen from Figure 4 which isthe enlargement of the final step of the curves in Figure 3. Accord-―３６―Chromatography, Vol.23 No.1 (2002)

Chaiyavat Chaiyasut , Takao Tsuda , Udompun Khansuwan and Siriwan Ong-chaiinitial step were between 165 and 172 sec. The starting time of lysis process of the second step, which was marked by obvious
change in curve pattern from the initial step to the second step,
were almost the same among all observed erythrocytes with the difference within 15 sec range. The half-lives of the second step werevaried between 54 and 68 sec in this experiment.
According to Figure 3, in the initial step, the linear relationships between the apparent light density of red blood cell and time
were observed with the correlation factors between 0.999 and0.994 among all the observed curves. It implied that the alterationof apparent light density value by time at several observed points in
the initial step was approximately constant. For instance, the relationship between apparent light density of observed erythrocyte
and time in the initial step of curve B could be expressed as the linear relationship whose the slope value, Y-intercept and correlation
factor are 2.60 × 10-2, 32.55 and 0.999, respectively. Therefore,
over all range of initial step of curve B, the differential of apparent
Figure 4. Relationship between apparent light density of red blood
cell and time for the red blood cell lysis phenomenon observation. Enlargement from Figure 3. All conditions are
the same as in Figure 3.

light density value by time, d[apparent light density]/dt, is approximated to be a constant value of 2.60 × 10-2 unit/sec. The same assumption was introduced to initial step of curve A to F. With thementioned reasons, the alteration of apparent light density of red
blood cell of the initial period by time was probably analogous to

ing to Figure 3, apparent light density values of red blood cell were

zero order. However, the lysis processes of the second and third

gradually decreased in the initial step, following a period in which

steps were too complicated for simple estimation. Further work is

the profile was dramatically changed until complete erythrocyte ly-

required for unequivocal identification.

sis finally occurred. The initial step of the curve in Figure 3 is ex-

It is interesting to note that three different processes of eryth-pected to be associated with partial cell lysis. While the second

rocyte lysis induced by Fenton reaction was observed. In the light

step, the hydroxyl free radical caused a progressive change in theof these studies, it corresponded very well with the known reaction

deformability, and ultimately led to complete cell lysis in the lastof lipid peroxidation induced by active hydroxyl radical. The lipid

step. From Figure 4, the apparent light density curve pattern sud-of the erythrocyte membrane can be oxidized by a free radical

denly changed at the point whose the image of single erythrocyte

chain mechanism consisting of three steps: initiation, propagation

disappeared from the observed position. The rate of apparent lightand termination steps [14]. There are several parameters that may

density alteration of red blood cell was alleviated in the last step

be studied for the quantitative mechanism of this lysis process. We

comparing to the second step as clearly shown in Figure 4.

hope that the proposed technique after elaboration would be usefulfor further reaction mechanism study.

The single erythrocyte lysis time observed under microscopeCCD camera and through apparent light density of cell were com-

Conclusion

pared in Table 1. These referred to the time that the single red
blood cell image disappeared from the observed position and their

The using of mini electrophoresis apparatus has been pro-values corresponded with each other very well. Whereas, the com-posed to observe the behavior of erythrocyte lysis. Our experimentplete erythrocyte lysis time obtained by apparent light density

shows that hydrogen peroxide reacted with ferrous loading red cell

monitoring method indicates the time that the color intensity of thealtering red cell deformability. This led to inevitable and complete

observed single red cell photograph become approximate constant.

lysis through time. By using the erythrocyte apparent light density

This might be due to the total lysis of red blood cell fragments orvalue monitoring method, the results in estimation of red cell lysis

the complete washing the components of the red blood cell out oftime shows an extremely good correlation to the observation under

the observed position.

microscope-CCD camera method.

To see the kinetics of cell lysis process, the half-lives of the

By using this proposed technique, the starting time of the lysis

initial step and the second step of the apparent light density - time

reaction on each single red blood cell could be observed. In addi-

curve were estimated and shown in Table 1. The half-lives of thetion, each single red blood cell was surrounded with the freshly hy-―３７―Chromatography, Vol.23 No.1 (2002)

tion, CRC Press, Boca Raton, 1996, p. 495.

drogen peroxide in saline medium by the rinsing effect of the electroosmotic flow of the medium. Therefore, the surrounding solu-[3]

Kitagawa, S.; Tsuda, T.; Nozaki, O. Bunseki Kagaku 1998,[4]

Kitagawa, S.; Nozaki, O.; Tsuda, T. Electrophoresis 1999,[5]

Tsuda, T.; Yamauchi, N.; Kitagawa, S. Anal. Sci. 2000, 16,[6]

Cooper, B. R.; Jankowski, J. A.; Leszczyszyn, D. J.; Wight-

47, 355.

tion of each red blood cell was always nearly the same as of thebulk solutions. We believed that it stabilized the lysis reaction

20, 2560.

mechanism owing to the minimizing of the depletion effect of enveloped solvent layer of cell. The flat flow profile of electroos-847.

motic flow of lysis induced reagent also increased homogeneity ofthe enveloped solvent layer of the cells especially the cell that isman, M. R.; Jorgenson, J. W. Anal. Chem. 1992, 64, 691.

adjacent to the capillary wall comparing to the introduction of pres[7]Chien, J. B.; Wallingford, R. A.; Ewing, A. G. J. Neuro-[8]

Olefirowicz, T. M.; Ewing, A. G. J. Neurosci. Meth. 1990,

lisms, and reaction mechanisms at the single cell level. The devel-[9]

Olefirowicz, T. M.; Ewing, A. G. Chimia. 1991, 45, 106.

opment for the investigation in this area is now focused on the ob-[10] Ewing, A. G.; Wallingford, R. A.; Olefirowicz, T. M. Anal.

surized flow.

chem. 1990, 54, 633.

The technique described in this paper is not limited to thestudy of erythrocyte lysis alone. The proposed technique might be

34, 11.

very fruitful for the study of other phenomena, behaviors, metabo-Chem. 1989, 61, 292 A.

servation of the lysis or interaction of cell under different reaction[11] Olefirowicz, T. M.; Ewing, A. G. Anal. Chem. 1990, 62,

mechanisms. By using the improved miniaturization apparatus and1872.

protocol, the further cell reaction mechanism is potential to be examined.

[12] Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61,

Acknowledgment[13] Lillard, S. J.; Yeung, E. S.; Lautomo, R. M. A.; Mao, D. T.

436.
J. Chromatogr. A 1995, 718, 397.

C. C. gratefully acknowledges the Ministry of Education, Sci-

[14] Davies, K. J. A.; Goldberg, A. L. J. Bio. Chem. 1987, 262,

ence, Sports and Culture, Japan for the research assistant grant-in-

8220.

aid supported as a part of the research. C. C. also gratefully thanks[15] Chiu, D.; Lubin, B. Semin. Hematol. 1989, 26, 128.

to Dr. S. Kitagawa for the technical guidance and help.

[16] Saltman, P. Semin. Hematol. 1989, 26, 249.
[17] Tsuda, T.; Ikedo, M.; Jones, G.; Dadoo, R.; Zare, R. N. J.
Chromatogr. 1993, 632, 201.

References[1][2]

Kitagawa, S.; Kawaura C.; Hashimoto, O.; Takahashi, T.;[18] Zhu, A.; Chen, Y. J. Chromatogr. 1989, 470, 251.

Naoi, M.; Tsuda, T. Electrophoresis 1995, 16, 1364.

[19] Surgenor, D. M. The Red Blood Cell Second Edition;
Acedemic Press: New York, 1975.

Swanek, F. D.; Ferris, S. S.; Ewing, A. G. in: Lander, J. P.
(Ed), Handbook of Capillary Electrophoresis second edi-

―３８―