U.S. patent number 3,632,473 [Application Number 04/825,099] was granted by the patent office on 1972-01-04 for method and apparatus for preserving human organs extracorporeally.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Folkert O. Belzer, Chester W. Truman.
United States Patent |
3,632,473 |
Belzer , et al. |
January 4, 1972 |
**Please see images for:
( Certificate of Correction ) ** |
METHOD AND APPARATUS FOR PRESERVING HUMAN ORGANS
EXTRACORPOREALLY
Abstract
A human organ is stored, between removal from one body and
implantation in another, in an apparatus mounted on a wheeled cart.
The apparatus has a pulsatile pump for pumping plasma, a heat
exchanger connected to the outlet of the pump for cooling the
plasma to about 4.degree. to 8.degree. C., and a perfusion chamber
to which the cooled plasma is supplied. The perfusion chamber
includes a support for the organ and means for connecting the organ
to the pulsing flow of cold plasma. Venous effluent from the organ
is collected and conducted by gravity to a membrane oxygenator,
which returns oxygenated plasma to the pulsatile pump for
recirculation through the organ.
Inventors: |
Belzer; Folkert O. (Mill
Valley, CA), Truman; Chester W. (Daly City, CA) |
Assignee: |
The Regents of the University of
California (Berkeley, CA)
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Family
ID: |
25243103 |
Appl.
No.: |
04/825,099 |
Filed: |
April 21, 1969 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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727762 |
May 9, 1968 |
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Current U.S.
Class: |
435/1.2; 62/306;
607/105 |
Current CPC
Class: |
A01N
1/02 (20130101); A01N 1/0205 (20130101) |
Current International
Class: |
A01N
1/02 (20060101); A61k 017/00 () |
Field of
Search: |
;195/1.7 |
References Cited
[Referenced By]
U.S. Patent Documents
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3406531 |
October 1968 |
Swenson et al. |
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Primary Examiner: Huff; Richard L.
Parent Case Text
This application is a continuation-in-part of application, Ser. No.
727,762, filed May 9, 1968, now abandoned.
Claims
1. A method for the preservation of a human or animal organ during
the time between removal from a donor body and implantation into a
patient's body comprising supplying cold plasma in a series of
regular pulses into said organ for perfusion of said organ while
keeping pressure of the pulses of cold plasma substantially that at
which the organ is operated within a
2. The method of claim 1 wherein the plasma is kept at a
temperature above
3. The method of claim 1 wherein the plasma is cryoprecipitated
and
4. The method of claim 3 wherein the plasma is cryoprecipitated by
freezing
5. The method of claim 1 wherein the effluent from the organ
resulting from the supply of plasma to the organ is oxygenated and
recirculated to the
6. The method of claim 5 wherein the effluent is filtered after
oxygenation
7. A method of storing a human organ between the operation of
removal from one human body and implantation into another human
body, comprising
pumping in a pulsing manner a flow of plasma,
cooling the plasma to approximately 4.degree. C.,
delivering the cool plasma in a pulsing manner to the intake of the
organ,
collecting the effluent from the organ,
oxygenating the effluent, and
8. The method of claim 7 wherein the plasma is oxygenated,
cryoprecipitated
9. The method of claim 7 wherein the oxygenating is done without
letting
10. The method of claim 9 wherein the oxygen and plasma are
separated by an
11. The method of claim 7 wherein the plasma is supplied to the
organ at substantially the pressure at which blood is supplied to
that organ in a
12. The method of claim 11 wherein the pressure of the plasma is
regulated by opposing the pulses with a column of air and by
regulating the amount
13. The method of claim 7 wherein the effluent is filtered to
remove
14. The method of claim 7 wherein the pH of the plasma is
controlled at about normal blood pH level by adding controlled
amounts of carbon dioxide
15. The method of claim 7 wherein the effluent is collected and
supplied to the oxygenating and recirculating steps by gravity
movement thereof.
Description
This invention relates to method and apparatus for preserving human
organs outside the body.
The invention described herein was made in the performance of work
under research grants from the United States Public Health
Service.
Heretofore there have been many difficulties and inconveniences in
the process of transplanting human organs from one person to
another. For example, patients waiting to receive an unrelated
donor kidney have had to be on constant standby in a hospital,
sometimes for weeks. When the donor appeared, the timing was very
important, for the surgery had to be substantially simultaneous so
that immediately upon removal of the kidney from the donor, it
could be put into the patient. This meant that there had to be at
least two surgical teams working on the transplantation. The donor
and the patient had to be located very close to each other during
these operations, because there was no way of preserving the
kidneys for any substantial period of time after they had been
removed from the donor body and before they were put into the
patient' s body. The procedure was always therefore an emergency
procedure and was fraught with risks as well as difficulties.
Similar problems and the same difficulties have applied to the
transplantation of other organs, such as a heart or liver.
The present invention solves these problems by making it possible
to keep the organ alive for many hours up to several days after
removal from the donor body. This makes it possible to use cadaver
kidneys, hearts, and livers and to have the removal operation and
the transplant operation spaced apart by several days. The
transplantation, therefore, can be an elective rather than an
emergency procedure.
Since additional time is available, it is possible to match the
donor and recipient by tissue-typing, for unrelated donors who are
proved compatible by tissue-typing are generally as successful as
donors who are related to the recipient.
Also, with the new apparatus of this invention available, it is
possible for the recipient to wait at home until the correctly
matched kidney or kidneys are available. Similar procedure is
possible for other organ transplantation.
This invention also enables a single team of surgeons to do the
removal operation and the transplanting operation, and the surgery
can be spaced apart by several days if necessary. Or the use of two
teams is still possible, but they need not be close to each other
at the time, for the organ can be moved substantial distances
during the time when the organ is out of both bodies.
In the present invention the system incorporates transfer of the
kidney, heart, liver or other organ from the donor' s body into a
perfusion chamber where human plasma, kept in constant supply and
preferably fortified with hormones and other substances, is pumped
through the organ. The organ functions generally as it would in the
body; for example, kidneys in the perfusion chamber produce urine;
however, it is important in the invention to perform the perfusion
at low temperatures, so that the organ' s activity is kept at a
minimum. The plasma is recirculated and oxygenated, and its pH is
adjusted as by a supply of carbon dioxide. Careful filtering
enables the plasma to be kept free from foreign matter. The pumping
of the plasma through the organ is done by a pulsatile pump, so
that pulses similar to those produced by the human heart are
employed to force the cold plasma through the organ. Pressure is
controlled with the aid of a damper having an air spring. The
operation of the apparatus thus resembles the operation in the
human body but differs from it in being conducted at a very low
temperature and in the type of control involved. Also, in a kidney,
for example, it is not necessary to free the recirculated plasma
from the small amount of urine produced during storage, for the
freeing of the kidney from the urine can take place later in the
patient' s body after transplant. Pressures maintained on the organ
are substantially those met by the organ in the human body, and the
flow of plasma through the organ is controlled in accordance with
the pressure desired.
Another significant feature of the method of this invention is its
use, at least with kidneys, of cryoprecipitated filtered
plasma.
Other objects and advantages of the invention, as well as other
features, will appear from the following description of a preferred
embodiment of the invention.
In the drawings:
FIG. 1 is a view in side elevation of an organ preservation
apparatus embodying the principles of the invention. This
particular apparatus is used for preserving two human kidneys at
once, and it is incorporated in a portable supporting cart. Some
conduits and wires are omitted to avoid needless confusion.
FIG. 2 is a top plan view of the apparatus of FIG. 1.
FIG. 3 is an enlarged view in section, taken along the line 3-- 3
in FIG. 1, of the perfusion chamber in which two kidneys may be
simultaneously maintained.
FIG. 4 is a fragmentary enlarged view in end elevation of a portion
of the apparatus in FIG. 1. The oxygenator is shown in one of its
positions in solid lines and in another of its positions in broken
lines, while the perfusion chamber is shown above the oxygenator
with its lid swung open.
FIG. 5 is a plan view of the oxygenator, broken in the middle to
conserve space and with layers stripped away selectively to show
the different layers and to illustrate the flow of air or oxygen
therethrough.
FIG. 6 is a view in side elevation of the pulsatile pump apparatus
embodying the principles of the invention, employing a micrometer
for enabling delicate adjustment. The eccentric cam and
reciprocating pump actuator are shown in one extreme position in
solid lines and in another extreme position in broken lines.
FIG. 7 is a top plan view of the pulsatile pump apparatus of FIG.
6.
FIG. 8 is an enlarged fragmentary view in section taken along the
line 8-- 8 in FIGS. 6 and 7, but showing the pump actuator in the
position shown in broken lines in FIG. 6.
FIG. 9 is an enlarged fragmentary view in section taken along the
line 9-- 9 in FIG. 6. The ball outlet valve is shown in its open
position in solid lines, while broken lines show its closed
position.
FIG. 10 is a view in front elevation of a control panel for the
apparatus of FIG. 1, a portion being broken away to save space.
FIG. 11 is a flow diagram of the plasma, the coolant, and the
oxygenating gases used in the system of FIG. 1.
FIG. 12 is an electrical circuit diagram of the apparatus of FIG.
1.
While applicable to various human organs, the invention will be
illustrated by the following example of apparatus 20 for preserving
human kidneys outside the bodies. Since an important feature of the
invention is its ability to move the organ from one place to
another, the preferred embodiment 20 shown in FIGS. 1 and 2
comprises a cart 21 having wheels 22 and caster wheels 23
supporting a frame 24, which may include several auxiliary
supporting decks or other support facilities. The entire unit 20
may be mounted on this cart 21, and it is preferably provided with
a double electrical system as shown in FIG. 12. One electrical
system uses plug-in current, such as a 115-volt alternating current
input 25 obtained from base plugs commonly found in hospitals; the
other electrical system relies on a battery 26. The battery system
is used while the cart 21 is moved from one location to another
carrying a live organ and also, if and when it is necessary, for
standby, as during power failures. Normally, in a fixed location
the alternating current system is used.
The apparatus 20 is preferably put together very compactly, and the
parts are so closely related that sometimes in the drawings they
obscure each other; hence, many of the cords and tubes have been
omitted from the drawings. It will be helpful during the following
description for the reader to make frequent reference to the flow
diagram of FIG. 11 and the electrical circuit diagram of FIG.
12.
As shown in FIG. 11, fresh human plasma 30 is added to an inlet 31
of an arterial reservoir 32, which has a recirculation inlet
conduit 33 for recirculated plasma. As described later the plasma
30 is preferably cryoprecipitated and filtered plasma. The plasma
30 may include additives such as hormones, steroids, penicillin,
magnesia, or insulin. An outlet conduit 34 from the arterial
reservoir 32 enables withdrawal of arterial samples 35 from time to
time through an outlet 36, and it conducts the plasma 30 to a
filter 37 which delivers the plasma 30 in a state free from any
foreign particles through a conduit 38 to the inlet 39 of a
pulsatile pump 40. The filter 37 may use two layers of fine silk
mesh.
The pulsatile pump 40 is a very important part of the apparatus and
is shown especially well in FIGS. 6- 9. It employs a flexible
deformable tube 41, preferably of transparent plastic, which is
used in many parts of this machine, because the plastic is a good
heat insulator and also because it enables direct observation. The
tube 41 has an inlet valve 42 at its inlet 39, preferably at the
bottom end, and an outlet valve 43, preferably at its top end. The
tube 41 is supported, preferably vertically, as by a lower bracket
44 secured beneath a deck 45, by a vertical anvil wall 46 that is
supported on the deck 45, and by an upper bracket 47 secured to the
anvil wall 46. The brackets 44 and 47 maintain closure of the upper
and lower ends when pressure is exerted on the walls of the tube 41
and prevent movement of the tube ends. As will be seen, the anvil
wall 46 is movable relative to the deck 45 by means of a micrometer
screw 48 having a handle 49. The anvil wall 46 at all positions
supports one side of the tube 41.
The pump is pulsed by a motor 50, which may be equipped with a
right-angle drive 51 to rotate a cam 52 which is shaped so as to
impart reciprocating motion to a shaft 53. The shaft 53 is provided
with a return spring 54 and a cam follower 55 to enable it to
reciprocate as the cam 52 is rotated, and it extends through a
guide 56 to an outer end 57, where it supports a bar 58. The bar 58
has a projection or link 59 that pivotally supports a plate 60 by a
pin 61 and link 62, and a pair of springs 63 provide a yielding
cushion between the plate 60 and the bar 58. The plate 60 comprises
the pump actuator and preferably has a plastic face 64, and
reciprocation of the shaft 53 results in reciprocation of the plate
60. The plate or actuator 60 is so located that it is able to bear
against the sidewall of the tube 41 opposite the anvil wall 46.
The tube 41 may be positioned relative to the actuator 60 by
movement of the micrometer 48, which is very similar to those used
on a lathe, to give a very delicate adjustment of the position of
the tube 41. If the handle 49 is turned so that the anvil wall 46
is sufficiently distant from the plate 60, then the plate 60 cannot
come into contact with the tube 41 at all and there is no pumping.
If the micrometer handle 49 is then turned slowly, it is possible
to adjust very accurately the maximum pressure exerted by the plate
60 on the flexible wall of the tube 41. Outward movement of the
plate 60 in each cycle then presses in one side of the tube wall,
opposite the anvil 46, and exerts pressure which sends a charge of
plasma 30 out through the upper valve 43; similarly, withdrawal of
the plate 60 in each cycle then results not only in closure of the
outlet valve 43, which acts then as a check valve, but also in
drawing in a charge plasma 30 through the inlet valve 42. During
each cycle, the outward movement of the plate or actuator 60 acts
through the liquid in the tube 41 to force the inlet valve 42 down
to its closed position, so that it then acts as a check valve,
while opening the valve 43 and exhausting a charge dependent in
volume on the relative position of the plate 60 and tube 41. The
stroke of the plate 60 remains constant unless the cam 52 is
changed, and the springs 63 help to give some resiliency. Thus, by
the micrometer 48 and the position of the anvil wall 46, the
pulsatile pump 40 of this invention gives very fine control of the
pumping pressure of the plasma 30, in conjunction with a damper 96.
The speed of the motor 50 determines the pulse rate, as it too is
adjustable. Thus, volume of plasma pumped and the pulse rate are
determined by the pump 40, and plasma pressure is, in part,
controlled by it.
A conduit 65 leads from an outlet 66 above the valve 43 to the
inlet 69 of a heat exchanger 70. As stated before, it is quite
important in this invention that the perfusion be conducted at very
cold temperatures, about as near to freezing as one can get without
actually freezing the plasma at any point of the cycle. This will
usually mean cooling the plasma 30 to about 4.degree. to 8.degree.
C. To get the plasma much colder might result in its freezing in
the heat exchanger 70. The heat exchanger 70 may be of any desired
type, but should be suitable for use with a pulsing system without
affecting the pressure of the plasma when the coolant changes
pressure. By way of example, cold alcohol or cold water may be
forced through a central conduit around which is a conduit carrying
the plasma. Preferably, two coolers are used in conjunction with
the heat exchanger 70. A main cooler 71 employs an electrical
refrigerating unit to circulate cold alcohol to the heat exchanger
70; this cooler 71 is used during stationary operation and only
with the plug-in circuit. A portable cooler 72, comprising a simple
chamber of ice water and a small circulating pump, is used
principally during transportation and at other times when the
operation is on battery power, but is made to be operable also by
plug-in current in emergencies, as if the cooler 71 should fail to
operate properly. Both coolers 71 and 72 send their cold liquid
through a tube 73 to the heat exchanger 70, from which it is
returned through a tube 74. From the heat exchanger 70, the cooled
plasma passes by a conduit 75 to a tee 79 in a perfusion chamber
80.
The perfusion chamber 80 may be generally cylindrical, having a
stationary lower housing portion 81 and a lidlike upper housing
member 82 held by a hinge 83 to the portion 81. The chamber 80 is
normally filled with air at ambient pressure and temperature. As
shown in FIG. 1, the perfusion chamber 80 is located on a slant so
that the plasma effluent from the kidney or other organ flows
downhill along a bottom wall 84. Approximately midway up the
chamber is a support member 85, which may be a screen to enable
full exposure otherwise, and on which a kidney 86 or other organ
may be placed. Preferably, the perfusion chamber 80 is divided by a
central wall 87 into two sections, each with its own screen 85 but
with the bottom wall 84 continuing straight through from one
section to the other, through an opening 88. This enables the
handling of kidneys 86 in pairs, as that is the way they, of
course, occur in the body. Into this central partition 87 is
brought the pulsing flow of cold plasma by the tee 79, and a pair
of outlets 90 and 91 are provided, to each one of which is
connected the proper portion of a kidney 86, which rests on its
screen 85. No additional refrigeration of the organ 86 is
needed.
Thus, the arterial portion of the plasma circulation cycle is
completed as the cold plasma is pumped by the pulsatile pump 40
(which represents the arterial delivery part of the human heart),
to the kidneys 86 via the heat exchanger 70. The effluent of venous
plasma is collected on the bottom wall 84 and flows down to an
outlet 92 at the lower end of the perfusion chamber 80, whence it
is then carried by a conduit 93 into a venous reservoir 94. From
there, the venous plasma passes by a pair or conduits 95 to a
membrane oxygenator 100.
Pressure of the plasma supplied to the kidneys 86 may be regulated
with the aid of a manifold-pulse dampener 96, which is connected to
the tee 79 by a conduit 89. A thick-walled transparent cylinder,
the dampener 96 has a lower inlet 97 for plasma and a cushion of
air is retained at the upper end of the cylinder, making an air
spring. A connection 98 is secured to the upper end wall 99 and
enables addition or subtraction of air from the dampener 96. Thus,
if the pressure is too low, more air is added to the dampener
cylinder 96 by the syringe, adding to the air pressure that must be
opposed by the plasma in its delivery pulses, so that less of the
pressure imposed by the pump 41 is taken up by the air cushion. If
the pressure is too high, the air spring is made less forceful by
letting some of the air out of the cylinder 96 through the
connection 98. The dampener 96 thus cooperates with the pump 40,
which also affects the pressure as its displacement is varied.
The oxygenator 100 preferably comprises a flat support screenlike
member 101, which gives rigid support and is mounted in a frame
102, and a membranous member which provides two outer walls 103 and
104 and two inner walls 105 and 106. Between each outer wall 103
and 104 and its adjacent inner wall 105, 106 a thin layer of the
venous plasma is introduced from the conduits 95, and in between
the two inner walls 105 and 106 a current of air, either alone or
admixed with oxygen or with carbon dioxide or with both, is
introduced. The gaseous oxygen is thus separated from the venous
plasma by an oxygen-penetratable membrane. The air may be supplied
by a blower 107, or by a bellows-type pump that provides
low-pressure compressed air, through a conduit 108 to a common
conduit 110. This blower 107 (or the bellows-type pump) is, as is
shown in FIG. 11, operated by the electrical circuit and is of
rather light horsepower so that a steady flow of air at low
pressure is obtained whether the battery 26 or the 115-volt input
25 is being used. Oxygen may be supplied from a cylinder 111
through a regulator 111 a and a conduit 112 to the common conduit
110. Carbon dioxide may be supplied from a cylinder 113, through a
regulator 114 and a conduit 115 to the common conduit 110, the
conduits 110 may be in pairs, and the oxygenator 100 may be
exhausted by gas outlets 116. These gas outlets 116 serve to
regulate the air or gas pressure in the oxygenator 100, by
relieving the interior.
The frame 102 is rocked back and forth by a motor 117, eccentric
118, and linkage 119, in order to circulate air to both outer walls
103 and 104 of the membranes, while the mixture of gases is
circulated to the inner membrane wall 105 and 106. The air for the
walls 103 and 104 may be ordinary room air, or a housing may be
placed around the oxygenator 100 and the atmosphere may then be
specially mixed, if that is desired. The rocking back and forth of
the oxygenator 100 corresponds to the action of the lungs in the
human body, and by mixing the venous plasma with oxygen, the plasma
is suitable for use again as arterial plasma. It will be noted that
gravity is substituted for one of the heart chambers, rather than
having a second pump, and the membrane oxygenator 100 supplies the
arterial reservoir 32 through the conduits 33.
When the plasma 30 flows by gravity from the membrane oxygenator
100 to the arterial reservoir 32, the cycle starts again. The same
plasma can be used indefinitely with some makeup plasma to replace
what is withdrawn for samples. Since each kidney 86 functions very
slowly, insofar as production of urine is concerned, due to the
cold temperature, there is no need to do anything about the
accumulation of urine over periods of several days, for the amount
is small. If it should be deemed advisable to do this, the supply
of plasma may be withdrawn from the oxygenator 100 or even from the
venous reservoir 94 and discarded, and a fresh supply added in the
meantime to the arterial supply reservoir 32.
As shown in FIG. 12, the battery 26 may be used in conjunction with
an inverter 120 and is placed in the circuit by a switch 121, so
that it operates the pulsatile pump motor 50, the blower 107, the
oxygenator motor 117, and the portable cooler 72. On the other
hand, when the 115-volt input is used, the switch 121 is thrown to
the other side, the battery 26 is then not in use, and a charger
122 attached to the 115-volt input 25 is used to recharge the
battery 26 while the same motors are used as before except for the
portable cooler 72, which is replaced in this instance by the
regular cooler 71. Similar results can be obtained by DC operation,
using the battery 26 without an inverter and rectifying the AC to
the input 25. As shown in FIG. 10, a control panel 125 carries the
switch 121 as well as a set of other switches, so that each motor
is separately turned on or off: namely, a refrigerator switch 126
for the cooler 71, a switch 127 for the motor 50, a switch 128 for
the blower 107, a switch 129 for oxygen addition, a switch 130 for
the cooler 72, a switch 131 for a recorder 132, and a switch 133
for the battery charger 135. Lights 134 indicate which elements are
turned on.
The recorder 132 is used to record the various data and may be used
for automatic or manual control of several factors. Thus, one
important factor is the pressure of the plasma as supplied to the
kidneys 86. In this purpose, a conduit 137 leads from the dampener
96 (and therefore via the dampener 96 from the input to the kidneys
86) to a pressure gauge 138 at the recorder 132 which records that
pressure. The information is used to adjust the pressure shown by
the gauge 138 to the normal body pressure, by using the syringe 98
to vary the amount of air in the air-spring or dampener 96. The
plasma temperature may also be recorded by a thermometric device
and watched to control the circulating pump of either cooler 71 or
72, or control is made automatic to keep the plasma temperature
constant.
The oxygenator 100 is carefully controlled to give desired results.
For example, upon its removal from a cadaver, the kidney ordinarily
is suffering from lack of oxygen in the blood; therefore, the
plasma may then give pure oxygen from the oxygen cylinder 111.
After a few minutes of this, some air can be mixed in and the
amount of oxygen reduced. The pH is observed by use of a pH meter
(not shown) which is from time to time supplied with a sample 35 of
arterial blood removed from the arterial sampler 36; this enables
the operator in charge to see whether additional carbon dioxide
should be added to reduce the pH or whether the carbon dioxide
should be reduced to increase the pH level.
A unique feature of this method has been the high flow rate,
without a rise in perfusion pressure and the absence of tissue
edema.
The role of platelet and blood cell aggregates, if whole blood is
used, was well established and led to the initial selection of
plasma as the perfusate in kidney transplantation. If the plasma
was diluted in a ratio of 1:3 of electrolyte solution, and the
osmolarity was maintained between 300 and 340 milliosmoles, edema
of the kidney was minimal and perfusion pressure rose only slightly
over a 24 -hour period of perfusion. However, on reimplantation of
these organs, function was greatly impaired and none of the animals
survived autotransplantation with immediate contralateral
nephrectomy. When undiluted plasma was used in tests, there was a
recurrence of the rising perfusion pressure, severe edema, and
tissue destruction. This was ameliorated, but not eliminated, by
the use of the pulsatile pump and the membrane oxygenator. Under
these circumstances, conventional microscopic studies showed no
evidence of thrombi. However, when frozen sections were taken of
the perfused kidney, fat stains revealed multiple small emboli in
the renal arterioles, and fat droplets in the tubules and
intratubular cells.
It appeared that the rising perfusion pressure was due to blockage
of the vessels by lipid components liberated into the perfusate by
denaturation.
Therefore, the present invention preferably employs a filtered
cryoprecipitated plasma, obtained, as described in a copending
patent application, Ser. No. 727,762, filed May 9, 1968, by
preliminary denaturation of the lipoproteins by freezing and quick
thawing. This may be done by storing the plasma at minus 20.degree.
C. for 12 to 24 hours, followed by rapid thawing in water at
60.degree. to 70.degree. C. Particular care is preferably taken not
to warm the plasma to a temperature higher than 38.degree. C. With
thawing, a flocculation appears in the plasma, which is then
removed by serial filtration, for example, through micropore
filters with pore diameters of 1.2, 0.45, and 0.22 m.mu..
The residue on the filter paper has been analyzed by thin layer
chromatography and found to consist primarily of phospholipids,
namely lecithin, sphingomyelin, and lysolecithin.
Before the last filtration through the 0.22 m.mu. micropore filter,
the following substances may be added to the perfusate per liter of
ACD collected plasma. Detrose--50 percent, 5 ml.; Insulin-- 80
units; Hydrocortisone-- 100 mg.; Pencillin-- 200,000 units;
Magnesium Sulfate-- 8 mEq.; and Phenolsulfonphthalein-- 12 mg.
Perfusion of the kidney with this filtered plasma completely
eliminates the rising perfusion pressure. Fat stains show that the
previously seen lipid particles are completely eliminated. After 72
hours of perfusion, kidney function is proven by reimplantation
with immediate contralateral nephrectomy. The kidneys appear
normal, and urine production usually occurs within 5 minutes after
release of the vascular clamps. Postoperatively, all animals
produce copious amounts of urine. Blood urea nitrogen rise has been
noted in all animals, especially in the 72-hour group, but all
return to normal within 2 weeks in the 24 -hour group and withing 5
weeks in the 72 -hour group.
Animals in both the 24 and 72 -hour groups have been followed for
periods beyond 6 months, and studies at that time reveal normal
renal function and renal architecture with no evidence of
hypertension. Human transplantation has also proved successful.
The perfusate for the human perfusions is identical to that used in
the animal experiments, except that the plasma is obtained from the
blood bank as frozen plasma, AB+ . Filtration of the plasma and the
addition of the previously mentioned substances may be done at the
time of notification of a potential donor.
Nearly all of the lipid components in blood plasma are combined
with proteins, and the soluble lipoproteins are responsible for the
transport of lipids in blood. There are at least three major groups
of lipoproteins present in the plasma of mammals. These are the
high-density lipoproteins, the low-density lipoproteins, and the
chylomicrons. Certain factors effect the stability of lipoproteins
in plasma, mostly in the low-density group. Lipoproteins are
readily damaged by conditions that are usually hazardous to plasma
proteins, such as extremes of pH, and, in some cases, ionic
strength, heat, freezing, the presence of ethanol (except at low
temperature), and exposure to interphases such as gas-water or
air-water. All of these agents tends to disrupt the complex,
consisting of an aggregate of mixed lipids stabilized and limited
by a specific peptide chain. This leads to aggregation of the
lipids into larger particles, which greatly increases the turbidity
of the solution.
By deliberate preliminary denaturation of the lipoproteins and
subsequent filtration through micropore filters, the lipid
aggregates are removed, and a perfectly clear plasma solution is
obtained. Studies have shown that about only 30-- 35 percent of the
lipid components were removed, probably primarily the low-density
lipoprotein group because the high-density lipoproteins have a
higher proportion of peptide-to-lipid molecule linkage and thus
have a greater stability to temperature variations. It had been
shown earlier that the low-density lipoproteins make up 40 percent
of the total phospholipids in plasma. With preliminary denaturation
and subsequent filtration, 75- 90 percent of the phospholipids can
be removed, primarily in the low-density group, thus eliminating
the problem of lipid aggregates. Even in 72 -hour perfusions, no
fat emboli could be found after this preliminary filtration. The
gentleness of the perfusion and the presence of a membrane
oxygenator appear to prevent further denaturation of the more
stable residual lipoproteins.
Such additives to the perfusate as insulin, cortisone, etc., were
chosen on an empirical basis, and further studies may be required
to show their necessity. It has been suggested that calcium acts as
a membrane stabilizer. Because of the use of citrate in the
perfusate used here, no calcium is available to the tissues, and
the substitution of magnesium might counteract the absence of
calcium. In addition, elevations of serum magnesium appear to be
characteristic hibernation.
Penicillin was added because of its nontoxic properties and with
good surgical aseptic technic, infection has never been a problem
in the animal or human preservation experiments. Steroids were
added because of their theoretical advantage as tissue stabilizer.
Dextrose was used on the basis of work reported by Folkman et al.,
although even after 72 hours of perfusion we have been unable to
show a definite utilization of glucose. Probably the organ is at
such a low temperature that glucose is barely used. The elimination
of glucose, or perhaps the substitution of fructose, is presently
under investigation. Phenosulfonphthalein is used as a pH
indicator, and is of value as a rough estimate of pH during
perfusion.
To those skilled in the art to which this invention relates, many
changes in construction and widely differing embodiments and
applications of the invention will suggest themselves without
departing from the spirit and scope of the invention. The
disclosures and the description herein are purely illustrative and
are not intended to be in any sense limiting.
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