THE EFFECT ‘OF THE SURFACE ACTIVE
AGENT PLURONIC F-68 ON
RED BLOOD CELL HEMOLYSIS IN VITRO
AND DURING CARDIOIPULMONARY BYPASS

Thesis for the Degree of M. S.
MICHIGAN STATE UNIVERSITY
DOROTHY TAO-- LIEN YAO
1972

 

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ABSTRACT
THE EFFECT OF THE SURFACE ACTIVE AGENT PLURONIC F-68
ON RED BLOOD CELL HEMOLYSIS IN VITRO AND DURING

CARDIOPULMONARY BYPASS

By

Dorothy Tao—Lien Yao

Pluronic F-68 is a nonionic polyol surfactant which greatly
increases the resistance of red blood cells in buffered saline to lysis
by mechanical trauma. Further studies revealed a similar effect on red
blood cells in plasma. There was no change in the osmotic fragility
of red blood cells treated with Pluronic F-68 in vitro or in viva.
However, an increase in osmotic resistance was observed to occur during
the bypass period, irrespective of the use of Pluronic F-68. This was
presumed to occur as the result of lysis of less resistant cells in the
extracorporeal pump-oxygenator. In vitro studies indicated that the
inhibition of hemolysis by Pluronic F-68 was dose related. Although
the value of Pluronic F—68 during cardiopulmonary bypass is still not
conclusively demonstrated, it is most probable that the concentration
of Pluronic F-68 (0.35 mg./ml. of blood) in experiments in viva was too

low in these animals to have exerted a significant effect.

THE EFFECT OF THE SURFACE ACTIVE AGENT PLURONIC F-68
ON RED BLOOD CELL HEMOLYSIS IN VITRO AND

DURING CARDIOPULMONARY BYPASS

By

Dorothy Tao-Lien Yao

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology

1972

Mr.

In memory of my parents

and Mrs. Yao, Boen Ling

ii

ACKNOWLEDGEMENTS

I wish to express my appreciation and gratitude to Dr. C. C.
Morrill, my major professor, for his advice and support during my course
of study.

To Dr. R. L. Michel, my thesis advisor, I wish to express my grati-
tude for his counseling and his guidance in the preparation of this
thesis.

My sincere gratitude to Dr. A. J. Bowdler for his guidance through-
out my research project and for his instruction in scientific writing.

I am thankful to Dr. R. W. Bull and Dr. G. E. Eyster for serving
on my committee and giving useful comments and suggestions.

My thanks are extended to Mrs. M. T. Thomas for her words of
encouragement and assistance.

To the Department of Pathology, the Department of Human Medicine,
the Cardiovascular Surgery Team, and the Veterinary Clinical Laboratory
staff, I wish to express my appreciation for their provision of facili-
ties and for their c00peration in collecting specimens for the
experiments.

I am grateful for the financial support from the Public Health
Service Grant l—AOZ-AH00237-Ol which was available to me during my

graduate study.

iii

TABLE OF CONTENTS
Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . . . . . . . 3

The Physical and Surface-Active Preperties
of the Pluronic Polyols. . . . . . . . . . . . . . o . .

LO

Molecular Weight . . . . . . . . . . . . . . . . o .
Taste and Odor . . . . . . . . . . . . . . . . . . .
Chemical Stability . . . . . . . . . . . . . . . . .
Hygroscopicity . . . . . . . . . . . . . . . . . . .
Solubility . . . . . . . . . . . . . . . . . . . . .
Surface-Active Properties. . . . . . . . . . . . . .
Toxicologic PrOperties . . . . . . . . . . . . . . .
Metabolic Reactions. . . . . . . . . . . . . . . .

b53>bbwww

Experimental Use of Pluronic F—68 . . . . . . . . . . . . . 5

Intravenous Infusion of Fat Emulsions. . . . . . . . 5
Complication of Cardiopulmonary Bypass . . . . . . . 7
Splenic Vascular Perfusion . . . . . . . . . o . . o 10
Experimentally Induced Amniotic Fluid Embolism . . . 10
Cardiac Assist Devices . . . . . . . . . o . o . . . lO
Hemorrhagic Shock. . . . . . . . . . . . . . . . . . ll

MTERIALS AND METHODS O C O 0 O O O O O O I C! O O O 0 3 6 O 0 e O O 12
In vitm StUdieS O O O f 9 O B O O D O 9 O I I O O O O O I n 12

Mechanical Fragility of Red Blood Cells. . . . . . . 12
Osmotic Fragility of Red Blood Cells . . . . . . . . 16

In vivo Studies . o . . . . . . . . . . . . . . . . . . . . 17
Red Blood Cell Studies . . . . . . . . . . . . . . . 20

Plasma Hemoglobin . . . . . . . . . . . . . . 20

Osmotic Fragility of Red Blood Cells. . . . . 21

Plasma Haptoglobino . . . . . . . . . . . o . 21

RESULTS 0 O I O O O 0 O O O C O C h f O 0 O 0 O O O O O O O O I O 9 23

iv

Page
In vitra Studies. . . . . . . . . . . . . . . . . . . . . . 23

Mechanical Fragility of Red Blood Cells. . . . . . . 23
Osmotic Fragility of Red Blood Cells . . . . . . . . 27

In viva Studies . . . . . . . . . . . . . . . . . . . . . . 35

Red Blood Cell Studies . . . . . . . . . . . . . . . 35

Plasma Hemoglobin . . . . . . . . .

Osmotic Fragility of Red Blood Cells. . . . . 35

Plasma Haptoglobin. . . . . . . . . . . . . . 35

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . . 47
In vitra Studies. . . . . . . . . . . . . . . . . . . . . . 47

In viva Studies . . . . . . . . . . . . . . . . . . . . . . 48

REFERENCES I O O O O O O O O O F O O O O O O O C O O O O O O O O O 49

APPENDICES O O O O O 9 O O O O O O O O O O O O O O I O O O O O 0 O 52

A Reagents O O O O O O O O O O O O O O O O O O O O O O 52
B Calibration of Standard Curve. . . . . . . . . . . . 54
C Calculation Formula. . . . . . . . . . . . . . . . . 57

Table

LIST OF TABLES

The effects of different air pressures and concentra-
tions of Pluronic F-68 on the mechanical fragility of
red blood cells in vitra (mean percent hemolysis,
standard deviation and probability for 5 dogs). . . . . .

Red blood cell mechanical fragility in vitra: comparison
of the effects of Pluronic F-68 on red blood cells sub-
jected to mechanical stress at 100 mm. Hg. air pressure
(summary from 5 dogs) . . . . . . . . . . . . . . . . . .

Effects of various concentrations of Pluronic F-68 on

in vitna mechanical fragility of red blood cells in
plasma: summary of data from 5 dogs (air pressure -

500 mm. Hg.). . . . . . . . . . . . . . . . . . . . . . .

Comparison of plasma hemoglobin values during cardio-
pulmonary bypass with and without Pluronic F-68 in
perfus ion flUidBO I O O O G O O O O O O I I O O O l O 9 O

Osmotic fragility in viva. Comparison of the saline
concentration giving 50% hemolysis with blood samples
incubated at 37 C. for 24 hours following in viva

bypass experiments;without addition of Pluronic F-68

to perfusion fluids . . . . . . . . . . . . . . . . . . .

Osmotic fragility in viva. Comparison of the saline
concentration giving 501 hemolysis in blood samples
incubated at 37 C. for 24 hours following in viva bypass
experiments; Pluronic F-68 added to perfusion fluids. . .

vi

Page

28

30

32

37

40

41

Figure

10

11

12

13

14

LIST OF FIGURES

Apparatus for determination of mechanical fragility . . .
The pump table, front view. . . . . . . . . . . . . . . .

Mechanical fragility (in vitro): the effect of varying
air pressure and different concentrations of Pluronic
F-68 on red blood cells in buffered saline (Dog A). . . .

Mechanical fragility (in vitra): the effect of varying
air pressure and different concentrations of Pluronic
F-68 on red blood cells in buffered saline (Dog B). . . .

Mechanical fragility (in vitra): regression calculated
on data from 5 dogs; red blood cells in saline. . . . . .

Mechanical fragility (in vitro): protective effects of
Pluronic F-68 on red blood cells subjected to mechanical
stress at 100 mm. Hg. air pressure; summary data from

5 dogs. . . . . . . . . . . . . . . . . . . . . . . I . .

Mechanical fragility (in vitro): the effect of varying
Pluronic F-68 concentrations on red blood cells in plasma

Osmotic fragility (in vitro) before incubation: mean
i l SIDI in 5 dogs. I I I O I I I I O I I I I I I I I O I

Osmotic fragility (in vitro) after 24 hours incubation
at 37 0.: mean i'l S.D. in 5 dogs. . . . . . . . . . . .

Plasma hemoglobin values in 9 dogs as a function of
bypass tim I I I I I I I I I I I I I I I I I I I I I I I

Osmotic fragility (in viva) without Pluronic F-68 after
24 hours incubation at 37 C.. . . . . . . . . . . . . . .

Osmotic fragility (in vivo) with Pluronic F-68 after 24
hours incubation at 37 C I I I I I I I I I I I I I I I I I

Average haptoglobin levels during cardiopulmonary bypass
in 9 dogs I I I I I I I I I I I I I I O I I I I I I I I I

Haptoglobin correction for the effect of hemodilution

'during cardiOpulmonary bypass . . . . . . . . . . . . . .

Page
l4

19

24

25

26

29

31

33

34

36

38

39

42

42

Figure

B-2

3-3

Page

Plasma hemoglobin standard curve. . . . . . . . . . . . . . 54

Calibration curve for estimation of haptoglobin
concentration . . . . . . .

I I I I I I I I I I I I I I I I 55

Haptoglobin calibration curve (comparison human pooled
serum and dog pooled plasma). . . . . . . . . . . . . . . . 56

viii

INTRODUCTION

The maintenance of cardiOpulmonary circulation by an artificial
apparatus is essential to certain types of cardiovascular surgery.

These include the replacement of heart valves and the implantation of
artificial heart-assist devices.

Up to the present time there are still many problems associated
with cardiOpulmonary bypass. In previous clinical and experimental
studies it has been demonstrated that blood perfusion systems cause
destruction of a fraction of the red cells with the immediate appearance
of free plasma hemoglobin. Other red cells, while escaping rapid
destruction, may nonetheless sustain a significant degree of injury
resulting in shortened cell life span and premature senescence.. As a
consequence, an extracorporeal perfusion system may result in anemia
continuing for days or weeks after an Operation. This condition has
been characterized by reduction of the packed cell volume and total
blood hemoglobin and an increased reticulocyte count. Mbrphologic
changes of the red blood cells has been observed, including anisocytosis,
spherocytosis and fragmentation associated with increased mechanical
fragility of the red blood cells. In addition, high plasma hemoglobin
levels have been associated with impairment of renal function (Brinsfield
et al., 1962; Galletti et aZ., 1965; Kusserow et aZ., 1965, 1966).

Pluronic F-68 has been used previously to stabilize fat emulsions
for intravenous use, to prevent fat embolism and to diminish hemolysis

during cardiOpulmonary bypass. This investigation was designed (a) to

l

2
determine the effectiveness of Pluronic F-68 in reducing hemolysis
consequent to experimental surgery in the dog involving the use of
cardiopulmonary bypass devices, and (b) to investigate in vitra the

possible mechanism by which Pluronic F-68 acts to stabilize erythrocytes.

LITERATURE REVIEW

The Pluronic polyols are ethylene oxide-polypropylene glycol
condensation products. These comprise a series of closely related
polymers with a unique molecular structure and with the general empirical

formula: H 0 (C H C H O)a (C H C H

2 2
CH3

2 O)b (C H2 C H2 O)CH.

These are nonionic surfactant polyols. Because the hydrophobic-
hydrOphilic ratio of the Pluronic polyols can be varied by small, con—
trollable amounts, the resulting graded structures provide a wide range
of properties. This variability makes it possible to meet particular
surface-active requirements (BASF Wyandotte Corporation, Publication,

1971a).

The Physical and Surface—Active Properties of the Pluronic Polyols

 

Molecular Weight. A unique preperty of the Pluronic polyols is their

 

unusually high molecular weight compared to many common surface-active

agents. The approximate molecular weight of Pluronic F-68 is 8,350.

Taste and Odor. Pluronic F-68 is unique among surface-active agents in

 

being practically tasteless and odorless. Animal feeding tests show

that Pluronic F-68 is readily accepted by animals when mixed with feed.

Chemical Stabiligy. Due to the stable ether linkage in the Pluronic

 

molecule, these materials are extremely stable to acids and are not pre-
cipitated by most metallic ions. They are also stable in alkaline

solutions.

4
Hygroscopicity. Pluronic F-68 is relatively non-hygrosc0pic and is free-
flowing even after being exposed to air for long periods of time. This

is typical of solid Pluronic polyols.

Solubility. The Pluronic polyols are more soluble in cold water than
in hot, owing to hydrogen—bonding of water with the many ether oxygen
atoms. They are more soluble in dilute mineral acid than in water
because of the formation of oxonium ions. Pluronic polyols can be
dissolved in organic solvents, in which they are normally insoluble,

by the use of a coupling agent.

Surface-Active Properties. Pluronic F—68 in solution has unusual surface-
active prOperties. Its surface tension is lower than that of water
combined with the best wetting agents. This characteristic holds over

a wide temperature range and is unaffected by change in pH. Pluronic
polyols exhibit a strong emulsifying effect and act as excellent

defoamers.

Toxicologic Properties. Studies have been made of the acute toxicity
of Pluronic F-68 in dogs, rats, mice, rabbits and guinea pigs, to which
aqueous solutions were administered by stomach tube. The data indicate
that Pluronic F-68 does not produce acute toxic effects. Additional
tests, carried out to assess possible chronic oral toxic effects over a

period of 2 years, showed no toxic manifestation (Carr, 1951, 1952).

Metabolic Reactions. In vitra and in viva experiments with human beings,
dogs and rats have shown no alteration in the molecular weight or
structure of Pluronic F-68. The Pluronic F-68 was recovered unchanged
in the urine; recovery was essentially quantitative within 24 hours

after administration. When ingested orally, Pluronic F-68 was recovered

5
quantitatively from the feces, showing that the Pluronic F-68 is neither
absorbed into the body nor is it metabolized (May, 1958). Pluronic F—68
had no effect on the absorption of the anionic dye, phenol red, from the
colon of anesthetized rats (Meyer at aZ., 1957).

Information accumulated since 1952 indicates that the toxicologic
prOperties of these products also vary in a regular and predictable
manner, according to the relative size of the polyoxyethylene and
polyoxypropylene portions of the molecule and the total molecular weight.
The prOperties of the Pluronic polyols suggest their use in several
fields where low toxicity is necessary. These include cosmetics, pharma-
ceuticals, drugs and certain foods. The effect of grade F-68, one of
the oldest and best known of the Pluronic series, has been studied in a
number of medical applications (BASF Wyandotte Corporation, Publication,

1971b).

Experimental Use of Pluronic F-68

 

l. Intravenous infusion of fat emulsions.
2. Complications of cardiOpulmonary bypass, including:
a. Fat embolism
b. Hemolysis
c. Blood viscosity
d. Platelet aggregation and adhesiveness
3w Splenic vascular perfusion.
4. EXperimentally induced amniotic fluid embolism.

5. Cardiac assist devices.

6. Hemorrhagic shock.

Intravenous Infusion of Fat Emulsions. Many emulsifying agents have
been utilized in the intravenous administration of fat. However, these

agents have proved to be unsatisfactory. Soya phosphatides ("lecithin")

6

were introduced by Stare in 1943 as an emulsifying agent in place of
egg phosphatide. Shortly thereafter it was found that the use of a small
amount of a co-emulsifier permitted a reduction in the amount of phospha-
tide required. In 1955 Pluronic F—68 was selected as the co—emulsifier.
It is a synthetic, nonionic, extremely water-soluble compound which
enhances the physical stability of the emulsion (Meyer at aZ., 1955;
Waddell et al., 1955).

In 1957, Waddell and his co-workers initiated the clinical use of
a nonphosphatide (Pluronic F-68-alcohol) emulsion. Their results showed
an unexpectedly high incidence of the immediate or "colloid" reaction
which was manifested by clinical signs such as back pain, dyspnea,
flushing, and abdominal pain. This "colloid" reaction can be eliminated
by the infusion of a mixture of Pluronic F-68, alcohol and glycerol
prior to the first fat infusion. A fat emulsion stabilized with Pluronic
F-68 for intravenous use does not cause this reaction.

Walter et al. (1957), in studying the effect of intravenous admin-
istration of Pluronic F-68 in the dog, stated that this agent produced
no change in the electrophoretic mobility of serum lipoproteins and no
prolongation of clotting time of the blood. Experimentally, combined
"Phosphatide-Pluronic" preparations given intravenously resulted in
an increase in the speed of electrophoretic migration of the lipoproteins
and prolongation of blood-clotting time. Since intravenous administra-
tion of Pluronic alone caused none of these changes, it was concluded
that the soybean phosphatide was responsible for the observed changes.

A side effect frequently reported following daily infusions of
intravenous fat emulsion is a significant drop in hemoglobin concentra-
tion. The severity of the anemia produced in this way has been

correlated with the emulsifier system utilized. Hartwig (1961) carried

an"

 

 

'q
.1:
I

7
out an in vitra comparison of hemolysis following incubation of erythro-
cytes in experimental fat emulsions. Pluronic F-68 consistently pro-

duced less hemolysis than any of the other emulsifier systems.
Complication of CardioPulmonary Bypass

Fat embolism. Pluronic F-68 has both a nonionic quality and excel-
lent emulsifying prOperties and has been employed to stabilize parenteral
fat emulsions. Adam at al. (1959) reported a study testing the effective-
ness of Pluronic F-68 in preventing the formation of fat emboli during
prolonged cardiOpulmonary bypass. Their results indicated that fat
emboli and lipuria can be largely prevented if a nonionic surface-active
agent (Pluronic F-68) is employed. They also demonstrated that Pluronic
F-68 has no effect either physiologically or on the function of the
pump-oxygenator.

Wright at al. (1963) indicated that fat globulinemia which develOps
during cardiOpulmonary bypass can be minimized by the addition of

Pluronic F-68 to the circulation.

Hemolysis. O'Neill and Collins (1965) designed a group of experi-
ments to evaluatethe effects of various agents on hemolysis occurring
in vitra during the use of the bypass apparatus. Pluronic F-68 in a
concentration of 0.6 mg./m1. of blood was added to the prime solution.
The total perfusion time was 2 hours. The results of these studies
indicated that the effect of Pluronic F—68 on the level of plasma hemo-
globin was inconsistent. A lower concentration of plasma hemoglobin
was noted, but the difference was not statistically significant.

Miyauchi at al. (1966) and Hymes at al. (1968) reported experimental
studies in which they compared the effect of various agents in the prime

solutions with Pluronic F-68. All of the data indicated that the

8
administration of Pluronic F-68 after the end of persufion markedly
improved capillary circulation and caused an immediate increase both in
venous return and arterial pressure. The most significant difference
between Pluronic and non-Pluronic groups was noted in plasma hemoglobin
levels. Miyauchi also demonstrated that fat embolism and fibrin deposi-
tion on the filter of the oxygenator were greatly reduced by use of
Pluronic F-68.

Wells et a1. (1968) performed studies in vitra to evaluate the
effects of polymethylsiloxane as a defoaming agent. They showed that
it greatly reduced the resistance of red blood cells to lysis by mechanical
trauma. The addition of the surfactant agent (Pluronic F-68) prevented
the hemolytic process without interfering with the action of the anti-
foam material. Similar studies of plasma showed the reduction of hemo-
lytic effects of the Pluronic F—68 was to some extent mediated by the
plasma.

In 1970 the effects of Pluronic F—68 were evaluated in 103 patients
during cardioPulmonary bypass. The patients were divided into control
and treatment groups. The final concentration of Pluronic in the pump
prime was approximately 1 mg./ml. The results indicated that Pluronic
F-68 significantly protected the red blood cells from hemolysis at all
times during perfusion up to 90 minutes. After that time the effect
diminished gradually. Serum potassium levels were significantly lower
in the Pluronic group, possibly reflecting the decrease in hemolysis
(Danielson et al., 1970).

During the past few years, vitamin E and Pluronic F-68 have been
employed in intracardiac surgery by Tamiya and his associates (1970),
wmo noted their marked suppressive effects on hemolysis. They stated

that vitamin E, when directly added to the pump perfusion, was

9
ineffective. However, daily intramuscular administration prior to surgery
reduced the hemolysis significantly. Various doses of Pluronic F-68
were employed during perfusion in human subjects. These included con-
centrations of 0.6 mg./m1., 1.0 mg./ml. and 2.0 mg./ml. of the circulating
blood volume. Hemolysis was reduced to about 80% of that observed in
the control in the 0.6 mg./m1. group. It was reduced even further in
the 1.0 mg./ml. and 2.0 mg./ml. groups. Postoperative laboratory studies
generally revealed advantages in the Pluronic group, with the exception
that higher blood urea nitrogen levels were observed in some members of
the 2.0 mg./m1. group. Therefore, it was recommended that the level of
Pluronic F-68 not exceed 1.0 mg./ml. of circulating blood volume during

cardiopulmonary bypass.

Blood Viscosity. Increased blood viscosity during bypass reflects
increased microaggregation, alteration in the physical properties of
erythrocytes, increased fibrinogen, and hemoconcentration (Replogle at
al., 1967). EXperimental studies by Grover at al. (1969) indicated that
the intravenous injection of Pluronic F-68 prior to bypass lowered blood
viscosity. Further reduction in viscosity occurred due to the hemo-
dilution produced by the solution used to prime the bypass apparatus and
persisted during the first hour of perfusion with no change in hemato-

crit values.

Platelet Aggregation and Adhesiveness. Platelet aggregation is an
important causative factor in the development of microembolism.whichv
commonly occurs during extracorporeal circulation. Grover at al. (1969)
gave Pluronic F-68 by slow intravenous infusion at the rate of 25 mg./kg./
hour to Open heart surgery patients. Platelet adhesiveness was reduced

from 53% prior to intravenous administration to 15% ten minutes afterward.

10
Grover concluded, therefore, that Pluronic F-68 should decrease platelet
aggregation during perfusion.
Tamiya at al. (1970) indicated that this agent is also effective in

preventing a reduction in the platelet count during perfusion.

Splenic Vascular Perfusion. P1uronic_F-68 has been shown to decrease

 

vascular resistance in a series of 10 porcine spleens perfused with
human blood. The reduction in vascular resistance during heterologous
perfusion is comparable to that noted during homologous perfusion

(Moore, 1968).

Experimentally Induced Amniotic Fluid Embolism. The effect of intra-

 

arterial human amniotic fluid embolism on the mesenteric microcirculation
of the rabbit has been studied. Amniotic cellular debris results in an
aggregation of cellular material that may obstruct the microcirculation
and cause a drOp in blood pressure. Infusion of Pluronic F-68 prior to
human amniotic fluid embolism prevented impairment of the microcircula—

tion and shock (Hymes at aZ., 1970‘.

Cardiac Assist Devices. Pluronic F-68, chemically coupled with heparin

 

and epoxy, is one of the most promising anti-thrombic agents which has
been developed. This compound has been used to coat surfaces in
circulatory-assist devices. The mechanical characteristics of this
compound show the advantages of extreme durability and compatibility
with saline. The chemically incorporated heparin epoxy polymers of this
material have been shown to be nonhemolytic, harmless to platelets and
blood cells, to have no effect on plasma proteins, and to have only a
small effect on plasma enzymes. Most significant was the fact that the
heparin was not readily depleted from these polymers. Attempts at leach-

ing it were ineffective (Salyer et al., 1971; Gott at al., 1971).

11

Hemorrhagic Shock. The treatment of hemorrhagic shock is directed toward

 

restoration of the microcirculation. Pluronic F-68 has been demonstrated
to increase the percentage of animals surviving experimentally induced
shock syndromes. Restoration of renal function occurred in a signifi-
cantly higher percentage of these animals than in the untreated control
groups. It was reported in these studies that blood surface tension was

markedly reduced in the animals treated with Pluronic F-68 (Hyme at aZ.,

1971).

MATERIALS AND METHODS

The experimental studies were divided into 2 categories: (1) evalua-
tion of the effects of Pluronic F-68 on red blood cells in vitra, and
(2) evaluation of the effects of Pluronic F-68 on red blood cells during

cardiopulmonary bypass.

In vitra Studies

 

Mechanical Fragility of Red Cells. The mechanical fragility of the red
cells was measured by an adaptation of the method of Adreason (1964) in
which a dilute suspension of red cells is forced under selected condi-
tions through the orifice of a chromatography spray.1 As with other
methods of measuring mechanical fragility, the precise forces leading
to destruction of red cells are incompletely understood. Hemolysis is.
presumed to be the result of turbulent flow at the orifice. Mechanical
resistance was evaluated by measuring the percentage of hemolysis which

occurred following stress.

Source of Specimens. Fresh blood samples were obtained from 5 dogs

 

in sterile heparinized syringes. Packed cell volumes were determined
immediately using the microhematocrit method with centrifugation at
approximately 10,000 g for 5 minutes in an International Micro-Capillary

Centrifuge.2 Ten percent suspensions in phosphate—buffered saline were

 

1Arthur H. Thomas Co., Philadelphia, Pa., U.S.A.
2International Equipment Co., Needham Hts., Mass., U.S.A.

12

13
then made by appropriate dilution. All determinations were made at

ambient room temperature, approximately 24.: 2 C.

Experiment I. Four l-ml. aliquots of each of the 10% cell suspensionsi

 

were further diluted to 100 ml. with phosphate-buffered saline solution
giving final dilution of 1 in 1000. Pluronic F-68 was added to each of
these to yield final concentrations of: (a) 1.0 mg./ml. Pluronic F—68,
(b) 3.0 mg./ml. Pluronic F-68, and (c) 5.0 mg./ml. Pluronic F-68. The

red blood cells suspended in buffered saline alone served as a control.

The apparatus which was used to determine mechanical fragility is
illustrated in Figure 1.

The air line tubing was connected both to a mercury manometer and
to the spray tube. This spray tube, made of borosilicate.g1ass, is
permanently attached to a molded phenolic plastic screw cap which fits
commercial narrow-mouth bottles.

A thumb—controlled air vent is located in the rear of the spray
tube. This allows instantaneous control of the spray by variable closure
of the vent. The spray tube projects a few millimeters inside the
mouth of the separatory funnel which delivers its contents into a test
tube.

To provide a measure of the relationship between traumatic hemolysis
and air pressure, the latter was adjusted in various experiments to 100,
200, 300, 400 and 500 mm. Hg. The diluted red blood cell suspensions,
were well mixed before each spraying. Each sample was sprayed into the
separatory funnel, and about 5 ml. were collected in the test tube.
Triplicate samples were run, and the fraction of the cells lysed was

determined from the measured hemoglobin liberated into the supernatant

14

 

 

L_____—__ , __,i_-,_,_ __ _l --.___ __-

Figure 1. Apparatus for determina-
tion of mechanical fragility. (A) air
valve; (B) air line tubing to compressed
air; (C) mercury manometer; (D) spray
apparatus; (E) separatory funnel.

15
obtained by centrifuging each sample at 2000 rpm for 7 minutes in the
International Centrifuge.l
Damage produced by mechanical stress to the red blood cells was

measured as follows:

(a) The supernatant from the unstressed red blood cell suspension
served as a blank.

(b) The test sample was the supernatant from the air-sprayed
suspension.

(c) 100% hemolysis was estimated by adding 3 drOps of ammonia
to a sample of the untreated initial cell suspension and
centrifuging as above.
Aliquots of blank, sample, and total hemolysis samples were measured
for absorbance at 417 nm. using a Beckman D.B. SpectrOphotometer.2

Mechanical fragility was calculated according to Shen at al. (1944)

as follows:

Mechanical Fragility - log-Z-a) %, where

a - absorbance of blank, b - absorbance of sample, and c - absorbance of

the totally hemolyzed sample.

Experiment 11. Red blood cells were prepared in 3 ways for determina-

tion of mechanical fragility:
(a) Suspended in phosphate-buffered saline solution.

(b) Suspended in 5 mg./ml. of Pluronic F-68 with phosphate-
buffered saline solution.

(c) Suspended in 5 mg./m1. of Pluronic F-68 for 3 hours at
which time they were washed twice in isotonic saline and
resuspended in phosphate-buffered saline.

The red blood cell suspensions were sprayed using an air pressure

of 100 mm. Hg.

 

1International Equipment Co., Needham Hts., Mass., U.S.A.

2Beckman Instrument Co., Palo Alto, Calif., U.S.A.

 

III!

‘ 1

ML.

16

Experiment III. Red blood cells were suspended in plasma for
determination of mechanical fragility as follows:

(a) Suspended in plasma.

(b) Suspended in plasma containing 1.0 mg./m1. Pluronic F-68.

(c) Suspended in plasma containing 3.0 mg./m1. Pluronic F—68.

(d) Suspended in plasma containing 5.0 mg./ml. Pluronic F—68.
These plasma suspensions were sprayed at an air pressure of 500 mm. Hg.
In Experiments II and III, mechanical fragility was evaluated in the

same way as in Experiment I.

Osmotic Fragility of Red Blood Cells. Osmotic fragility of the red blood

 

cells was determined by a modification of the method of Dacie (1938).
The resistance of the red blood cells to hemolysis was measured by
suspending the cells in decreasing concentrations of phosphate-buffered
saline solution and subsequently determining the percentage of hemolysis

from the supernatant hemoglobin.

Source of Specimens. Five normal dogs were used as a source of

 

blood. Twenty milliliters of heparinized blood were obtained from each
by venipuncture using sterile technique.

Quantities of 5.0, 15.0, and 25.0 mg. of Pluronic F—68 were dissolved,
respectively, in 3 tubes, each containing 0.5 ml. of phosphate-buffered
saline. A fourth tube of the saline served as a control. Four and one-
half milliliters of blood were transferred into each of these 4 tubes_
to give final concentrations of 0, 1.0, 3.0 and 5.0 mg./m1. These tubes
werezmixed'well by gentle inversion and allowed to stand at room temperature
for’ 30 minutes. The osmotic fragility of each of these samples was

determined both immediately and after incubation at 37 C. for 24 hours.

17-

Method. The range of concentration of buffered saline varied from
the osmotic equivalents of 0.10% to 1.0% NaCl-PO4 in 0.05% increments.
The concentration of each buffered saline solution was determined by
freezing—point depression using an osmometer.l

The test was carried out by adding 0.1 m1. of blood to 5 m1. of
each buffered saline solution. The tubes were mixed well and incubated
at room temperature for 1-1/2 hours. They were subsequently centrifuged
at 2,000 rpm for 7 minutes. The absorbance of each supernate wa8~
measured at 540 nm. in a Coleman Junior SpectrOphotometer.2 One percent
saline was used as a blank. The tube containing 0.1 ml. blood in water
represented 100% hemolysis.

The percentage of hemolysis in each tube was calculated as follows:

Absorbance of supernate
Absorbance of 100% hemolysis

 

Percent Hemolysis - x 100.

Percent hemolysis versus concentration of buffered saline was then plotted

for each specimen.

In viva Studies

 

Source of Specimens. Experimental dogs used for the surgical pro-
cedures were generally young mongrels weighing between 20 and 35 kg.
They were examined clinically and determined to be in good health.

The dogs were anesthetized with a gas anesthetic mixture of halo-
thane, N20, and 02. A slow intravenous drip of 5% dextrose in water was

given by catheter into the cephalic vein, and this route was used.also

for drug administration. Heparin, 3.5 mg./kg., was used as an

 

1Advanced Instruments, Inc., Newton Highland, Mass., U.S.A.

2Coleman Instruments, Inc., Maywood, 111., U.S.A.

l8
anticoagulant. Large-size catheters for venous drainage were placed in
the superior vena cava and inferior vena cava through the right atrium.
The left femoral artery was cannulated for arterial perfusion and the
right femoral artery for continuous blood-pressure recordings. Heart
action was monitored continuously with a DR8 Recorder.l Electrocardio-
graphic tracings were obtained at lS-minute intervals.

The arterial and venous cannulae were connected to the arterial and
venous lines from the heart-lung machine. The blood flowed by gravity
into a disposable miniprine oxygenator.2 The roller type Med-Science
Electronic3 was used as a blood pump (Figure 2).

Oxygen was bubbled freely into the oxygenator column of blood at

3 liters OZ/liter blood flow, and CO at 300 cc./minute. The perfusion

2
flow rate was maintained at 80 ml./kg. of body weight/minute. During
cardiOpulmonary bypass, all lost blood was aspirated by auction and
returned to the heart-lung machine in order to minimize blood loss from
the system.

In all experiments the priming solution consisted of 1000 m1. of
lactated Ringer's solution containing 1 Gm. of Pluronic F—68 and 20 ml.
bicarbonate. This was equivalent to 0.35 i 0.05 mg./ml. of Pluronic
F-68 when the priming volume was added to the circulating blood volume.

Pluronic F-68 was used in 6 of the perfusions while the other 3
procedures served as controls.

Heparainized blood was taken from the arterial catheter to determine

blood pH, pCO packed cell volume, and hemoglobin concentration,

2’ p02’

 

1Electronics for Medicine, Inc., White Plains, N.Y., U.S.A.
2Artificial Organs Division, Travenol Labs, Inc., Morton Grove,
IllI’ UISIAI

3Med-Science Electronic, St. Louis, Mo., U.S.A.

 

l9

 

 

 

 

 

 

 

 

 

 

Figure 2. The pump table, front
view. Resting on the pump table are
the 3 pumps. A disposable oxygenator
bag hangs in the frame.’

20
and esophageal temperature was measured at intervals throughout the

experimental procedure.

Red Blood Cell Studies. In order to evaluate the effect of Pluronic F—68

 

on the red blood cells, blood samples were obtained prior to perfusion
and during perfusion at lS-minute intervals throughout the bypass for
1 hour. Subsequent samples were obtained at 30-minute intervals.

The following determinations were carried out:

Plasma Hemoglobin. Plasma hemoglobin was determined by Fielding's

 

method (1958). The procedure is based on the use of an Orthotolidine
reagent.1 Colorless orthotolidine acts as an oxygen acceptor and is
oxidized to a blue reaction product by the peroxidase activity of

hemoglobin.

Method. A quantity of0.02m1. plasma was added to 10 m1. of Ortho-
tolidine solution and mixed well. In exactly 8 minutes, the absorbance
of the mixture was measured against a distilled water blank at.630 nm.
in a Coleman Junior Spectrophotometer. The concentration of plasma

hemoglobin was derived from a prepared standard curve.

Calibration Standard Curve. The concentration of hemoglobin in a

 

completely hemolyzed red blood cell mixture was determined by the cyan?
methemoglobin method. This mixture was then diluted to apprOpriate con—
centrations with distilled water which ranged from 10 to 100 mg./d1.
Plasma hemoglobin determination was made on each of the standard dilutiOns
as described above and a standard curve was constructed by plotting

absorbance against known hemoglobin concentration.

 

1Hematest tablet R, manufactured by Ames Company, Inc., Elkhart,
Ind., U.S.A.

21
Osmotic Fragilipy of Red Blood Cells. The osmotic fragility of red
blood cells was determined by a modification of the method of Dacie

(1938) as described previously.

Plasma Haptoglobin. Plasma haptoglobin was measured using the method
of Owen (1960). This is a simple colorimetric procedure based on the

peroxidase activity of haptoglobin-methemoglobin complexes.

Method. This procedure was performed at room temperature (24 to
25 C.). One volume of plasma was diluted with 4 volumes of saline. One
milliliter of the diluted plasma was added to 1 m1. of methemoglobin
solution for the unknown and 1 ml. to 1 m1. of water for the blank,
mixing well. One tenth milliliter of the plasma-methemoglobin mixture
was added to 5 m1. guaiacol reagent. For the blank, 0.1 m1. of the
plasma-water mixture was added to 5 ml. of guaiacol reagent. One milli-
liter of freshly made 0.05 M hydrogen peroxide was then added to each
tube. The tubes were thoroughly mixed quickly and placed in the dark
for color development. This is important as the color fades slowly when
exposed to bright daylight. After exactly 8 minutes, the absorbance of
the unknown was read against its blank in a Beckman DB Spectrophotometer
at a wavelength of 470 nm. These readings should be completed within
4 minutes after the 8-minute reaction time. The concentration of hapto-
globin was obtained from a calibration curve and was expressed in terms

of bound methemoglobin.

Construction of a Calibration Curve. One milliliter of methemoglobin

 

solution was placed into each of 11 tubes. Pooled normal plasma, free
from hemoglobin, was added to each tube, respectively, in increasing

amounts of 0.0, 0.1, 0.2...to 1.0 ml. in the last tube. The volume in

22
each of the 11 tubes was made up to 2.0 ml. with saline. Each sample
and a blank prepared with 0.1 m1. NaCl and 5 m1. guaiacol was then
submitted to the procedure described above for the unknown. Absorbance
was plotted against plasma content for each tube. The inflection point
indicates the amount of undiluted pooled plasma which just binds all
the methemoglobin present. Thus the reading at this point corresponds
to a methemoglobin-binding capacity of 50 mg./dl. This was marked on
the abscissa, and the distance between this point and the origin was
recalibrated proportionately to give readings of haptoglobin concentra—
tion. Samples of test plasma, however, were normally diluted 1 in 5
so that, with these, the reading which corresponds to haptoglobin content

should be multiplied by 5.

RESULTS

In vitra Studies

 

Mechanical Fragility of Red Blood Cells

Experiment I. The results of mechanical fragility tests on Dog A
and Dog B using various concentrations of Pluronic F-68 and air pressure
are summarized in Figures 3 and 4. In Dog A red blood cell mechanical
fragility was related to both the concentration of Pluronic F-68 and
air pressure. When plotted on probability paper, the curves relating
hemolysis to air pressure for Dog A appeared linear, suggesting that
the cumulative hemolysis curve was a simple sigmoid function, and that
mechanical fragility was distributed through this population as a normal
(Gaussian) distribution. The curves for Dog B were similar except that
they tended to plateau at the higher pressures, suggesting that the red
blood cell pOpulation in that individual was less uniform with respect
to mechanical fragility. In the remaining 3 dogs, the curves plotted
on probability paper were predominantly linear, but showed a tendency
to plateau formation at higher pressures of a lesser degree than with
Dog B.

The results of the whole series are recorded in Figure 5. The
mechanical fragility regression line was calculated from the percent
probit of the data for 5 dogs. This indicated that Pluronic F-68 in

phosphate-buffered saline significantly protected the red blood cells.

23

24

80 n

60*-
50*
40*

20 P

% Hemolysis

 

2 ' ,-'. ./ —-— Control
/' .mg-.. 1 mg./ml.
0.5 " -°°o--- 3 mg./m1.

0.2 . -".—" 5 mgo/mlo

0.1 P
0.05-

 

0.01

 

100 200 300 400 500
mm. Hg.

Air Pressure

Figure 3. Mechanical fragility (in vitra): the effect of varying
air pressure and different concentrations of Pluronic F-68 on red blood

cells in buffered saline (Dog A).

90

80

70

60
50

40

30

20

10

% Hemolysis

0.5

0.2

001
0.05

0.01

Figure 4.

 

25

 

 

/ . /". -.-' Control
/ ‘I /.'
j; .' “"“ l mg./m1.
I 4" o/
I..°../ ""0". 3 mg./ml.
7 '°-"“- 5 mg./ml.
4 I I l———‘-——--
100 200 300 400 500

mm. Hg.
Air Pressure

Mechanical fragility (in vitra): the effect of varying

air pressure and different concentrations of Pluronic F-68 on red blood
cells in buffered saline (Dog B).

26

 

 

 

I
90 "
80 '
70 .
60 p
50 -
40 r
30 r
3 20 -
m
>.
F1
é .
:3 10
{J
H
e s '
H
9.;
es
2 P
1 b '-—- Control
0 5 . ‘”"' l mg./m1. Pluronic F-68
0 2 , '“°” 3 mg./m1. Pluronic F-68
0.1 - -°-— 5 mg./m1. Pluronic F-68
0.05 .
0.01 .5 a n . .
100 200 300 400 500
mm. Hg.

Air Pressure

;Figure 5. Mechanical fragility (in vitra): regression calculated
on data from 5 dogs; red blood cells in saline.

27
The coefficient of correlation of these regression lines ranged from
0.90 to 0.94.
The significance of the effects of different air pressures and
concentrations of Pluronic F-68 was calculated by Students' 5 test: the
data showed a significant difference between the untreated and treated

red blood cells as indicated in Table 1.

Experiment II. Figure 6 and Table 2 show the effect on mechanical
hemolysis of variations in the suspension medium. Red blood cells
suspended in Pluronic F-68 in buffered saline were protected to a sig-
nificant degree. By contrast, red blood cells which were suspended.in
Pluronic F-68 and buffered saline for 3 hours and then washed with iso-
tonic saline before being subjected to mechanical stress were not signifi-
cantly protected. Although there was some reduction in hemolysis in the
latter cells as compared with controls, the differences were not

significant (P>O.l).

Experiment III. The results of mechanical fragility testing of red

 

blood cells suspended in plasma are given in Figure 7 and Table 3. A
solution of Pluronic F-68 in concentrations of 3.0 mg./ml. or 5.0 mg./m1.
of plasma significantly reduced the mechanical fragility of red blood
cells as compared with untreated controls (both P<0.001). Much less
protection was afforded by a concentration of 1.0 mg./m1. of plasma

(P-0.05).

Osmotic Fragility of Red Blood Cells. The results of in vitra osmotic

 

fragility tests are given in Figures 8 and 9. There was no significant
difference in the osmotic fragility of cells treated with Pluronic F-68

as compared with controls when tests were performed soon after treatment

Table l.

28

The effects of different air pressures and concentrations of

Pluronic F—68 on the mechanical fragility of red blood cells
in vitra (mean percent hemolysis, standard deviation and
probability for 5 dogs)

 

Pluronic F-68

Air Pressure (mm. Hg.)

 

 

Concentration 100 200 300 400 500
Control % Hemolysis 11.20 25.76 39.72 55.14 63.10
S.D. 2.32 7.60 9.88 7.44 7.48
1.0 mg./ml. % Hemolysis 3.26 12.50 24.52 40.60 49.62
S.D. 0.84 6.94 13.22 9.69 8.70
t 7.19 2.88 2.06 2.66 2.63
P P<0.001 0.05>P> 0.1>P> 0.05>P> 0.05>P>
0.02 0.05 0.02 0.02
3.0 mg./m1. % Hemolysis 2.72 10.00 21.16 33.44 44.10
S.D. 0.76 7.36 11.76 11.33 12.96
t 6.23 3.33 2.71 3.58 4.65
P P<L001 0.01>P> 0.S>P> 0.01>P> 0.01>P>
0.001 0.02 0.001 0.001
5.0 mg./m1. % Hemolysis 2.04 6.06 14.96 22.86 30.24
S.D. 0.68 2.63 4.46 7.88 9.08
t 8.47 5.48 5.11 6.67 4.74
P P<0.001 P<0.001 P<0.001 P<0.001 0.01>P>
0.001

 

Degrees of Freedom - 8.
S.D. - Standard Deviation.

t - as determined by Students' p test.

P - Probability.

 

29

 

 

 

40 '
T
35 ’
30 '
25 - I?
m
-H
(D
33’ .
a 20
:1: x
as
15 »
.II.
10 '
5 - -7 I
0
F—**—d Cells suspended in buffered saline (control).
|—-X-—4 Cells suspended in Pluronic F-68 (5.0 mg./m1. in buf-
fered saline) for 3 hours and saline-washed twice before
being subjected to mechanical stress.
t—«o—q Cells suspended in Pluronic F-68 (5.0 mg./ml. in buffered

saline).

Figure 6. Mechanical fragility (in vitra):' protective effects of
Pluronic F-68 on red blood cells subjected to mechanical stress at 100
mm. Hg. air pressure; summary data from 5 dogs.

30

Red blood cell mechanical fragility in vitra: comparison of

the effects of Pluronic F-68 on red blood cells subjected to
mechanical stress at 100 mm. Hg. air pressure (summary from

5 dogs)

Table 2.

 

 

5.0 mg./ml.
.F—68-saline 5.0 mg./m1. F-68
wash before in suspension
Control test medium
Pheaaxn % Hemolysis 25.28 18.50 4.24
S.D- 13.28 13.01 1.14
t 0.8155 3.5298
c1..1E . 8 8
1’ P>0.l 0.01>P>0.001
No Yes

S :L gnificant

 

31

 

 

 

100 . J
90 ' 7h , :13.D.__
‘I'
80 ' I. T—
._ r
70 I -u
60 r
U)
H
U)
53‘ so _
O
8
:1:
N 40 »
3o .
20 »
10 I
o

 

 

 

 

 

 

 

 

 

 

Control 1 mg./m1. 3 mg./ml. 5 mg./m1.

Figure 7. Mechanical fragility (in vitra): the effect of varying
Pluronic F-68 concentrations on red blood cells in plasma (air pressure
3 500 mm. Hg.).

32

 

 

Table 3.‘ Effects of various concentrations of Pluronic F—68 on in vitra
mechanical fragility of red blood cells in plasma: summary
of data from 5 dogs (air pressure = 500 mm. Hg.)

Control 1.0 mg./ml. 3.0 mg./ml. 5.0 mg./m1.
Mean % Hemolysis 94.82 89.22 79.96 75.08
S .D. 4.32 3.57 6.58 5.33
t 2.308 6.066 6.432
d . f . 8 8 8
P P=0.05 P<0.001 P<0.001
Significant Yes Yes Yes

 

 

 

 

33

 

 

 

 

 

 

 

 

 

100 -
n
I F"—4 Control
90 ~ 1 A
F—{*—{ l mg./m1. Pluronic F—68
-I
P—A-4 3 mg./m1. Pluronic F-68
80 »
I—-X-—1 5 mg./m1. Pluronic F-68
70 I 1'
C
60 ' O A; x
m
‘74
U)
>.
'3 5 .IN. L
E, 50 - 1
m L . dL
N C-
? .'
40 ' T
a
0 n
30 > ' T
20 . ' '1 I_
I II I
10 I 1’ 1 ]:‘I
0 ‘ ‘ 4‘ ‘ 4 EL;—
71 0.40 0.45 0.50 0.55 0.60
% NaCl—PO4

Figure 8. Osmotic fragility (in vitra) before incubation: mean

i 1 S.D. in 5 dogs.

100' p

 

34

 

 

 

 

 

 

 

 

 

I I-—-I—-I Control
90 I'
1 J. T I—O—‘I 1 mg./m1. Pluronic F-68
" F—e-d 3 mg./m1. Pluronic F—68
80 I
. I-—-X—-i 5 mg./m1. Pluronic F-68
0
ll
70 -
X
.IL I
co I-
"<3 60
>~.
r-4
8 .-
a)
m 1
N I"
so '
I
- I
40 ‘ ’
I
A
X
30 I _L L
I.
20 D
I
10 - I T
I 1 Iii
0 __. 1 up? L I
0.40 0.45 0.50 0.55 0.60
7. NaCl-PO4
Figure 9. Osmotic fragility (in vitra) after 24 hours incubation

at 37 C.: mean :I-_ l S.D. in 5 dogs.

35
(P>0.l) (Figure 8). If cells were incubated with Pluronic F-68 for 24
hours at 37 C., osmotic fragility was somewhat reduced, but the differ-

ences were not statistically significant (P>0.l) (Figure 9).

In viva Studies

 

Red Blood Cell Studies

 

Plasma Hemoglobin. The average plasma hemoglobin concentrations

 

throughout the period of cardiopulmonary bypass are presented for 9 dogs
(Figure 10). There was no significant difference between plasma hemo-
globin values in animals treated with Pluronic F-68 and those in untreated

controls (Table 4).

Osmotic Fragility of Red Blood Cells. The results of tests of osmotic
fragility during cardiOpulmonary bypass indicated that Pluronic F-68 had
no effect. However, if the blood samples were incubated for 24 hours at
37 C. before testing, Pluronic-treated cells exhibited greater resistance
to osmotic hemolysis than did untreated controls (Figures 11 and 12). A
comparison of these 2 groups was calculated using Students"p test for

50% hemolysis as demonstrated in Tables 5 and 6.

Plasma Haptoglobin. Data for average plasma haptoglobin concentra-
tion during cardiOpulmonary bypass are presented for 9 dogs (Figure 13).
Plasma haptoglobin concentration fell rapidly during the first 15 minutes
as a result of the diluting effect of the priming solution. In untreated
controls, with bypass times in excess of 30 minutes, a subsequent fall
was found. In Pluronic-treated animals, however, plasma haptoglobin con-
centrations began to rise after 30 minutes and continued to rise for
another 30 minutes. However, as shown in Figure 14, when the data for

plasma haptoglobin were corrected for the hemodilution effect of the

35
(P>0.1) (Figure 8). If cells were incubated with Pluronic F-68 for 24
hours at 37 C., osmotic fragility was somewhat reduced, but the differ-

ences were not statistically significant (P>0.1) (Figure 9).

In viva Studies

 

Red Blood Cell Studies

 

Plasma Hemoglobin. The average plasma hemoglobin concentrations

 

throughout the period of cardiopulmonary bypass are presented for 9 dogs
(Figure 10). There was no significant difference between plasma hemo-
globin values in animals treated with Pluronic F-68 and those in untreated

controls (Table 4).

Osmotic Fragilityfiof Red Blood Cells. The results of tests of osmotic
fragility during cardiOpulmonary bypass indicated that Pluronic F-68 had
no effect. However, if the blood samples were incubated for 24 hours at
37 C. before testing, Pluronic-treated cells exhibited greater resistance
to osmotic hemolysis than did untreated controls (Figures 11 and 12). A
comparison of these 2 groups was calculated using Students' 3 test for

50% hemolysis as demonstrated in Tables 5 and 6.

Plasma Haptoglobin. Data for average plasma haptoglobin concentra-
tion during cardiOpulmonary bypass are presented for 9 dogs (Figure 13).
Plasma haptoglobin concentration fell rapidly during the first 15 minutes
as a result of the diluting effect of the priming solution. In untreated
controls, with bypass times in excess of 30 minutes, a subsequent fall
was found. In Pluronic-treated animals, however, plasma haptoglobin con-
centrations began to rise after 30 minutes and continued to rise for
another 30 minutes. However, as shown in Figure 14, when the data for

plasma haptoglobin were corrected for the hemodilution effect of the

36

 

 

 

40 t
x
/
l
‘ " Pluronic F—68 (6 dogs) /
/
/
x----X No Pluronic F-68 (3 dogs) ’
/
/
/
30 b X/
/
I
l o
I
I
/
I
/
/
l I
20 I x’
I
at /
o /
é” , ’
*I
/
I
l
/
I
I
10 ' ’
-———,——’-/+ I i I
, P>0.10 P>0.lO P>O.10 P>0.10
xI
O A _:f t I l 1
Pre-B.P. 15 30 ' 45 60

Perfusion Time in Minutes

Figure 10. Plasma hemoglobin values in 9 dogs as a function of
bypass time (P represents degree of significance of differences of
means between 2 groups).

37

Table 4. Comparison of plasma hemoglobin values during cardiOpulmonary

bypass with and without Pluronic F-68 in perfusion fluids

 

 

 

 

 

Initial 15 min. 30 min. 45 min. 60 min.
Sample B.P. B.P. B.P. B.P.
No Pluronic F-68
Mean Z Hemolysis 6.33 14.83 19.37 29.50 38.77
S.D. 0.29 4.54 4.02 2.50 5.32
Pluronic F-68
Mean 2 Hemolysis 8.78 9.47 13.50 21.12 27.20
S.D. 3.89 5.13 7.78 11.16 10.48
Comparison
t 1.525 1.199 1.243 1.759
d.f. 7 7 7 7
P P>0.10 P>O.10 P>O.10 P>0.10
Significant No No No No

 

38

 

 

100 I
A
90 ' o -' - Control
—0— 15 min. bypass
'30 ‘ —X— 30 min. bypass
_A_ 45 min. bypass
70 F . —-I— 60 min. bypass
60 ’
4‘3
«1 1
>.
F4 1
a 50 '
:2
ac
4O . A
30 P
20 .
f
10 b
O
O n n a J a
0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75
2 NaCl-PO4

Figure 11. Osmotic fragility (in viva) without Pluronic F—68 after
24 hours incubation at 37 C.

  
 

39

 

 

 

100
90 b \x -—o-—Control
-o-15 min. bypass
30 - -—x—-30 min. bypass
""'45 min. bypass
70 L —'_ 60 min. bypass
. -A— 90 min. bypass
60 - —+-l80 min. bypass.
.3
a)
>.
E
:3 so -
we
40 -
O
30 y- 5
20 r .\
\a -
10 - , ' .
+§‘\:§o\
\‘ o
o . . . i\t\';iԤ2\\t
.40 .45 .50 .55 .60 .65 .70 .75 .80
Z NaCl-PO4
Figure 12. Osmotic fragility (in vivo) with Pluronic F-68 after 24

hours incubation at 37 C.

Table 6. Osmotic fragility in viva.

41

Comparison of the saline concen-

tration giving 50% hemolysis in blood samples incubated at

37 C. for 24 hours following in viva bypass experiments;

Pluronic F-68 added to perfusion fluids

 

 

Initial 15 min. 30 min. 45 min. 60 min.
Sample B.P. B.P. B.P. B.P.
Z NaCl-PO4 0.542 0.490 0.475 0.485 0.482
S.D. 0.0402 0.0297 0.0423 0.0281 0.0223
t 2.544 2.809 2.845 3.198
P 0:05>P>0.02 0.02>P>0.01 0.02>P>0.01 0.01>P>
0.001
d.fI 10 10 10 10
Significant Yes Yes Yes Yes

 

42

  
  

 

 

140.0 b
o—-—-‘ Pluronic F—68 (6 dogs)
%\
xP-—X No Pluronic F-68 (3 dogs)
120.0 .
Sf
on
X
100.0 .
J-
0] 4* :: é ‘ 1 ”L
Pre-B.P. 15 30 45 60

Perfusion Time in Minutes
Figure 13. Average haptoglobin levels during cardiOpulmonary bypass

in 9 dogs.

 

 

4.0 -
4.; x -"’"'- — x“‘~
o ‘,’ """" x--
:1: l” !./<:;
E, 300 5*, t
5.
g 2.0 P r———4 Pluronic F-68 (6 dogs)
N:
00
z 1.0 > x—-—x No Pluronic F-68 (3 dogs)
0 L :: l I l A

 

 

Pre-B.P. 15 30 45 6O
Perfusion Time in Minutes

Figure 14. Haptoglobin correction for the effect of hemodilution
during cardiopulmonary bypass (haptoglobin level divided by hematocrit
concentration).

43
priming fluids by dividing each value by the hematocrit of the sample,

the haptoglobin values showed no significant differences during the

first hour. Insufficient data for bypass times in excess of 60 minutes

were available for further analysis.

DISCUSSION

The results of these studies lend further support to previously
published work suggesting that the nonionic surfactant, Pluronic F-68,
reduces the mechanical fragility of erythrocytes (Wells at al., 1968;
Tamiya et al., 1970). The data from in vitra studies of mechanical
fragility indicate that the degree of mechanically-induced hemolysis
is diminished by Pluronic F—68, the effect being dependent upon the
concentration of the surfactant. This effect is demonstrable both with
red blood cells suspended in buffered saline solution and with red
blood cells suspended in plasma.

The mechanical fragility of erythrocytes which had been incubated
with Pluronic F-68 for 3 hours and then washed was somewhat less than
that of untreated cells, but the difference was not statistically sig-
nificant. This shows that the protective effect with respect to mechanical
fragility is dependent upon the surfactant's being present in the sur-
rounding medium and that, if it acts by adsorption to the cell surface,
it must be very loosely bound and unable to enert its effect in the
absence of significant concentrations in the suspending fluid.

Tests of erythrocyte osmotic fragility were conducted with Pluronic
F-68 in the hypotonic media. There was no difference in osmotic fra-
gility between control cells tested in media lacking the surfactant and
the treated cells. After 24 hours incubation with Pluronic F-68 at 37 C.,
the treated red blood cells showed slightly increased resistance to

osmotic lysis but the differences were not significant. It therefore

44

 

”.5

45

appears that the protective effect of Pluronic F-68 on red blood cell
lysis does not arise from changes in the relationship between the surface
area of the red blood cell and its osmotically active contents.

Data derived from in viva experiments regarding changes in plasma
hemoglobin level are difficult to interpret. The differences in plasma
hemoglobin levels between the untreated and treated animals were small
and not statistically significant. The series was too small to exclude
minor differences, but it seems most probable that the concentrations
of Pluronic F-68 in viva (0.35 mg./ml. blood) were too low in these
animals to have exerted a significant effect. In vitra studies indi-
cated that the inhibition of hemolysis by Pluronic was dose related.

In many previous experimental studies, Pluronic F-68 has been employed
in considerably higher concentrations. Tamiya at al. (1970) suggested
that a dose of Pluronic F-68 in a concentration of 1.0 mg./ml. in the
circulating blood during perfusion would be effective in preventing
hemolysis. The same investigator found that a Pluronic concentration
of 2.0 mg./m1. of blood, though more effective in reducing hemolysis,
was associated with postoperative elevation of blood urea nitrogen con-
centration and concluded that this concentration was not to be
recommended.

Pluronic F-68 had no significant effect on the osmotic fragility
of red blood cells in blood samples collected during cardiopulmonary
bypass. However, if the blood was incubated for 24 hours at 37 C. before
testing, it was possible to demonstrate a significant increase in osmotic
resistance in samples collected later in the bypass period. This effect
was noted irrespective of treatment with Pluronic F-68 and in some cases
corresponded to an increase in plasma hemoglobin. A possible explanation

is that there had been a subtle selective process during the Operation

mWhIALI DIRK-,7
.
9
M;
If
r

 

a...

46

of the bypass, such that those cells which would be more fragile on
incubation had been removed from the circulation.

Plasma haptoglobin concentrations were investigated during cardio—
pulmonary bypass to compare the behavior of free hemoglobin and the
hemoglobin-haptoglobin complex with respect to plasma. In general,
increased hemolysis is associated with lowering the plasma haptoglobin
levels and often its complete disappearance (Brus and Lewis, 1959). In
these experiments, Pluronic—treated animals had a higher plasma hapto-
globin and lower plasma hemoglobin level as compared to the untreated
animals at the end of perfusion, but there were no significant differ-
ences between these 2 groups of animals. This is further evidence that
the Pluronic F-68 in the concentration used failed to modify in viva
hemolysis.

Although the value of Pluronic F-68 during cardioPulmonary bypass
is still not conclusively demonstrated, in vitra studies indicated marked

reduction in mechanical fragility of Pluronic-treated cells.

 

SUMMARY AND CONCLUSIONS

The Pluronic polyols are ethylene oxide-polypropylene glycol con-
densation products. Pluronic F-68 is a nonionic polyol surfactant of
molecular weight 8,350. This agent has been subjected to extensive
toxicologic studies which indicate that it is non-toxic and suggest
that it is not metabolized. This surfactant has been used in total
body perfusion experimentally and clinically to protect blood cells
from trauma. The present investigation is a further study of the
effects of Pluronic F-68 both in vitra and during cardiOpulmonary
bypass.

The protective effects of Pluronic F-68 on red blood cells can

be summarized as follows:

In vitra Studies

 

l. Pluronic F-68 in phosphate buffered saline significantly pro-
tects red blood cells against mechanical hemolysis.

2. Pluronic F-68 in plasma also significantly increased the
resistance of red blood cells to mechanical hemolysis.

3. When erythrocytes were suspended in Pluronic F-68 and then
washed with isotonic saline prior to exposure to mechanical stress, a
slight reduction in the degree of hemolysis as compared with untreated
cells was observed but was not significant (P>0.1).

4. Pluronic F—68 had no immediate effects on the osmotic fragility

of red blood cells.

47

 

48
5. There was a slight but statiStically insignificant.(P>0.l) reduc-
tion in the osmotic fragility of erythrocytes incubated with Pluronic

F-68 for 24 hours prior to testing.

In viva Studies

 

1. There was no significant difference between the plasma hemo-
globin and haptoglobin levels of Pluronic F-68 treated animals with blood
concentrations of approximately 0.35 mg./ml. and those of untreated con-
trols during cardiOpulmonary bypass.

2. There was no immediate change in the osmotic fragility of
erythrocytes of Pluronic F-68 treated animals during cardiopulmonary
bypass.

3. If blood was incubated at 37 C. for 24 hours prior to testing
for osmotic fragility, the osmotic resistance of samples drawn during
the later stages of cardiOpulmonary bypass was significantly greater than
that of blood collected early in the bypass period whether Pluronic F-68
was used or not. This suggested that there was a selective loss of the
potentially more fragile cells during passage through the bypass
equipment.

Pluronic F-68 has been widely studied by numerous investigators.

It possesses certain properties which make it a uniquely suitable additive
to the perfusion system. The use of Pluronic F-68 is a safe and effective
means of minimizing the adverse effects of the pump oxygenator on red

blood cells during extracorporeal circulation.

REFERENCES

Adams, J. E., Owens, G., Mann, 6., Headrick, J. R., Munoz, A., and Scott,
H. W., Jr.: Experimental evaluation of Pluronic F-68 (a nonionic
detergent) as a method of diminishing systemic fat emboli result-
ing from prolonged cardiopulmonary bypass. Surg. Forum, 10,
(1959): 585-589.

Andreasen, F.: A new method for the estimation of the mechanical resist-
ance of the red blood corpuscles. Scand. J. Clin. Lab. Invest.,
16, (1964): 503-510.

Brinsfield, D. E., Hopf, M. A., Geering, R. B., and Galletti, P. M.:
Hematological changes in long-term perfusion. J. Appl. Physiol.,
17, (1962): 531-534.

Brus, 1., and Lewis, S. M.: The haptoglobin content of serum in hemo-
lytic anemia. Brit. J. Hemat., 5, (1959): 348-355.

Carr, C. J.: Acute toxicity studies with Pluronic F-68. Report to
Wyandotte Chemical Corporation, June 29, 1951.

Carr, C. J.: Chronic toxicity studies with Pluronic F-68. Report to
Wyandotte Chemical Corporation, May 19, 1952.

Dacie, J. V., and Vaughan, J. M.: The fragility of the red blood cells:
its measurement and significance. J. Path. Bact., 46, (1938):
341-3560

Danielson, G. K., Dubilier, L. D., and Bryant, L. R.: Use of Pluronic
F-68 to diminish fat emboli and hemolysis during cardiopulmonary
bypass. J. Thorac. and Cardiovasc. Surg., 59, (1970): 178-184.

Fielding, H. E., and Langley, P. E.: A simple method for measurement of
hemoglobin in serum and urine. Am. J. Clin. Path., 30, (1958):
528—529.

Galletti, P. M.: Laboratory experience with 24 hour partial heart-lung
bypass. J. Surg. Res., 5, (1965): 97-104.

Grover, F. L., Heron, M. W., Newman, M. M., and Paton, B. C.: Effect of
a nonionic surface-active agent on blood viscosity and platelet
adhesiveness. Circulation, Supp. 1, 39, (1969): I249-252.

Hartwig, Q. L.: Factors influencing hemoglobin changes associated with
daily infusions of intravenous fat emulsions. Metabolism, 10,
(1961): 41-49.

49

 

50

Hymes, A. C., Safavian, M. H., Arbulu, A., and Baute, P.: A comparison
of Pluronic F-68, low molecular weight dextra, mannitol, and
saline as priming agents in the heart—lung apparatus. Part I.
Pluronic F-68: first use as a plasma substitute. J. Thorac. and
Cardiovasc. Surg., 56, (1968): 16-22.

Hymes, A. C., Robb, H. J., and Margulis, R. R.: Influence of an industrial
surfactant (Pluronic F-68) on human amniotic fluid embolism. Am.
J. Obstet. Gynec., 107, (1970): 1217-1222.

Hymes, A. C., Safavian, M. H., and Gunther, T.: The influence of an
industrial surfactant Pluronic F-68, in the treatment of hemor-
rhagic shock. J. Surg. Res., 11, (1971): 191-197.

Kusserow, B. K., Machanic, B., Collins, F. M., Jr., and Clapp, J. F. III.:
Changes observed in blood corpuscles after prolonged perfusions
with two types of blood pumps. Trans. Am. Soc. Artif. Int. Org.,
11, (1965): 122-126.

Kusserow, B. K., and Clapp, J. F. III.: Red blood cell survival after
prolonged perfusion with a blood pump. Trans. Am. Soc. Artif.
Int. Org., 12, (1966): 121-123.

May, L. E.: A new aid in the medical management of diverticulitis (300
cases). Am. Geriatrics Soc. J., 6, (1958): 814-817.

Meyer, C. E., Fancher, J. A., Schurr, P. E., and Webster, H. D.: Compo-
sition, preparation and testing of an intravenous fat emulsion.
J. Lab. Clin. Med., 45, (1957): 591-595.

Miyauchi, Y., Inoue, T., and Paton, B. C.: Adjunctive use of a surface-
active agent in extracorporeal circulation. Circulation, Suppl.
I, 33-34, (1966): 171-77.

Moore, A. R., Paton, B. C., and Eiseman, B.: Reduction of splenic
vascular resistance during perfusion by Pluronic F-68. J. Surg.
Res., 8, (1968): 563-566.

O'Neill, J. A., Jr., and Collins, H. A.: The effect of various blood
additives on hemolysis during extracorporeal circulation. Ann.
Thorac. Surg., 1, (1965): 769-774.

Owen, J. A., Better, F. C., and Hoban, J.: A simple method for the
determination of serum haptoglobins. J. Clin. Path., 13, (1960):

Parpart, A. K., Lorenz, P. B., Parpart, E. R., Gregg, J. R., and Chase,
A. M.: The osmotic resistance (fragility) of human red cells.
J. Clin. Invest., 26, (1947): 636-640.

Pluranic PalyaZs Ibmicity and.Irritatian Data. BASF Wyandotte Corpora-
tion, Wyandotte, Mich., 1971(b), pp. 1-10.

Replogle, R. L., Meiselman, H. J., and Merrill, E. W.: Clinical implica-
tions of blood rheology studies. Circulation, 36, (1967): 148—160.

 

51

Shen, S. C., Castle, W. B., and Fleming, E. M.: Experimental and clinical
observations on increased mechanical fragility of erythrocytes.
Science, 100, (1944): 387-389.

Salyer, I. 0., Blardinelli, A. J., Ball, G. L., Weesner, W. E., Gott,
V. L., Ramos, M. D., and Furuse, A.: New blood-compatible polymers
for artificial heart applications. J. Biomed. Mater. Res. Symp.,
1, (1971): 105-127.

Tamiya, T.: A preventive measure for destruction of the blood during
extracorporeal circulation, with particular reference to the
vitamin E and the Pluronic F-68. Tateno, 23, (1970): 275-282.

Waddell, W. R., Geyer, R. P., Olsen, F. R., Andres, S. B., and Stare,
F. J.: Clinical experience with intravenous infusion of emulsi-
fied fat. J. Clin. Med., 45, (1955): 697-700.

Waddell, W. R., Geyer, R. P., Olsen, F. R., and Stare, F. J.: Clinical
observations on the use of nonphosphatide (Pluronic) fat emulsions.
Metabolism, 6, (1957): 815-821.

Walter, F. L., and Bronius, 3.: Effects of intravenous administration
of fat emulsions and their emulsifying agents. J. Invest.
Dermatol., 28, (1957): 317-320.

Wells, R., Bygdeman, M. S., Shahriari, A. A., and Matloff, J. M.:
Influence of a defoaming agent upon the hematological complica-
tions of pump oxygenators. Circulation, 37, (1968): 638-647.

Wonderful World of'Pluranic Polyols, The. BASF Wyandotte Corporation,
Wyandotte, Mich., 1971(a), pp. 4, 23.1

Wright, E. S., Sarkozy, E., Dobell, A. R. C., and Murphy, D. R.: Fat

globulinemia in extracorporeal circulation. Surgery, 53, (1963):
500-5040

General References

 

Greenwalt, T. J., and Jamieson, G. A.: Formation and Destruction of
Blood Cells. J. B. Lippincott Company, Philadelphia, 1970.

Harris, J. W.: The Red Cell: Production, Metabolism, Destruction,
Normal and'Abnarmal. Harvard University Press, Cambridge, Mass.,
1963.

Wintrobe, M. M.: Clinical Hematology, 6th ed. Lea and Febiger,
Philadelphia, 1967.

APPENDICES

APPENDIX A

REAGENTS

in):

wmw- "l I‘JLL :‘

 

APPENDIX A

Reagents

Fielding Method for Plasma Hemoglobin Determination

 

*
Orthotolidine Reagent. Crush 1 Hematest reagent tablet and dilute
with 25 ml. of distilled water; mix well and allow to stand 30 minutes
before filtering. This reagent should be crystal-clear, and stored in

the refrigerator.
Modified Owen Method for Plasma Haptoglobin Determination

Guaiacol Reagent. Guaiacol (A.R. l Gm./ml.) 3.72 ml. are added in

 

700 ml. water to which are added 100 m1. of].bfacetic acid. The pH is
adjusted to 4.0 by the addition of 1 N NaOH, using a glass-electrode pH

meter. The volume is finally made up to 1 liter.

Hydrogen Peroxide 0.05 M. This is prepared immediately before use

 

by diluting a stock solution with distilled water to a concentration of

0.05 M.

Methemoglobin Solution. To 8 volumes of thrice-washed packed red

 

blood cells are added 3 volumes of water and 1 volume of ether. The
mixture is shaken and centrifuged. The hemolysate is pipetted off, and

its hemoglobin concentration determined by ordinary clinical

 

*
Ames Company, Inc., Elkhart, Indiana, U.S.A.

52

53

hemoglobinometry (Drabkin's method). It is then diluted until the hemo-
globin concentration is 1 Gm./100 m1. To 25 ml. of this solution are
added 10 ml. of potassium ferricyanide (100 mg./100 ml.) to convert the
hemoglobin to methemoglobin. After 10 minutes the volume is made up to

500 ml. The solution can be stored at 4 C. for several weeks.

APPENDIX B

CALIBRATION OF STANDARD CURVE

 

Optical Density

54

1.00A‘

V

0.900

0.800L

T

0.700

I

0.600

T

0.500

0.400}

0.300?

0.200}

0.100’

 

 

0 I I L J A I L I I j

10 2o 30 40 so 60 7o 80 90 100
Mg./100 m1.

Figure B-l. Plasma hemoglobin standard curve.

 

55

 

 

0.300 I

0.200

Optical Density

0.100

 

 

I
I
I
I
I
I
I
I
I
I
l
I
I
I
I
I
I
I
I
I
l
I
I
I
I
I
I
I
I
I
I
I
I
I
j

Plasma (m1.)
. 2 0.03 0.04 0.05
1 I I I I L j

 

10 20 30 40 50 Haptoglobin (mg./100 m1.)

Figure B-2. Calibration curve for estimation of haptoglobin concen-
tration (the abscissa shows at the point of change of gradient the normal
pooled plasma contains 50 mg. haptoglobin per 100 m1.).

 

56

I

0.30I

0.20

Optical Density

0.10

 

'_' Pooled human serum

x """ x Pooled dog plasma

Serum or Plasma Dilution (m1./tube)
0.03 0.04 0.05

 

 

' I
l
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I

l A

10 20 3O 4O 50 60 70 80 90 100
Haptoglobin (mg./100 ml.)

1 4 g ; L

Figure B—3. Haptoglobin calibration curve (comparison human pooled
serum and dog pooled plasma).

APPENDIX C

CALCULATION FORMULA

APPENDIX C

Calculation Formula

 

Comparison of Two Means
To obtain 2 sets of numbers and to test whether the mean.of one set-
is significantly different from the mean of the other set, or whether the
2 sets can be regarded as drawn from 1 p0pulation.
1 I I
Let X1, X2...an, and X 1, x 2...X n2 be the 2 group samples.

Calculate as following terms

 

ESZKX) , E_X(X')
n1 1“2

I 2 2
02 = [2:(x'2) - Q5710)“, 2(x2) - lit—IEELI/(n1 + n2 - 2)
2 1

Having found the value of t, this enters the table of t with degree
of freedom (n1 + n2 - 2). If the value of t exceeds 5% level, this may
assure that the p0pulations are different.

Where - Mean
. Summation

- Number

- Variance.
Standard deviation
= Student_£ test

n x n

Standard error of the difference in
- the mean of the 2 group samples
Degree of freedom

I- Id
0
h) h) n~q nap has“
I

I n + n

(nl + n2 - 2)

57

I‘IICHIGQN STRTE UNIV. LIBRQRIES

 

2931030515