Jpn J Extra-Corporeal Technology 43（4）：339 − 345, 2016

Alkalemic conditions result in blood clottingin the circuit soon after initiating cardiopulmonary bypass
Tomonori Soyama 1, 2），Yukihiro Takahashi 2），Hideto Yoshida 3）
Tohru Sakoda 4），Tomokazu Niitsuma 4），Toshiya Nishikubo 2）
Abstract
Purpose : This study investigated the relationship between blood clotting in the circuit soon after initiating cardiopulmonary bypass (CPB) and echinocytes that appear with alkalemia, using a recirculation circuit filled withheparinized bovine blood.
Methods : Alkalemic conditions in the recirculation circuit were prepared by adding various concentrations of
NaHCO3 to the priming fluid. Albumin was also added to confirm its inhibitory effect on blood clotting. Blood pH,
hold-up, the pressure gradient, and red blood cell ( RBC) reduction rate were monitored. Blood clots were examined
microscopically.
Results : Although blood pH was elevated under all experimental conditions, clotting in the circuit increased with-increased concentrations of HCO3 . Albumin inhibited the clotting under the same alkalemic conditions. Microscopic
findings revealed echinocytes in the blood clots.
Conclusions : The shape of echinocytes was transformed by a reduction in the Donnan equilibrium ratio becauseof changes in pH inside and outside the RBC membrane. Blood clotting in the circuit soon after initiating CPB may
be caused by echinocytes that appear under alkalemic conditions. This was inhibited by albumin, suggesting thatthe addition of albumin to the priming fluid may prevent such clotting in the circuit after initiating CPB.
Key words : alkalemic conditions, blood clotting, echinocyte, Donnan equilibrium ratio, albumin

Ⅰ．Introduction

tion/thrombus formation2-5) and agglutination of the

Blood clotting has occasionally been reported in theechinocytes that appear with alkalemia.6, 7 ) However,cardiopulmonary bypass (CPB) circuit soon after initi-

the underlying mechanisms have yet to be elucidated.

ating CPB despite adequate anticoagulation manage-

Prior to the initiation of CPB, priming fluid is re-This can cause an obstruction incirculated to evacuate any air from the recirculation

the venous reservoir filter and membrane oxygenator,

circuit and to prevent a reduction in temperature.

and replacement of these parts may become necessary

However, oxygen insufflation and the addition ofduring surgery. Two theories have been proposed tosodium bicarbonate to the priming fluid may result in

explain this clotting in the circuit : platelet aggrega-extreme alkalization.

ment with heparin.

1-7 )1）Health Care Education and Research Center, Tenri Health Care University2）Division of Neonatal Intensive Care, Nara Medical University Hospital3）Department of Clinical Laboratory, Tenri Hospital4）Research Section 3, Central Research Laboratory, JMS Co., Ltd.
Corresponding Author : Tomonori Soyama
Health Care Education and Research Center, Tenri Health Care University
80-1, Bessho-cho, Tenri city, Nara, 632-0018, Japan[Received September 10, 2016 : Accepted October 21, 2016]

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It has been postulated that blood clotting in the

To confirm whether any increase of blood clotting

circuit soon after initiating CPB is caused by echi-

in the circuit was because of the appearance of echi-

nocytes that form as a result of contact between thenocytes under alkalemic conditions, and to reduce

alkalized priming fluid and blood. We have previously

the clotting, albumin was added under alkalemic

investigated in vitro the frequency of echinocytes,

conditions. Three sets involved different volumes

platelet aggregation, and blood coagulation abilities

of NaHCO3 : no addition (control), 100 mL ( NaHCO3

under alkalemic conditions,demonstrating that100), and 200 mL ( NaHCO3 200). Three further sets

the frequency of echinocytes increased under suchincluded different volumes of albumin (50 mL [Alb 50],

conditions.

In contrast, platelet aggregation and100 mL [Alb 100] , and 200 mL [Alb 200] ) after the

blood coagulation were not enhanced under these

addition of 100 mL NaHCO3, resulting in final albumin

8)

8, 9)

conditions. We also confirmed that the presence ofconcentrations of 1.6, 2.6, and 4.1 g/dL, respectively.

albumin under alkalemic conditions inhibited the ap-

These were combined with lactated Ringer's solution

pearance of echinocytes. This suggested that bloodto adjust the priming fluid volume to a total of 1,000

clotting in the circuit soon after initiating CPB could

mL.

9)

For each of the six conditions, the priming liquid

be prevented with albumin. 8)
The aim of the present study was to confirm thesewas added into the circuit, which was then stabilized

previous findings by investigating the relationshipat a blood flow of 2 L/min at a 37℃ solution tempera-

between various alkalemic conditions and the appear-

ture for 5 min. Subsequently, 400 mL of heparinizedance of clotting in the circuit during CPB, using abovine blood (with the heparin isolated from the intes-

recirculation circuit filled with heparinized bovine
blood.

Temperature

Ⅱ．Materials and Methods
An experimental recirculation circuit was prepared

Venous Reservoir

with a venous reservoir (OXIA RE40 ; JMS, Hiroshima,
Japan), a roller pump (Caps Roller Pump ; Stockert
GmbH, Freiburg, Germany), and a membrane oxygen-

Flow
Probe

Sampling port

ator (OXIA LP ; JMS, Hiroshima, Japan) (Fig.1).

Roller Pump

The priming fluid was prepared with a 7% NaHCO3
solution ( MEYLON Injection 7%, Otsuka Pharmaceuticals, Tokyo, Japan), 25% albumin solution (Alb ; Cohn
Fraction V, pH 7.0 ; Wako, Osaka, Japan ; dissolved in

Pressuresaline and the final concentration adjusted to 25%),

Gas inand lactated Ringer's solution ( Lactec Injection,
Otsuka Pharmaceutical, Tokyo, Japan). The experiment was performed under six different alkaline

Heat Exchanger

conditions involving different amounts of the priming
liquid in a total volume of 1,000 mL were examined( Table 1).

Priming fluid（mL）
25％ Albumin solution

Pressure

Experimental recirculation circuit

The experimental recirculation circuit comprised a venous reservoir,roller pump, and membrane oxygenator.

Table 1
7％ NaHCO3 solution

Fig. 1

Membrane
Oxygenator

ControlPriming fluid

NaHCO3 100

NaHCO3 200

Alb 50

Alb 100

Alb 200

100

200

100

100

100
200

0
0

0

0

50

100

Lactated Ringer's solution

1,000

900

800

850

800

700

Total Volume

1,000

1,000

1,000

1,000

1,000

1,000

NaHCO3： 7％ NaHCO3； Alb： 25％ albumin solution

340

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Jpn J Extra-Corporeal Technology 43（4），2016

Pre HU

Post HU⇨

Fig. 2

HU

Hold up

Blood clotting in the circuit results in a difference in the liquid level（the “hold up,” HU）between the inside and outsideof the venous reservoir filter.

tinal mucosa of pig at a concentration of 15,000 IU/L),Japan).

stored overnight at 4℃ after blood collection, was

2．Albumin concentration

circulated under each condition. At the same time,

To confirm that albumin reduced the grade of blood

oxygen insufflation of the membrane oxygenator wasclotting in the circuit under alkalemic conditions,started at 1 L/min.

albumin concentrations (g/dL) were measured at theAfter initiating CPB, the following items were

initiation of CPB using a microplate reader ( Infinite

measured for a 120-min recirculation period (or for 60

M200 ; Tecan Austria GmbH, Grödig, Austria).

min in the experiment with NaHCO3 200).

3．Prothrombin time, activated partial thrombopla­
stin time, and soluble fibrin monomer complex

1．CPB Parameters

To investigate whether the blood clot in the circuit

1) Blood pH
Because blood pH changes with the addition of

was caused by echinocytes, the prothrombin time

NaHCO3 to the priming liquid and oxygen insufflation( PT ; s), activated partial thromboplastin time (APTTof the membrane oxygenator, it was measured every; s), and soluble fibrin monomer complex (SFMC ; μg/

30 min using a pH meter ( F-55 ; HORIBA, Kyoto,mL) were measured at the initiation and completion

Japan).

of circulation using an automated blood coagulation

2) Hold up and pressure gradientanalyzer (Coapresta 2000, SEKISUI MEDICAL,

To evaluate the grade of blood clotting in theTokyo, Japan).

circuit, the difference in the liquid level (the “hold up,”

4．Electron microscopic analysis

HU ) (Fig.2) between inside and outside of the venous

The membrane oxygenator surface that resulted

reservoir filter was measured every 15 min (Fig.1), as

in a PG because of a blood clot in the circuit under

was the pressure gradient ( PG) across the membrane

alkalemic conditions (100 mL NaHCO3, 400 mL

oxygenator. HU was measured in mm using a ruler.

lactated Ringer's solution, and 200 mL heparinized

PG was measured using a digital pressure sensor

bovine blood) was analyzed using scanning electron(AP-34A ; KEYENCE, Osaka, Japan), and thephotomicrography. The membrane oxygenator wasunits were converted from kPa to mmHg. The PG

fixed with 2.5% glutaraldehyde for 1 h after washing(mmHg/L) per 1 L of flow was also calculated.

with saline and the sample was analyzed using a3) Red blood cell reduction rate

SEMEDX Type N scanning electron microscope

To confirm the protective effects of albumin on red( Hitachi, Tokyo, Japan).

blood cells ( RBCs), the RBCs were counted every 30

5．Statistical analysis

min, and the percentage reduction from the initial

The aim of this study was to confirm the in vitro

count was calculated ( RBC reduction rate ; RRR). The

findings of a previous study. Accordingly, this was a

RBC count was measured using an automated multi-

single-run experiment, and the effects of the control,channel blood cell analyzer (XT-1800i, Sysmex, Kobe,

NaHCO3 100, NaHCO3 200, Alb 50, Alb 100, and Alb

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200 conditions on blood pH, HU, PG, and RRR werevenous reservoir filter and membrane oxygenator cananalyzed using comparisons of two regression slopes.

become obstructed, and it may become necessary to

These analyses were performed using Microsoft

replace parts during surgery,1, 4) although a blood clot

Excel, and a P value of <0.05 was considered statisti-

in the circuit that appears soon after initiating CPB

cally significant.10, 11)

may reversibly disappear with time.4)
In this study, we hypothesized that blood clotting

Ⅲ．Results

in the circuit may be caused by echinocytes that form

The results for blood pH, HU, PG, and RRR are

because of alkalization of the priming fluid, as pre-shown in Fig.3. Because of the alkaline conditions,

viously reported.6) We therefore investigated blood

the blood pH slope increased with time across all six

clotting in the circuit soon after initiating CPB underconditions (control, NaHCO3 100, NaHCO3 200, Alb 50,

six different priming conditions.

Alb 100, and Alb 200), but there were no significant

In the control condition, there was no NaHCO3 but

differences in their slopes ( P>0.05). The blood pH of

the pH was elevated to 8.29 by oxygen insufflation of

the control condition only increased to 8.29, but thethe membrane oxygenator alone. A blood clot did not

pH increased above 9.0 in the other five conditions

form in the circuit in this condition. This suggests(Fig.3a).

that, without NaHCO3 in the priming fluid, oxygen

There were significant differences between the HU

insufflation of the membrane oxygenator alone may

slopes of all six conditions ( P<0.05) (Fig.3b). Notably,

not induce echinocyte-forming alkalemic conditions.

increasing albumin concentration reduced the HU. In

In contrast, a blood clot did appear in the circuit in

the NaHCO3 200 experiment, a circulation of 2 L/min

both the NaHCO3 100 and the NaHCO3 200 condi-

could not be maintained due to the increase in HU,

tions. Although the pH change was similar under

and the experiment was terminated after only 60 min.

these two conditions, the blood clot in the circuit was

PG did not differ significantly between the three

more prominent in the NaHCO3 200 condition.

albumin concentrations (Alb 50, Alb 100, and Alb

The cytoskeletal lining of the RBC membrane200), but there were significant differences with themaintains a wide extension in a planar form throughother three conditions ( P<0.05) (Fig.3c).

the binding of component proteins (e.g., spectrin and

The RRR results for both NaHCO3 100 and NaHCO3

actin) ; this lines the cytoplasmic side of the lipid

200 differed significantly from those of all the otherbilayer by coupling with the RBC transmembrane

five conditions ( P<0.05) ; however, there were no sig-

Band 3 protein ( Band 3).12, 13) The Band 3 conformationnificant differences between the RRR results for the(inward- or outward-facing) depends on the Donnan

control and Alb 50, Alb 100, and Alb 200 conditions

equilibrium ratio of Cl , HCO3 , OH , and H concen-(Fig.3d).

trations between the inside (i) and outside (o) of the

-

-

-

+

The albumin concentration was 0.6 g/dL in the

RBC membrane, as represented by the following

control, NaHCO3 100, and NaHCO3 200 conditions, 1.6

equation : [Cl ] i / [Cl ] o = [HCO3] i / [HCO3] o = [OH ] i

g/dL in Alb 50, 2.6 g/dL in Alb 100, and 4.1 g/dL in/ [OH ] o = [H ] o / [H ] i .14-16) Previous studies have

Alb 200.

reported reductions in this ratio with increased pH,

-

-

-

+

+

-

-

-

-

PT and APTT were prolonged at a level higher

and that higher concentrations of HCO3 outside thethan the detection limit ( PT>100 s ; APTT>300 s).

RBC membrane increase the amount of Band 3 with

SFMC was 0.4 μg/mL or lower for all conditions, in-

an inward-facing conformation.16, 17 ) This conformation

dicating that the blood's coagulation ability was not

induces a contraction of the membrane cytoskeleton

enhanced.

and induces the echinocyte shape.16, 17 ) Echinocytes

On electron microscopy, several echinocytes were

have previously been shown to be less deformable

observed on the membrane oxygenator surface (Fig.4).

than discocytes,18, 19) and so the obstruction of pores inthe venous reservoir filter and membrane oxygenator

Ⅳ．Discussion

may occur because of echinocytes. However, it is also

Blood does not readily coagulate during CPB

possible that a change in the electrostatic attraction

because of the application of continuous anticoagu-

on the surface of the echinocyte membrane could po-lant therapy. However, if a blood clot occurs, thetentially result in a blood clot in the circuit. 20) Consid-342

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Jpn J Extra-Corporeal Technology 43（4），2016

a：Blood pH
10

Blood pH

9.5

Blood pH

9

NaHCO3 100

NaHCO3 200

Alb 50

Alb 100

Alb 200

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

n.s.

NaHCO3 100

8.5

NaHCO3 200

8

Alb 50

7.5

Alb 100

7
6.5

Control

Controln.s.

Alb 200
0

1530

45 60 75 90 105 120
Time（min）

b：Hold up
160

HU

140

Control120
HU（mm）

Control

100

NaHCO3 100

80

NaHCO3 200

60

Alb 5040

Alb 100

20
0

NaHCO3 100

NaHCO3 200

Alb 50

Alb 100

Alb 200p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05

Alb 200
0

1530

45 60 75 90 105 120
Time（min）

c：Pressure gradient120

PG

PG（mmHg/L）

100

Control

80

NaHCO3 100

NaHCO3 200

Alb 50

Alb 100

Alb 200p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05n.s.

n.s.

NaHCO3 100
NaHCO3 200

60

Alb 5040

Alb 100

20
0

Controln.s.

Alb 200
0

1530

45 60 75 90 105 120
Time（min）

d：RBC reduction rate

RBC reduction rate（％）

0

RRR

−5

Control

Control

NaHCO3 100

NaHCO3 200

Alb 50

Alb 100

Alb 200p＜0.05p＜0.05

n.s.

n.s.

n.s.

p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05p＜0.05n.s.

n.s.

NaHCO3 100−10

NaHCO3 200−15

Alb 50
Alb 100−20n.s.

Alb 200
−25

0

15

30

45 60 75 90 105 120
Time（min）

Fig. 3

Change with time of six different alkaline conditions

The results for blood pH, hold up（HU），the pressure gradient（PG），and red blood cell reduction rate（RRR）．Increases in blood pH weresimilar under all the conditions tested（excluding the control condition），regardless of the volumes of NaHCO3 and albumin added. However, HU
and PG increased, and RRR was greatest with NaHCO3 200. The addition of albumin inhibited these changes to a greater extent than did NaHCO3
100. The increase in HU was inhibited as the albumin concentration increased.

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negative charge was enhanced under alkalemic conditions ; this negative charge in the albumin surround-ing the RBC membrane may have repelled HCO3 .17 ) In
our previous studies, we reported that the presenceof albumin under alkalemic conditions inhibited theappearance of echinocytes, 8) and this was morphologically confirmed in the present study. Furthermore,
RRR was inhibited more strongly by Alb 50, 100, and
200 than by NaHCO3 100 and 200, suggesting thatalbumin has a protective effect against hemolysis orpore obstruction of the venous reservoir by echinocytFig. 4Echinocytes on the membrane oxygenator surface

A representative scanning electron photomicrograph of themembrane oxygenator surface resulting in a pressure gradient due
to a blood clot in the circuit under alkalemic conditions. Manyechinocytes can be seen on the membrane oxygenator surface.

ic transformation. These findings therefore confirmed
the previous reports that the addition of albumin caninhibit the appearance of echinocytes in the circuit,perhaps due to an increase in the Donnan equilibrium ratio between the inside and outside of the RBC
membrane. 8, 16, 17, 23)

ering that PT and APTT were markedly prolonged
in the NaHCO3 100 and 200 conditions with a very

Ⅴ．Conclusion

low SFMC, and that platelet aggregation ability was

Blood clotting in the circuit soon after initiating

not enhanced under the alkalemic conditions in our

CPB may be caused by echinocytes that appear

previous study, it is unlikely that blood clotting in theunder alkalemic conditions. The addition of albumin

circuit with alkalemia was caused by enhanced bloodto the priming fluid may inhibit this echinocyte-in-coagulation ability or platelet aggregation.

duced clotting.

9, 21, 22 )We

also morphologically demonstrated an increase in thenumber of echinocytes under the alkalemic conditions<Conflicts of Interest>

used in our previous study, 8) an observation consistentThis was a cooperative study in which three institutions par­with the present findings.

ticipated : Nara Medical University, JMS Co., Ltd., and Tenri

Thus, the greatest degree of blood clotting observedin the circuit under the NaHCO3 200 condition may
have been due to an accumulation of echinocytes,
perhaps because of a decrease in the Donnan equi-

Hospital. The first author performed the study at the researchfacility of JMS Co., Ltd., using materials and instruments pro­vided by the facility. All remaining authors have declared noconflicts of interest.

librium ratio resulting from an increase in the con-centration of HCO3 outside the RBC membrane due

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