Evaluation of Blood Cell Destruction by Measuring Occlusion Distance
SHOTA KATO1,2, TADASHI HANDA1, JUN YOSHIOKA3, KAZUHIKO NAKADATE4,
YASUTOMO NOMURA2, HITOSHI KIJIMA1
1Department of Medical Technology and Clinical Engineering,
Gunma University of Health and Welfare, 191-1 Kawamagarimachi Maebashi Gunma 371-0823,
JAPAN
2Maebashi Institute of Technology Graduate School of Environment and Biotechnology,
460-1, Kamisadori Maebashi Gunma 371-0816,
JAPAN
3Sendai Red Cross Hospital Medical Technology Department,
Clinical Engineering Technology Division,
2-43-3, Hakugiyamahonchou Sendai Miyagi 982-8501,
JAPAN
4Department of Basic Science Educational and Research Center for Pharmacy,
Meiji Pharmaceutical University,
2-522-1Nojiri Kiyose Tokyo 204-8588,
JAPAN
Abstract: - Roller pumps are commonly used for electric motor-driven blood purification. Even the optimal
occlusion for a roller pump is stimulated in JIS (Japanese Industrial Standard) -T1603, the blood cells can be
destroyed if an applied pressure is too strong on the tube. On the other hand, the perfused blood volume might
decrease if the pressure becomes weaker. Therefore, skilled operation is required. However, as there are no
techniques to automatically measure occlusion, a highly reproducible method is urgently required to obtain an
optimal setting. In this study, we classified the occlusion specified in JIS-T1603 into five categories (3, 6, 9, 12,
and 15 drops/min) and measured those using a laser sensor. The distance between each occlusion was only a
few microns. Based on the microscopic observation of the blood cell morphology at each occlusion, the blood
cells with normal outlines were classified as normal blood cells, while those with protrusions were labeled as
acanthocytes. Further, we calculated the normalized milligram index of hemolysis (mgNIH) to confirm
hemolysis for each occlusion. By classifying occlusion into five categories and converting them into distances,
we derived a safe, easy, and highly reproducible method.
Key-Words: - occlusion, occlusion distance, mgNIH, acanthocytes, red blood cells, roller pump, laser sensor.
Received: August 7, 2022. Revised: October 12, 2023. Accepted: November 13, 2023. Published: December 12, 2023.
1 Introduction
Currently, available medical pumps, especially
infusion pumps, syringe pumps, and implantable
axial flow pumps are indispensable for treatment. In
particular, medical pumps used for extracorporeal
circulation, broadly classified into centrifugal and
roller pumps, are crucial for maintaining the body’s
physiological homeostasis, [1], [2], [3]. Roller
pumps are inexpensive, easy to operate, and provide
stable irrigation flow, [4].
According to Japanese Industrial Standards
(JIS)-T1603, the optimal occlusion setting for a
roller pump is obtained by clamping the inlet side of
the circuit tube, opening the outlet toward the
atmosphere, and connecting the infusion tube of the
infusion circuit at a height of 1m, [5]. However, this
adjustment method has a strong subjective element
and low reproducibility because the falling speed is
confirmed visually, [6], [7]. Moreover, changes in
occlusion alter the perfusion volume and blood cell
morphology, significantly impacting the therapy
during extracorporeal circulation, [8], [9]. Therefore,
there is a need for a highly reproducible and
quantitative occlusal adjustment method, [10]. One
method with high reproducibility is to use a laser
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DOI: 10.37394/23208.2023.20.32
Shota Kato, Tadashi Handa, Jun Yoshioka,
Kazuhiko Nakadate, Yasutomo Nomura,
Hitoshi Kijima
E-ISSN: 2224-2902
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sensor to convert the tiny occlusion gap into
distance. By quantifying the occlusion distance
using a laser, a quantitative and highly reproducible
method can be established.
In this study, to establish a highly reproducible
occlusion setting method, we used a laser sensor to
classify the occlusion into 3 drops/min, 6 drops/min,
9 drops/min, 12 drops/min, and 15 drops/min. The
distance was measured. At this time, when
quantified using mgNIH (Normalized Milligram
Index of Homolysis), which indicates haemolysis,
the percentage of acanthocytes was as high as
34.04% at the short distance of 3 drops/min, and the
average of mgNIH the value was 178.83mg/L,
which was a high percentage, [11]. On the other
hand, at the longer distance of 15 drops/min, the rate
of acanthocytes was low at 8.15%, and the average
value of mgNIH was low at 37.22 mg/L.
2 Materials and Methods
2.1 Five Occlusion Categories Settings
Setting methods using static and dynamic pressures
have been reported for roller pumps. Here, we
adopted the static pressure occlusion method by
clamping the tube outlet side, opening the inlet side
to the atmosphere, and dropping 5-10 drops/min
from a height of 1 m from the pump (6-13 drops/min
due to changes in the standards of the infusion
Fig. 1: The ZX2-LDL50 was installed at a position
50mm from Roller center point.
circuit), [12]. Based on JIS-T1603, which is
considered to be the optimum occlusion, we used
five occlusion categories: 3, 6, 9, 12, and 15
drops/min.
2.2 Sensor Performance
We fixed a ZX2-LDL50 device (Omron Corp.,
Kyoto, Japan) outside the housing 50 mm from the
center of the roller and constructed a system for
measuring the distance of occlusion. A cylindrical
hole (20 mm in diameter) was made on the housing
side to utilize the scattered light of the laser beam.
The sensor utilizes a complementary metal oxide
film semiconductor method with a triangular side
distance method to detect the light intensity of the
pixel and convert it into the distance.
The ZX2-LDL50 irradiated visible
semiconductor laser of 660 nm was used to measure
the occlusion distance. The measurement center
distance was 10 mm out of 50 mm, the resolution
was 1.5 μm, and the linearity was 0.05% full scale
(measurement range). This sensor can also irradiate
a linear beam and convert the amount of light in the
pixel into a distance with a temperature
characteristic of 0.02% full scale/°C.
The ZX2-LDL50 has a measurable range of 50
mm ± 10 mm, and it can measure the collapsed
distance of the tube when the roller moves from the
center point toward the housing. This device can
also automatically measure the occlusion distance of
up to 10 μ units (Figure 1), [12], [13].
2.3 Blood Circulation
Pig blood (Tokyo Shibaura Organ Co. Ltd, Tokyo,
Japan), Meiron (115 mEq； Fuso Pharmaceutical
Industries, Ltd. Osaka, Japan), heparin (7.7 U/kg；
Nipuro Corp., Osaka, Japan) and MAP solution (D-
Mannitol 14.57 g/L, Adenine 0.14 g/L, Sodium
phosphate monobasic 0.94 g/L, sodium citrate 1.5
g/L, citric acid 0.2 g/L, dextrose 7.21 g/L, sodium
chloride 4.97 g/L) were added to the circulation
circuit to create a perfusate solution, which was
circulated within the Excelline H3/8-inch tube
(MERA Corp., Tokyo, Japan) using a HAS-150
roller pump (MERA Corp., Tokyo, Japan). Before
starting the experiment, the distances for each of the
five occlusion categories were measured. Thereafter,
it was circulated for 180 minutes at a rotational
speed of 150 rpm, and blood was collected every 30
minutes. At the same time, the volumetric flow rate
was measured using a magnetic flowmeter MIM
(Kobold Messring GmbH., Nordring Germany).
Figure 2 shows the circuit diagram of the above
experiment.
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Fig. 2: Experimental closed-circuit diagram
2.4 Determination of Blood Cell Morphology
To confirm the blood cell morphology for all five
occlusion categories, a 1 mL sample was collected
from the blood circuit, and a smear specimen was
prepared. The morphology of the prepared smears
was observed using an optical microscope (Olympus
Co. Ltd, Tokyo, Japan) at 1000x magnification and
after 0 and 180 min. The red blood cells with round
morphology were classified as normal, while those
with protrusions were classified as acanthocytes.
Further, we performed Wright-Giemsa staining
following the manufacturer’s protocol (Muto Pure
Chemical Ltd., Tokyo, Japan) with the required
staining reagents (Muto Pure Chemical Ltd.).
2.4.1 Calculation of mgNIH
To calculate the mgNIH, we collected the blood in
the circulation circuit and centrifuged it at 1500 rpm
for 10 minutes. We measured the free hemoglobin
in the supernatant using Plasma HemoCue
(HemoCue AB, AMCO Inc., Japan). Then, we
measured the RBCs (Red blood cells), hemoglobin
(Hb), and hematocrit (Hct) using the MEK-7300
Celltac Es hemocytometer (Nihon Kohden Co. Ltd,
Tokyo, Japan). After extracting these items, the
mgNIH was calculated as follows, [14].
ΔPfHb : increment of plasma free hemoglobin
concentration (mg/dL)
V : whole blood volume in flow loop (mL)
Hct : hematocrit%
Q : flow rate (L/min)
T : sampling period (min)
2.4.2 Evaluating the Relationship between the
Occlusion Distance and mgNIH
To investigate the relationship between blood cell
destruction and occlusion, we measured the
occlusion distance and mgNIH at 3, 6, 9, 12, and 15
drops/min. We performed Welch’s T-test at p < 0.01
using Excel statistics to identify the significant
differences between occlusion distance and mgNIH.
3 Results
3.1 Occlusion Distance
Figure 1 shows occlusions at 3, 6, 9, 12, and 15
drops/min converted to distance. At 3 drops/min, the
distance and dispersion are higher than other
occlusions. Also, at 6 drops/min, the distance and
dispersion were higher than at 9, 12, and 15
drops/min. The mean values and standard deviations
of occlusion distances are shown in Table 1. From
Table 1, it can be seen that 3 drops/min, and 6
drops/min are larger than the dispersion of 9
drops/min, 12 drops/min, and 15 drops/min.
Table 1. Mean value and standard deviations (SD)
of occlusion distance when repeated 5 times
Drops
rate
3
6
9
12
n
5
5
5
5
Mean
(mm)
1.2051
1.191
1.1818
1.1771
SD
(mm)
0.0188
0.0097
0.0039
0.0038
3.2 Relationship between Occlusion Distance
at 0 and 180 min and Blood Cell
Morphology
Figure 3 shows the morphology of blood cells in
five occlusions. At the start time of 0 min, the
percentage of acanthocytes in all occlusions was
less than 10%. Figure 4 shows the morphology of
each blood cell at five occlusion categories after 180
min. The number of acanthocytes was high at 3
drops/min and 6 drops/min, and there was no
significant change at 9 drops/min, 12 drops/min, and
15 drops/min. Table 2 lists the respective occlusion
and acanthocyte percentages at 0 min. Table 3 lists
the respective occlusion and acanthocyte
percentages at 180 min.
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Fig. 3: Five occlusions’ categories and blood cell
morphology at 0 min
Fig. 4: Five occlusions’ categories and blood cell
morphology at 180 min
Table 2. Percentage of acanthocytes, mean and
standard deviation (SD)
Percentage of acanthocyte at 0 min (%)
Drops rate
3
6
9
12
15
1st
2.20
3.88
8.76
9.03
4.60
2nd
3.16
3.79
7.39
8.36
5.42
3rd
4.83
6.46
8.47
7.45
4.68
4th
4.25
5.32
6.87
5.13
8.12
5th
6.21
5.81
8.58
4.72
7.42
Mean
4.13
5.05
8.01
6.94
6.05
SD
1.38
1.06
0.75
1.72
1.45
Table 3. Percentage of acanthocytes, mean and
standard deviation (SD)
Percentage of acanthocyte at 180 min (%)
Drops rate
3
6
9
12
15
1st
35.14
22.83
9.14
7.88
8.31
2nd
29.92
25.94
11.77
11.12
7.81
3rd
31.22
24.27
10.2
9.57
7.40
4th
37.75
24.16
12.54
8.23
9.12
5th
36.17
21.92
12.23
9.08
8.09
Mean
34.04
23.82
11.18
9.18
8.15
SD
2.98
1.37
1.30
1.14
0.57
3.3 Relationship between Occlusion Distance
and Blood Cell Destruction
We observed a large distribution for mgNIH at 3
drops/min, and the average value was 178.83 mg/L
(Figure 5). Whereas the mgNIH was low at 15
drops/min, with an average value of 37.22 mg/L.
Also, no significant difference could be confirmed
for 3 and 6, but significant differences could be
confirmed for the other 9, 12, and 15 drops/min (p <
0.01).
Further, although the initial values of mgNIH
for the five occlusion categories are similar, the
standard deviations for mgNIH at 3 and 6 drops/min
were much higher than those at 9,12, and 15
drops/min. These values indicated that 3 and 6
drops/min had higher hemolytic indices than 9,12
and 15 drops/min.
Fig. 5. Relationship with Plasma free Hb each
occlusion
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4 Discussion
4.1 Occlusion Distance for all Five Occlusion
Categories
In extracorporeal circulation, a quantitative and
highly reproducible setting method is required rather
than setting the occlusion based on skill and
experience. Here, we converted the five occlusion
categories into distances using a laser sensor. Since
the distance of the converted occlusion is extremely
small, with a difference of several tens of microns,
precise manipulation was required. Moreover, the
reproducibility of the settings based on intuitive
manipulation might be low. Based on these points,
we evaluated the occlusion changes from two points
of view, morphological and numerical.
4.2 Relationship between Occlusion Distance
and Blood Cell Morphology
After 180 minutes, the rate of acanthocytes
increased at 3, and 6, drops/min, but was similar at
9,12, and 15 drops/min. This might be because of
the decrease in the amount of sialic acid due to the
prolonged force on the blood cells, which might
change the internal structure, [15]. The surface of
red blood cells (RBCs) is negatively charged, and
the amount of sialic acid determines the repulsive
force between adjacent blood cells. In addition, the
decrease in the amount of sialic acid and loss of
other proteins decrease the repulsive force between
blood cells and increase the rate of red blood cell
aggregate formation. This reduction in the repulsive
force might also reduce the viscosity of the plasma
component of the Newtonian fluid.
When RBCs pass through narrow blood vessels,
such as capillaries, their morphology changes due to
the Tancred movement in the presence of certain
conditions, including low Reynolds number,
pressure, and velocity. Based on this theory, the
morphology of the RBCs might change when they
pass through the rollers of the pump. As a
characteristic of Tancred movement, the biconcave
shape can be changed vertically and horizontally to
enable passage through blood vessels whose
diameters are smaller than blood cells. Even though
RBCs are subjected to uniform pressure as they pass
through the narrow path, the membrane itself has a
high restoring force and returns to its original
biconcave shape. However, at 3 drops/min, the yield
value limit of the RBCs’ membranes might have
exceeded as the pressure on the tube is higher than
that at 15 drops/min. Originally, the RBCs’
membranes were thought to undergo hemolysis due
to high shear stress. However, reports have shown
that the RBCs’ membranes do not rupture
immediately and are highly contractile over a wide
range. In other words, when high pressure is applied
to the RBCs, they do not immediately deform as
they can withstand a few stretching cycles. After
that, it can be assumed that the RBCs lose their
shape and turn into acanthocytes.
4.3 Relationship between Occlusion Distance
and mgNIH
Originally, when blood encounters the air, it
undergoes anaerobic metabolism, which promotes
the destruction of RBCs. Furthermore, the
application of strong forces, such as shear stress,
damages RBCs, which seems to be the case in this
study.
From Figure 5, the average distance for 3, 6, 9,
12, and 15 drops/min is 1.2051, 1.191, 1.181,
1.1771, and 1.174 mm, respectively. There was a
maximum difference of 0.031 mm between 3 and 15
drops/min, which is half the thickness of European
and American hair. The occlusion distance of 3
drops/min against 15 drops/min is 0.03 mm when
pushed into the housing, which might be due to the
strong pressure applied at 3 drops/min that also
increases the mgNIH.
Considering the difference in occlusion distance
(mean value) between 3 drops/min and 15
drops/min, the momentum imparted to the RBCs
when the roller is translated can be substituted into
the following equation, where p is the power, m is
the mass and v is the angular velocity calculate.
p = m × v (1)
It can be expressed by the formula of (1)
Let r be the position of the mass from the origin and
the angular momentum vector measured from the
origin can be expressed by the following equation.
L ≡ r × p (2)
Substitute the formula of (1) into (2).
L ≡ m × r × v (3)
Based on angular momentum, v rotates n times per
minute. n = 1 / t = ω / 2π
Therefore,
ω = 2πn
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It can represent.
L ≡ m × r2 × 2πn (4)
Also, the impulse generated when the roller
encounters the RBCs through the tube from the
moment of inertia has been considered (ignoring the
elastic force of the tube).
I = FΔt (5)
I = m × R2 × Δt (6)
p : Linear momentum
L : Angular momentum
v : angular velocity
r : Radius
t : Second
ω : Angular velocity
F : Impulse
I : Moment of inertia
R : Distance of roller movement from roller
center to housing 0.00064 m
m: Roller mass 0.375 kg
t : 180 × 60 sec
At occlusion of 3 and 15 drops/min, the average
distances to the housing side were 1.2051 mm and
1.1740 mm, respectively. Substituting these into
equation (6), the difference in thrust between 3 and
15 drops/min is 1.54×10−9 kg ･m. RBCs are
deformed by shear stress. The limit value of
membrane plasticity is based on the following
equation, [16].
1.6 × 10−2 dyn / cm ≤ T0 ≤ 8 × 10−2 dyn / cm
(7)
This surface tension unit can be expressed as
N/m = dyn/cm = kgf/m, and to convert the obtained
impulse to dyn/cm, it should be divided by the
product of mass and time.
The moment of force on the RBCs is 116.25
dyn/cm, which is approximately 1400-7200 times
the yield limit of the RBCs’ membranes. However,
considering the elastic resistance of the circuit tube,
although the actual pressure applied to the blood is
reduced, the long-term accumulation of force might
cause blood cell destruction. Therefore, considering
the moment of force, at 3 drops/min, the applied
pressure is approximately 1400 to 7200 times higher
than that of 15 drops/min, which might accelerate
the rupture of the RBCs. Also, in terms of impulse,
the RBCs go around a circuit of 1000 mm once
every 2 seconds. Therefore, the blood damage might
be massive under a 1400 to 7200 times higher
pressure multiplied by 90 minutes x 60 seconds.
4.4 Clinical Indication
In our occlusion experiments, mgNIH values were
lower at 9, 12, and 15 drops/min than at 3 and 6
drops/min. However, according to JIS-1603,
occlusion at 6 to 13 drops/min is considered optimal.
This is because as the occlusion loosens, the volume
flow circulating in the circuit decreases, and the
cannula tip may collide with arterial pressure,
making it impossible to maintain the target
perfusion rate. Considering the above, 6 to 12
drops/min is considered optimal.
5 Conclusion
When using a roller pump for extracorporeal
circulation, too strong an occlusion pressure may
promote blood cell destruction, and a weak
occlusion pressure may reduce the perfused blood
volume. Therefore, there is an urgent need for a
method to achieve optimal and highly reproducible
occlusion. In this study, we developed a safe, easy,
and highly reproducible method by classifying
occlusal conditions into five categories and
converting them into distances using a laser sensor.
The main results are as follows:
(1) To establish a highly reproducible occlusion
setting method, we used a laser sensor to convert
five occlusion categories into 3 drops/min, 6
drops/min, 9 drops/min, 12 drops/min, and 15
drops/min.
(2) The laser sensor can also automatically measure
the distance the roller moves from the center point
toward the housing.
(3) The results suggested that occlusion at 3
drops/min caused higher blood cell destruction than
that at 15 drops/min. However, considering the
perfusion pressure of the human body at 13
drops/min, the perfusion amount may decrease.
(4) We concluded that the occlusion in the range of
6 drops/min to 12 drops/min is optimal.
(5) In the future, we would like to perform reflux at
the same pulse pressure as in the human body and
measure the amount of sialic acid at that time.
Additionally, we would like to establish a roller
pump that causes less blood cell destruction
compared to centrifugal pumps.
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Contribution of Individual Authors to the
Creation of a Scientific Article (Ghostwriting
Policy)
- Shota Kato conducted all of the experiments and
wrote the submitted paper.
- Tadashi Handa performed the staining of the
smear specimens.
- Jun Yoshioka created a blood cell preservation
solution.
- Kazuhiko Nakadate prepared and analyzed the
blood smears.
- Yasutomo Nomura performed the physical
analysis of blood cell destruction and constructed
the entire experiment.
The entire study was supervised by Hitoshi Kijima.
Sources of Funding for Research Presented in a
Scientific Article or Scientific Article Itself
No funding was received for conducting this study.
Conflict of Interest
The authors have no conflicts of interest to declare.
Creative Commons Attribution License 4.0
(Attribution 4.0 International, CC BY 4.0)
This article is published under the terms of the
Creative Commons Attribution License 4.0
https://creativecommons.org/licenses/by/4.0/deed.en
_US
WSEAS TRANSACTIONS on BIOLOGY and BIOMEDICINE
DOI: 10.37394/23208.2023.20.32
Shota Kato, Tadashi Handa, Jun Yoshioka,
Kazuhiko Nakadate, Yasutomo Nomura,
Hitoshi Kijima
E-ISSN: 2224-2902
320
Volume 20, 2023