U.S. patent application number 09/325244 was filed with the patent office on 2002-02-28 for plasma-like solution.
Invention is credited to SEGALL, JUDITH M., SEGALL, PAUL E., STERNBERG, HAL, WAITZ, HAROLD D..
Application Number | 20020025562 09/325244 |
Document ID | / |
Family ID | 26752331 |
Filed Date | 2002-02-28 |
United States Patent
Application |
20020025562 |
Kind Code |
A1 |
SEGALL, PAUL E. ; et
al. |
February 28, 2002 |
PLASMA-LIKE SOLUTION
Abstract
Aqueous solutions comprising a polysaccharide oncotic agent, a
physiologically compatible buffer, a simple hexose sugar, dissolved
chloride salts of calcium, sodium and magnesium, and a dissolved
organic salt of sodium are disclosed. The solutions are effective
substitutes for blood and may be used to preserve the biological
integrity of the organs of a mammalian donor organism as shown by
superior anatomical integrity of cryopreserved organs and tissues
of subjects perfused with the solution. The solutions may be used
for maintaining a partially or substantially completely
exsanguinated subject at normal temperatures and at temperatures
substantially below those normally maintained by a mammal and may
be used in conjunction with hypobaric environments to maintain such
partially or completed exsanguinated subjects alive without
infusing blood back into the subject.
Inventors: |
SEGALL, PAUL E.; (BERKELEY,
CA) ; STERNBERG, HAL; (BERKELEY, CA) ; WAITZ,
HAROLD D.; (BERKELEY, CA) ; SEGALL, JUDITH M.;
(BERKELEY, CA) |
Correspondence
Address: |
CAROL L. FRANCIS
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD ROAD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
26752331 |
Appl. No.: |
09/325244 |
Filed: |
June 3, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09325244 |
Jun 3, 1999 |
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09052827 |
Mar 31, 1998 |
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09052827 |
Mar 31, 1998 |
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08839021 |
Apr 23, 1997 |
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5968726 |
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08839021 |
Apr 23, 1997 |
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08133527 |
Oct 7, 1993 |
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08133527 |
Oct 7, 1993 |
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08071533 |
Jun 4, 1993 |
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5407428 |
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Current U.S.
Class: |
435/100 |
Current CPC
Class: |
Y10S 514/833 20130101;
A61K 33/14 20130101; A61K 33/14 20130101; Y10S 514/832 20130101;
A61K 33/10 20130101; A61K 33/14 20130101; A61K 33/10 20130101; A61K
33/14 20130101; A61K 33/10 20130101; A61K 33/14 20130101; A61K
31/715 20130101; A61K 31/19 20130101; A61K 31/715 20130101; A61K
33/14 20130101; A61K 31/7004 20130101; A61K 31/715 20130101; A61K
31/22 20130101; A61K 31/715 20130101; A61K 2300/00 20130101; A61K
31/7004 20130101; A61K 33/10 20130101; A61K 31/715 20130101; A61K
31/715 20130101; A61K 2300/00 20130101; A61K 31/7004 20130101; A61P
7/08 20180101; A61K 31/715 20130101; A61K 33/10 20130101; A61K
33/14 20130101; A61K 31/19 20130101; A01N 1/0284 20130101; A61K
31/7004 20130101; A61K 31/22 20130101; A61K 31/715 20130101; A61K
33/10 20130101; A61K 33/14 20130101; A01N 1/0221 20130101; A61K
33/14 20130101; A61K 33/14 20130101; A61K 9/0026 20130101 |
Class at
Publication: |
435/100 |
International
Class: |
C12P 019/12 |
Claims
1. An aqueous-based blood substitute solution, wherein said
solution includes an oncotic agent, and does not include more than
5 mM K.sup.+, and does not include a conventional biological
buffer.
2. The solution of claim 1 further comprising Na* and an organic
carboxylic acid, salt, or ester thereof.
3. A solution suitable for use as a blood substitute, comprising:
0-5 mM K.sup.+; concentrations of Na.sup.+, Mg.sup.++, Ca.sup.++,
Cl.sup.-, which are physiological or subphysiological
concentrations; a macromolecular oncotic agent; an organic
carboxylic acid, salt, or ester thereof; and a sugar, with the
proviso that said solution does not include more than 5 mM K.sup.+,
and with the further proviso that said solution does not include a
conventional biological buffer.
4. The solution of claim 3 wherein K.sup.+ is present in the
concentration range of 2-3 mM.
5. The solution of claim 3 wherein Na.sup.+ is present in the
concentration range of 130-150 mM.
6. The solution of claim 3 wherein Mg.sup.++ is present in the
concentration range of 0.20-0.45 mM.
7. The solution of claim 3 wherein Ca.sup.++ is present in the
concentration of 2.0-2.5 mM.
8. The solution of claim 3 wherein said sugar is a simple hexose
sugar selected from the group consisting of glucose, fructose, or
galactose, or a mixture thereof.
9. The solution of claim 3 wherein said organic carboxylic acid,
salt or ester thereof is represented by the formula RCOOX, wherein
R is an alkyl, alkenyl, or aryl, having a branched or straight
chain containing 1 to 30 carbons which carbons may be substituted;
and X is a hydrogen or sodium or other biologically compatible ion
substituent which can attach at the oxygen position, or is a short
straight or branched chain alkyl containing 1 to 4 carbons.
10. The solution of claim 9 wherein said organic carboxylic acid is
selected from the group consisting of lactate, acetate, pyruvate,
or citrate.
11. The solution of claim 3 further comprising NaCO.sub.3.
12. A method for maintaining a partially or substantially
completely exsanguinated subject alive under hypothermic
conditions, comprising substituting a solution comprising a
macromolecular oncotic agent, Ca.sup.++, and which does not contain
a conventional biological buffer.
13. The method of claim 12 further comprising glucose.
14. A method for maintaining a partially or substantially
completely exsanguinated subject alive under hypothermic
conditions, comprising substituting a solution comprised of 0-5 mM
K.sup.+, concentrations of Na.sup.+, Mg.sup.++, Ca.sup.++, Cl.sup.-
at physiological or subphysiological levels, a macromolecular
oncotic agent, an organic carboxylic acid, salt, or ester thereof,
a sugar, and NaHCO.sub.3, with the proviso that said solution does
not include more than 5 mM K.sup.+, and with the further proviso
that said solution does not include a conventional biological
buffer.
15. The method of claim 12 wherein said solution does not contain
K.sup.+.
16. A method for maintaining the biological integrity of a subject
or cells, tissues or organs from said subject, comprising perfusing
said subject or cells, tissues or organs from said subject with a
solution comprising 0-5 mM K.sup.+, concentrations of Na.sup.+,
Mg.sup.++, Ca.sup.++, Cl.sup.- at physiological or subphysiological
levels, a macromolecular oncotic agent, an organic carboxylic acid,
salt or ester thereof, and a sugar, with the proviso that said
solution does not include more than 5 mM K.sup.+, and with the
further proviso that said solution does not include a conventional
biological buffer.
17. The method of claim 14 wherein said solution has substantially
no ability to buffer pH in the range of 7-8 in a cell-free
system.
18. The method of claim 14 wherein said solution does not contain a
buffer selected from the group consisting of HEPES, MOPS, TES,
EPPS, THAM, or TRIS.
19. A method for providing a heat sterilized blood substitute
comprising: placing a solution comprised of 0-5 mM K.sup.+,
concentrations of Na.sup.+, Mg.sup.++, Ca.sup.++, Cl.sup.- at
physiological or subphysiological levels, a macromolecular oncotic
agent, an organic carboxylic acid, salt or ester thereof, and a
sugar, in a heat-sterilizable container; and raising the
temperature of said solution under pressure and for a period of
time sufficient to kill all or substantially all bacteria and
inactivate all or substantially all viruses in the solution.
20. The method of claim 17 wherein said temperature of said
solution is raised under pressure to 120.degree. C. for 15
minutes.
21. The method of claim 17 wherein said solution is placed in an
autoclave.
22. The method of claim 17 wherein said solution is infused into a
subject as a blood substitute or plasma extender.
23. The method of claim 17 with the proviso that said solution does
not include more than 5 mM K.sup.+, and with the further proviso
that said solution does not include a conventional biological
buffer.
24. A method for perfusing a subject prepared for circulatory
perfusion in need thereof, comprising the steps of: reducing the
subject's temperature to a temperature below normal; circulating
into the subject a solution comprising 0-5 mM K.sup.+,
concentrations of Na.sup.+, Mg.sup.++, Ca.sup.++, Cl.sup.- at
physiological or subphysiological levels, a macromolecular oncotic
agent, an organic carboxylic acid or salt thereof, a sugar, and
NaHCO.sub.3; and subsequently returning blood to the subject.
25. The method of claim 22 with the proviso that said solution does
not include more than 5 mM K.sup.+, and with the further proviso
that said solution does not include a conventional biological
buffer.
26. The method of claim 22, comprising the additional step of
placing the subject in a hyperbaric oxygen atmosphere after
reducing the subject's temperature to a temperature below normal,
and further comprising the step of allowing the subject to
regenerate a normal blood level in a hyperbaric oxygen atmosphere.
Description
CROSS-REFERENCES
[0001] This patent application is a continuation-in-part of
application Ser. No. 08/133,527 filed Oct. 7, 1993, which is a
continuation-in-part of application Ser. No. 08/071,533, filed Jun.
4, 1993, which applications are incorporated herein by reference
and to which applications we claim priority under 35 U.S.C. .sctn.
120.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
aqueous solutions such as plasma-like solutions used to perfuse a
living subject in need of perfusion and which act as effective
substitutes for blood. The invention also relates to methods of
preserving the biological integrity of the organs of a mammalian
donor organism (as shown by superior anatomical integrity of
cryopreserved organs and tissues of subjects perfused with the
solution of the invention) and to methods of maintaining a
partially or substantially completely exsanguinated subject at
temperatures substantially below those normally maintained by a
mammal.
BACKGROUND OF THE INVENTION
[0003] Two clinically applied preservation methods for organs are
known: (1) initial perfusion for about 5 min with subsequent cold
storage (2.degree. C.), and (2) continuous perfusion using
solutions containing albumin or plasma.
[0004] Many of the solutions used for initial perfusion with
subsequent cold storage are based on the solutions of Collins et
al. (1969) Lancet 2:1219 and Sacks et.a. (1973) Lancet 1:1024. Ross
et al. (1976) Transplantation 21:498 compared canine renal
preservation following flushing and storage for 72 hours in various
solutions. It was found that only kidneys preserved in a hypertonic
citrate (HC) solution (comprising in part 80 mM K.sup.+, 55 mM
citrate, 400 mOsmol/kg, pH 7.1) survived after 72 hours. The
Collins and Sacks solutions in part contained 115-126 mM K.sup.+,
290-430 mOsmol/kg, pH 7.0-7.3. Wall et al. (1977) Transplantation
23:210 reports the hypothermic preservation of human livers for up
to about 4 hours in a solution in part comprising 250 mg dextrose,
and 15 mEq potassium phosphate. Bishop & Ross (1978)
Transplantation 25:235 reported that renal function was preserved
best in the HC solution of Ross et al. (1976) supra, rather than
other available solutions. Fischer et al. (1985) Transplantation
39:122 found a new preservation solution for hypothermic ischemic
storage (comprising in part 110 mM Na.sup.+, 115 mM K.sup.+, 400
mOsm/kg, solvent D.sub.2O, 110 mM HEPES) to be superior to other
solutions in clinical use, including Collins, Sacks, and HC.
[0005] Among the solutions used for continuous organ perfusion,
Belzer et al. (1985) Transplantation 39:118 reported a newly
developed solution which preserved renal function when kidneys were
perfused for 48 hours and stored for 24 hours (comprising in part
80 mM sodium gluconate, 22 mEq/l K.sup.+, 128 mEq/l Na.sup.+, 4.9
mM adenosine, 10 mM HEPES, 3.0 mM glutathione, 3.75 g % albumin, pH
7.45). Kallerhoff et al. (1985) Transplantation 39:485 examined the
effect of temperature on pH of organs continuously perfused with
two different solutions (Euro-Collins: 10 mM Na.sup.+, 115 mM
K.sup.+, 198 mM glucose, 406 mOsm/L, pH 7.2 at 20.degree. C.; HTK:
15 mM Na.sup.+, 10 mM K.sup.+, 2.0 mM tryptophan, 180 mM histidine,
30 mM mannitol, 310 mOsm/L, pH 7.3 at 8.degree. C.) At incubation
temperatures between 5.degree. C.-35.degree. C., HTK solution
maintained pH at consistently higher values than Euro-Collins
solution.
[0006] Klebanoff & Phillips (1969) Cryobiology 6:121 describe
hypothermic asanguinous perfusion of dogs perfused with buffered
Ringer's lactate at 7.1 to 16.degree. C. Segall et al. (U.S. Pat.
No. 4,923,442) describe a blood substitute capable of maintaining a
subject and its organs at temperatures below 20.degree. C. having
four different solutions--a base solution, a cardioplegia-inducing
solution, a cardioplegia-maintaining solution, and a recovery
solution. The base solution contains electrolytes in physiological
concentration, a macromolecular oncotic agent, a conventional
biological buffer effective at physiological pH, sugar, and K.sup.+
ranging from 4-5 mEq. The cardioplegia-inducing solution had a
K.sup.+ concentration of 25-45 mEq; the cardioplegia-maintenance
solution had a K.sup.+ concentration of 15-45 mEq; and the recovery
solution had a K.sup.+ concentration of 6-10 mEq. Segall et al.
(U.S. Pat. No. 5,130,230) further described the four-solution
system, where the recovery solution contains 0-10 mEq K.sup.+.
SUMMARY OF THE INVENTION
[0007] This invention features methods of using a single solution
suitable to maintain a partially or substantially completely
exsanguinated subject alive at normal temperatures or at
temperatures substantially below those normally maintained by a
mammal, generally less than 37-38.degree. C. and greater than
-2.degree. C., comprising a sub- and/or physiological levels of
K.sup.+ and Mg.sup.++; physiological Na.sup.+, Ca.sup.++, Cl.sup.-;
a macromolecular oncotic agent; an organic carboxylic acid or salt
thereof; and a sugar.
[0008] The solution of the invention may be used as a plasma
extender at normal body temperature. The solution of the invention
is also useful to maintain the life or the biological integrity of
a perfused subject and/or its organs during and after exposure to
profound hypothermic conditions. The solution can also be used to
maintain a euthermic subject in a pressurized environment with
increased oxygen concentration up to 100% O.sub.2 for time periods
sufficient to permit adequate restoration of the subject's blood
components.
[0009] The solution according to the invention may be used to
perfuse and chill a mammalian subject to temperatures profoundly
hypothermic to the subject's normal temperature. The solution can
be used to maintain the subject in profound hypothermia for long
periods of time, usually exceeding an hour, from which an intact
subject can recover without apparent durable ill effects.
[0010] An important distinction of the solution of the present
invention is that it does not require multiple solutions for it to
be effectively administered to a subject for the purposes of blood
substitution, or low temperature maintenance of a mammalian
subject. The solution of the invention may be used at all phases of
plasma extension or blood substitution.
[0011] Another important distinction of the solution of the present
invention is the feature of a subphysiological amount of K.sup.+ at
all steps of administration. This requirement reduces the risk of
hyperkalemia-induced heart sufficiency resulting in blood
transfusion in primates and humans.
[0012] Another important distinction of the solution of the present
invention is the absence of a conventional biological buffer. The
absence of a conventional biological buffer in the solution confers
the important medical advantage of allowing the solution to be
terminally heat sterilized without degradation of solution
components.
[0013] The solution of the present invention requires the presence
of an organic carboxylic acid, salt, or short chain esters thereof.
The organic carboxylic acid, salt or ester thereof is a component
of the dynamic buffer system of the solution able to maintain a
biologically appropriate pH range when used in a mammal.
[0014] The solution of the present invention requires the presence
of a macromolecular oncotic agent sufficient to maintain
physiological osmotic pressure. The macromolecular oncotic agent
used in the solution of the present invention may be a protein(s)
or starch(es).
[0015] An advantage of the solution is that it can be used in a
mammalian subject during all phases of blood substitution from
initial washout of the subject's blood through full substitution of
all or substantially all circulating blood.
[0016] A feature of the invention is that it may be used to
maintain a mammal without blood and also during re-perfusion with
blood.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0017] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a formulation" includes mixtures of
different formulations and reference to "the method of treatment"
includes reference to equivalent steps and methods known to those
skilled in the art, and so forth.
[0018] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the invention, the
preferred methods and materials are now described. All publications
mentioned herein are incorporated herein by reference to describe
and disclose specific information for which the reference was cited
in connection with.
[0019] Red blood cells of primates contain high concentrations of
potassium ion (K.sup.+). When primate blood is stored (as is the
case with virtually all blood obtained from blood banks), even low
levels of lysis of the red blood cells generally result in high
potassium ion concentrations. This is due to release-of potassium
ion from inside the lysed primate red blood cells into the plasma
surrounding the cells. Accordingly, the blood will be hyperkalemic
when infused. The increased potassium level can be diffused if
blood is infused into patients with sufficient circulating blood
since the high potassium ion concentration is diluted. However, the
problem increases if primate blood is transfused into a maintenance
solution of the type described in U.S. Pat. No. 4,924,442, which
contains high concentrations of potassium. The potassium ion
concentration in the transfused blood will not be diluted to safe
levels. As a result, cardiac insufficiency may and frequently does
occur. Hyperkalemia is also associated with tissue damage resulting
from burns, accidents, surgery, chemotherapy, and other physical
traumas. The prior art teaches that organ preservation at low
temperatures requires the presence of high potassium ion
concentrations for the maintenance of tissue integrity.
[0020] The solution according to the present invention contains a
subphvsiological amount of potassium. Thus, the solution allows for
dilution of the potassium ion concentration in stored transfused
blood. As a result, high concentrations of potassium ion and
potential cardiac arrhythmias and cardiac insufficiency caused
thereby can be more easily controlled. The solution containing a
subphysiological amount of potassium is also useful for purposes of
blood substitution and low temperature maintenance of a subject. By
"subphysiological amount of potassium" is meant between 0-5 mEq/l
K.sup.+ (0-5 mM), preferably 2-3 mEq/l K.sup.+ (2-3 mM).
[0021] The solution of the present invention comprises a mixture of
materials which when placed in aqueous solution may be used to
perfuse a subject in need thereof. While the materials may be
provided as a dry mixture to which water is added prior to heat
sterilization, the solution is preferrably provided in the form of
a sterile aqueous solution.
[0022] The solution of the present invention may be used as a
single solution for all phases of procedures in which a subject's
blood is removed and replaced or a subject is cooled. Such phases
include hemodilution or plasma extension at normal body
temperatures, blood replacement and exchange at hypothermic body
temperatures, blood substitution at substantially hypothermic body
temperatures, and subject warming. "Hypothermic body temperatures"
are defined as 4-5.degree. C. below normal body temperatures of
37-38.degree. C. In other words, a hypothermic condition may be
considered to start at body temperatures of about 32-35.degree. C.
"Substantially hypothermic body temperatures" are defined as body
temperatures just below the freezing point (-2.degree. C.) to about
10.degree. C. Therefore, the term "hypothermic body temperature" or
"hypothermia" as used herein encompasses body temperatures of about
-2 to 3.degree. C. to about 32-35.degree. C.
[0023] The solution of the present invention does not include a
conventional biological buffer. By "conventional buffer" is meant a
compound which in solution, in vitro, maintains pH at a particular
range. By "conventional biological buffer" is meant a compound
which in a cell-free system maintains pH in the biological range of
7-8. Examples of conventional biological buffers include
N-2-Hydroxyethylpiperazine-N'-2-h- ydroxypropanesulfonic acid
(HEPES), 3-(N-Morpholino) propanesulfonic acid (MOPS),
2-([2-Hydroxy-1,1-bis(hydroxymethyl)ethyl]amino) ethanesulfonic
acid (TES),
3-[N-tris(Hydroxy-methyl)methylamino]-2-hydroxyethyl]-1-piper-
azinepropanesulfonic acid (EPPS), Tris[hydrolymethyl]-aminomethane
(THAM), and Tris[Hydroxylmethyl]methyl aminomethane (TRIS).
Conventional biological buffers function independently of normal
biological processes, e.g., the conventional buffer is not
metabolized in vivo, and are most potent in cell-free systems.
[0024] The solution of the present invention uses normal biological
components to maintain in vivo biological pH, a concept termed a
"dynamic buffering system". The dynamic buffering system concept
rests on the discovery by the inventors that compounds with no
intrinsic buffering capacity in the biological range, such as
lactate, capable of being metabolized in vivo, act with other
solution components to maintain a biologically appropriate pH in an
animal, even at hypothermic temperatures and at essentially
bloodless conditions. The dynamic buffering system of the present
invention depends in part on oxygenation and removal of carbon
dioxide (CO.sub.2); and allows but does not require additional
bicarbonate (NaHCO.sub.3). The dynamic buffer of the invention has
no or substantially no ability to act as a buffer outside of a
biological system, i.e., a dynamic buffer maintains pH in the
biological range in vivo but not in a cell free environment. A
component of the dynamic buffering system of the invention include
a carboxylic acid, salt or ester thereof. What is meant by a
carboxylic acid, salt or ester thereof is a compound having the
general structural formula RCOOX, where R is an alkyl, alkenyl, or
aryl, branched or straight chained, containing 1 to 30 carabons
which carbons may be substituted, and preferably one of the carbon
chains that compose the carbon chain of lactate, acetate, citrate,
pyruvate, or other biological metabolites; and X is hydrogen or
sodium or other biologically compatible ion substituent which can
attach at the oxygen position, or is a short straight or branched
chain alkyl containing 1-4 carbons, e.g., --CH.sub.3,
--CH.sub.2CH.sub.3.
[0025] As shown in Table 1, a typical conventional buffer solution
(25 mM TRIS) that has an initial pH of about 7.7, and maintains a
pH above 7.2 with the addition of up to 0.12 mls of a 1.25 M HCl
solution. By contrast, the pH of HLB solution (initial pH 7.7)
drops below 7.2 with the addition of about 0.01 ml of a 1.25 M HCl
solution.
[0026] When the solution of the present invention is used as a
blood substitute at hypothermic temperatures, medical grade sterile
NaHCO.sub.3 is added to the heat sterilized solution (HL solution).
The solution containing NaHCO.sub.3 is called HLB solution. The
buffering capacity of HLB solution relative to a conventional
biological buffer in a cell-free system is shown in Table 1. Under
in vivo conditions with oxygenation, HLB solution is shown to
maintain pH above 7.3 in temperatures ranging from 1.6-36.1.degree.
C. (Tables 2 and 3).
[0027] When the solution of the invention is used as a plasma
extender at normal body temperatures, in vivo pH is maintained in
the biological range without the addition of NaHCO.sub.3.
[0028] The absence of a conventional biological buffer in the
solution of the invention confers the important medical advantage
of allowing the solution to be terminally heat sterilized.
Generally, medical solutions are preferred to be terminally heat
sterilized prior to use in a patient. The term, "terminally heat
sterilized" or "heat sterilized" as used herein referes to the
process involving heating a solution to 120.degree. C. for 15
minutes under pressure, i.e., maintaining heat and pressure
conditions for a period of time sufficient to kill all or
substantially all bacteria and inactivate all or substantially all
viruses the solution. This procedure is normally performed in an
autoclave, and is also known as "autoclaving". The purpose of heat
sterilization is to kill possible infectious agents present in the
solution. Infectious agents are known to tolerate temperatures up
to 100.degree. C. It is generally considered by the art that
heating a solution under pressure to 120.degree. C. for about 15
minutes is sufficient to insure sterility.
[0029] All transplant or blood substitute solutions of which the
inventors are aware cannot tolerate terminal heat sterilization. It
is known that heat sterilizing a solution having a pH above 7.0
results in substantial degradation of other solution
components.
[0030] By contrast, the solution of the present invention is
designed to be heat sterilizable with minimal degradation of other
solution components, such as sugar. Solution HL is heat sterilized
prior to use. When it is desirable to add NaHCO.sub.3 to form HLB
solution, NaHCO.sub.3 is added as a commercially-available sterile
1 M solution to sterile HL solutoin. Generally, 5 mls of a 1 M
NaHCO.sub.3 solution is added per liter of HL solution to form 1 l
of HLB solution. However, more NaHCO, may be added.
[0031] The HLB solution of the present invention, or its buffering
organic acids and salts, may also be used to sustain cultured
tissues and cells in vitro. The dynamic buffering system of the
solution maintains cultured tissues and cells at the appropriate
biological pH. We have shown that the addition of lactate and
bicarbonate to cultured cells is sufficient to sustain normal cell
growth and morphology.
[0032] The solution of the present invention includes an organic
carboxylic acid or salt thereof. The term "organic carboxylic acid
or salt thereof" includes any carboxylic acid or carboxylic acid
derivative capable of being metabolized by the mammal. Examples of
carboxylic acids and carboxylic acid salts suitable for use in the
solution of the present invention include lactate and sodium
lactate, citrate and sodium citrate, gluconate and sodium
gluconate, pyruvate and sodium pyruvate, succinate and sodium
succinate, and acetate and sodium acetate. In the following
Examples describing the use of HLB solution, sodium lactate is
used. When metabolized in viva, lactate helps maintain bicarbonate
levels, and thereby functions as a component of the dynamic
buffering system of the solution to maintain an in viva biological
pH.
[0033] For purposes of the further description of the invention,
the mixture according to the invention will be discussed as an
aqueous solution. From the following description of the invention,
it is expected that one ordinarily skilled in the art would be
enabled to provide the mixture as a dry mixture and make the
adjustments to amounts of sodium chloride and organic salt of
sodium as necessary to accommodate the amounts of sodium chloride
found in normal saline solution, which may be used as a diluent for
the dry mixture according to the invention.
[0034] The amount of organic salts of sodium is calculated in a
manner so as to consider the concentration of sodium ions present
in the subject's blood as well as the sodium chloride concentration
of any solution to which dry components are added. An amount is
added so that the concentration of sodium ion obtained from the
organic salt of sodium is sufficient to bring the concentration of
sodium ion in the solution to a concentration about that of
physiologically normal plasma. Therefore, when taking into account
the amount or concentration of sodium ion obtained from the organic
salt of sodium and sodium chloride, the concentration of sodium ion
in the solution is about the concentration of sodium ion found in
physiologically normal plasma.
[0035] The solution also includes a concentration of calcium,
sodium and magnesium ion which is within the range of normal
physiological concentrations of said ions in plasma. In general,
the desired concentration of these ions is obtained from the
dissolved chloride salts of calcium, sodium and magnesium and in
the case of sodium from a dissolved organic salt of sodium which is
also in solution.
[0036] The sodium ion concentration is preferably in a range from
70 mM to about 160 mM, and preferably in a range of about 130 to
150 mM.
[0037] The concentration of calcium ion is in a range of about 0.5
mM to 4.0 mM, and preferably in a range of about 2.0 mM to 2.5
mM.
[0038] The concentration of magnesium ion is in a range of 0 to 10
mM, and preferably in a range of about 0.3 mM to 0.45 mM. It is
important not to include excessive amounts of magnesium ion in the
solution according to the invention because high magnesium ion
concentrations negatively affect the strength of cardiac
contractile activity. In a preferred embodiment of the invention,
the solution contains subphysiological amounts of Mg.sup.++.
[0039] The concentration of chloride ion is in the range of 70 mM
to 160 mM, preferably in the range of 110-125 mM Cl.sup.-.
[0040] The solution also includes an amount of simple hexose sugar
such as glucose, fructose and galactose, of which glucose is
preferred. In the preferred embodiment of the invention nutritive
hexose sugars are used and a mixture of sugars can be used. In
general, the concentration of sugar is in a range between 2 mM and
10 mM with concentration of glucose of 5 mM being preferred. At
times, it is desirable to increase the concentration of hexose
sugar in order to lower fluid retention in the tissues of a
subject. Thus the range of hexose sugar may be expanded up to about
50 mM if necessary to prevent or limit edema in the subject under
treatment.
[0041] The oncotic agent is comprised of molecules whose size is
sufficient to prevent their loss from the circulation by traversing
the fenestrations of the capillary bed into the interstitial spaces
of the tissues of the body. As a group, oncotic agents are
exemplified by blood plasma expanders.
[0042] Human serum albumin is a blood plasma protein used to expand
plasma volume. Also known are polysaccharides, generally
characterized as glucan polymers which are used as blood plasma
expanders. In general, it is preferred that the polysaccharide is
non-antigenic.
[0043] Hetastarch (McGaw, Inc.) is an artificial colloid derived
from a waxy starch composed almost entirely of amylopectin with
hydroxyethyl ether groups introduced into the alpha (1- - - 4)
linked glucose units. The colloid properties of a 6% solution
(wt/wt) of Hetastarch approximates that of human serum albumin.
Other polysaccharide derivatives may be suitable as oncotic agents
in the solutions according to the invention including hydroxymethyl
alpha (1- - - 4) or (1- - - 6) polymers. Cyclodextrins are suitable
oncotic agents.
[0044] D-glucose polymers may be used. For example, dextran, which
is D-glucose linked predominantly in alpha (1- - - 6) linkage, may
be used as the oncotic agent in the solution of the invention.
Polysaccharides such as dextran in a molecular weight range of
30,000 to 50,000 daltons (D) are preferred. Most preferred is
Dextran 40 having a molecular weight of about 40,000 D.
[0045] High molecular weight polysaccharides, such as Dextran 70,
having a molecular weight of about 70,000 D are generally less
preferred because they increase the viscosity of the colloidal
solution, thereby impairing high flow rates. However, for some
uses, high molecular weight dextran solutions are preferred in that
they are more effective in preventing tissue swelling due to their
lower rates of leakage from capillaries. Thus, such high molecular
weight dextran solutions are particularly useful in the treatment
of cerebral ischemia at hyperbaric oxygen tensions and in
effectively managing cerebral oedema. In such circumstances, it may
be desirable to use higher molecular weight polysaccharide such as
dextran in a molecular weight range of 50,000 to 70,000 D.
[0046] When Dextran 40 is used in the solutions according to the
invention, about 8% Dextran 40 (wt/wt) or about 80 grams (g) per
liter (1) of water is used. Molarity of the blood substitute
according to the invention will be in a range of about 290 to 330
milliMolar with a molarity of about 300 being preferred. Most
preferred is a final molarity of about 298 mM.
[0047] The concentration of the polysaccharide is sufficient to
achieve (when taken together with chloride salts of sodium, calcium
and magnesium, organic ion from the organic salt of sodium and
hexose sugar discussed above) colloid osmotic pressure
approximating that of normal human serum, about 28 mm Hg.
[0048] The solution may be used as a circulating solution in
conjunction with oxygen or hyperbaric oxygen at normal body
temperatures, or with or without hyperbaric oxygen in subjects
during procedures. The solution may also be used as a circulating
solution in subjects during procedures when the subject's body
temperature is reduced significantly below the subject's normal
temperature. When warm-blooded subjects are exposed to low
temperature conditions during surgical procedures and in cadaver
organ donation at low temperature, it is generally desirable to
replace the subject's blood with the cold circulating solution of
the invention, or the solution circulated for a time, designed to
perfuse and maintain the subject and its organs intact during the
procedure.
[0049] The solution of the present invention may be administered
intravenously or intraarterially to a euthermic subject which is
placed in a pressurized atmosphere of increased oxygen
concentration up to 100% oxygen or to such a subject undergoing a
procedure during which the subject's body temperature is reduced
significantly below the subject's normal temperature whether or not
hyperbaric oxygen is used. While the solution is being administered
to and circulated through the subject, various agents such as
cardioplegic agents may be administered either directly into the
subject's circulatory system, administered directly to the
subject's myocardium, or added to the circulating solution of the
present invention. These components are added to achieve desired
physiological effects such as maintaining regular cardiac
contractile activity, stopping cardiac fibrillation or completely
inhibiting contractile activity of the myocardium or heart
muscle.
[0050] Cardioplegic agents are materials that cause myocardial
contraction to cease and include anesthetics such as lidocaine,
procaine and novocaine and monovalent cations such as potassium ion
in concentrations sufficient to achieve myocardial contractile
inhibition. Concentrations of potassium ion sufficient to achieve
this effect are generally in excess of 15 mM.
[0051] During revival of a subject (after a period of subnormal
temperature or cryogenic maintenance using the solution according
to the invention to maintain the subject) the subject may be
reinfused with a mixture of the solution according to the invention
along with blood retained from the subject or obtained from blood
donors. As the subject is warmed, whole blood is infused until the
subject achieves an acceptable hematocrit, generally exceeding
hematocrits of about 30%. When an acceptable hematocrit is
achieved, perfusion is discontinued and the subject is revived
after closure of surgical wounds using conventional procedures.
[0052] In general, the solution according to the invention is
administered using an intravenous line (when the subject is at
normal temperature) or to a chilled subject using a pumped
circulating device such as a centrifugal pump, roller pump,
peristaltic pump or other known and available circulatory pump. The
circulating device is connected to the subject via cannulae
inserted surgically into appropriate veins and arteries. When the
solution is administered to a chilled subject, it is generally
administered via an arterial cannula and removed from the subject
via a venous cannula and discarded or stored.
[0053] The solution may be used in a variety of surgical settings
and procedures. It may be useful in delicate neurosurgery where
clear surgical fields are imperative and reduced central nervous
system activity may be desirable and achieved by performing the
procedure on a patient whose core temperature and/or cerebral
temperature has been substantially reduced.
[0054] The solution may be used to maintain a subject (which has
lost a significant amount of blood, e.g. 20% to 98% of its blood)
at normal body temperatures in a pressurized environment at
increased oxygen concentration above atmospheric oxygen tension up
to 100% oxygen. The subject is maintained in a high oxygen
concentration until enough blood components can be synthesized by
the subject to support life at atmospheric pressure and oxygen
concentration. The solution according to the invention may be used
to maintain a subject at temperatures lower than normal body
temperature and at a reduced rate of metabolism after traumatic
life threatening injury until appropriate supportive or corrective
surgical procedures can be performed. In addition the solution may
be used to maintain a patient having a rare blood or tissue type
until an appropriate matching donor can be found and replacement
blood units or other organ can be obtained.
[0055] Surprisingly it has been discovered that it is possible to
replace substantially all of a mammalian subject's circulating
blood with the solution according to the invention and to maintain
the subject alive without reinfusing blood into the subject.
Substantially all of a mammalian subject's circulating blood is
considered to be replaced when the subject's hematocrit drops below
10%. Hematocrit may be lower than 10% if O.sub.2 is provided to the
subject, or substantially lower than 10% in a hyperbaric O.sub.2
chamber. The solution according to the invention can of course be
used to maintain a subject having a hematocrit in excess of
10%.
[0056] The procedure for replacing substantially all of a mammalian
subject's circulating blood may be carried out with the mammalian
subject's body temperature being maintained at its substantially
normal temperature. In addition the procedure may be carried out
with cooling of the subject and reduction of the mammalian
subject's body temperature below that of its normal temperature.
Cooling may be accomplished by chilling the subject in an ice bath,
ice-salt slurry, or cooling blanket. The subject may be further
cooled by chilling the solution according to the invention prior to
perfusing the subject with the solution.
[0057] In the procedure according to the invention for replacing
substantially all of a mammalian subject's circulating blood, it is
preferred that the subject is chilled and perfused with the
solution, using an arterial catheter to deliver the solution to the
subject's circulatory system and a venous catheter to remove blood
and the perfusate from the subject. Substantially all of the
subject's circulating blood is removed in this manner as determined
by measurement of the hematocrit of the effluent from the venous
catheter. When substantially all of the subject's circulating blood
is removed, perfusion is stopped.
[0058] In addition, the procedure for replacing substantially all
of the subject's blood may be carried out with the aid of
hyperbaric O.sub.2. The subject is placed in a hyperbaric chamber
pressurized with oxygen at concentrations exceeding 20%, preferably
100% oxygen. The pressure of the hyperbaric chamber is maintained
during most of the procedure in a range between 0.5 pounds per
square inch over atmospheric pressure to pressures up to about
twice atmospheric pressure. In one embodiment, the procedure is
performed with the subject in a hyperbaric chamber at hyperbaric
pressures of about 0.07 to about 2 atmospheres over ambient
pressure (0.5-30 pounds per square inch [psi]) with 100% oxygen. If
necessary, the pressure of the hyperbaric chamber may be reduced to
atmospheric pressure during wound closure. The subject is
subsequently maintained at hyperbaric pressure at high oxygen
concentration. The pressure is gradually reduced to a lower
pressure but one still hyperbaric. Preferably the pressure is
maintained below 10 psi to about 5 psi for a number of hours to
several days. Subsequently, the pressure is again gradually lowered
below 1 psi and preferably to about 0.5 psi and is maintained at
this pressure for an additional period of time up to a day or
more.
[0059] The solution may also be used to maintain the physiological
integrity of an organ donor subject immediately after the
occurrence of brain death. The subject can be chilled, the
subject's blood removed and replaced with a circulating solution
maintained below 37.degree. C., or while circulating cold solution
according to the invention. Through this use of the solution,
ischemia of vital organs can be minimized. By circulating cold
solution according to the invention through the subject's
circulatory system at low temperature with or without placing the
subject in a hyperbaric oxygen chamber, vital organs can be
maintained for longer periods of time, thus maximizing the number
of organs that can be effectively used from one donor for potential
transplant recipients.
[0060] In another aspect of the invention, it has been discovered
that by using certain adducts, particularly propanediol and high
concentration glucose to augment the solution, it may be possible
to reduce the temperature of donor organs, and in particular donor
hearts, below the freezing point of water (0.degree. C.) and
recover them from freezing in a useful state, i.e. a state capable
of maintaining coordinated cardiac contraction. Furthermore by
using the solution according to the invention with such adducts, it
has been possible to reduce the temperature of intact mammalian
donor subjects below the freezing point of water (0.degree. C.) and
restore them from freezing in a state capable of maintaining
coordinated cardiac contraction. Other organ systems are also
believed to be maintained with a high degree of biological
integrity, i.e. in a physiological state capable of maintaining
life.
[0061] The adducts to the solution include low molecular weight
aliphatic polyalcohols. Diols, exemplified by ethylenediol,
propanediol, and butanediol are preferred. Of these diols
propanediol is particularly preferred. Other polyalcohols that may
be suitable as adducts for low temperature, sub-zero GC
preservation of organ and organ donor subjects are low molecular
weight polyethylene glycol. It is preferred in this aspect of the
invention that the adduct is added to the solution to a final
concentration in a range between about 0.2 Molar to 1 Molar. With
respect to propanediol, in particular a range of 0.2M to 0.6M is
preferred. A concentration of about 0.4M propanediol is most
preferred. 1,2 propanediol is preferred as the adduct to the
solution used for low temperature organ and donor preservation
according to the invention, although 1,3 propanediol may be
used.
[0062] The glucose concentration in the solution useful for
sub-zero .degree. C. preservation of organ and organ donor subjects
ranges between about 0.6M to about 1.4M. A concentration of about
1M glucose is preferred.
[0063] Another adduct that is useful in the solution for low
temperature and sub-zero .degree. C. preservation of organ and
organ donor tissues is trimethylamine oxide (TMAO). TMAO may be
added to the solution described immediately above to a final
concentration in a range between 0.2M and 7M. The solution
including TMAO when perfused into a subject leads to improved
biological integrity of the subject's tissues as evidenced by
superior anatomical preservation of the tissues.
[0064] The following Examples are intended to illustrate the
invention and its use, and are not intended by the inventors to be
limiting of the invention.
EXAMPLES
[0065] The following example is put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how to carry out the synthesis of the invention and
is not intended to limit the scope of what the inventors regard as
their invention. Efforts have been made to ensure accuracy with
respect to numbers used (e.g., amounts, temperature, etc.), but
some experimental error and deviation should be accounted for.
Unless indicated otherwise, parts are parts by weight, molecular
weight is weight average molecular weight, temperature is in
degrees Centigrade, and pressure is at or near atmospheric.
Example 1
[0066] Solution Preparation.
[0067] Preparation of 10 L of Solution A.
[0068] Into an appropriate container, add 80 g/L (or 800 g for 10
liters) of pyrogen-free Dextran 40 (Pharmachem or Pharmacia). Add
deionized water, bringing the volume up to 6-9 liters. Dissolve the
Dextran 40 completely by shaking. The following components may be
added in any order, dissolving each completely before the addition
of the next. The following reagents may be obtained from chemical
supply houses; in this example the listed reagents were obtained
from Sigma: NaCl (5.2 g/L), CaCl.sub.2 (0.29 g/L), MgCl.sub.2 (0.40
g/L), glucose (0.9 g/L), Tris (3.03 g/L), and sodium gluconate
(6.54 g/L).
[0069] Next, the solution is brought to pH 7.80 at room temperature
by the dropwise addition of 0.25M HCl while shaking and monitoring
with a pH meter. The solution is then brought to its final desired
volume (i.e. 10 liters) by the addition of more deionized
water.
[0070] Finally, the solution is pumped through a 0.2.mu. filter
(Gelman, Whatman, or ideally Pall filter units can be used) into
sterile containers or bags. The bottled and capped solution is
stored on ice until used.
[0071] The solution may then be prepared as a sterile dry powder in
containers suitable for preparation of sterile IV solutions after
freeze drying under appropriate conditions.
[0072] Preparation of Solution HL.
[0073] To prepare 50 liters of solution L (BioTime
Hextend.TM.-lactate), 3.0 kg of high molecular weight Hetastarch
(HES) is added to 25 liters of water. Sufficient NaCl is added to
bring the final NaCl concentration to 6.72 g/l. The solution is
stirred until both the HES and NaCl are dissolved. The solution may
be heated to 50.degree. C. if necessary. The total volume is
brought to 45 liters and the following components are added and
mixed until completely dissolved: CaCl.sub.2.2H.sub.2O 18.5 g;
MgCl.sub.2.6H.sub.2O 4.5 g; KCl 11.0 g; glucose 45.0 g; and sodium
lactate 4.03 ml of a 60% (wt/wt) solution. The solution is brought
up to a volume of 50 liters. The solution is filtered to remove
undissolved material and placed in autoclavable containers and
heated in an autoclave to a temperature of 120.degree. C. for 15
minutes.
[0074] Solution HLB.
[0075] To each heat sterilized liter or HL solution is added 5 ml
of a sterile 1 M solution of NaHCO.sub.3, medical grade, forming
HLB solution (BioTime Hextend.TM.-lactate-bicarbonate).
[0076] Solution L.
[0077] Solution L is prepared as described for HL solution above
without the addition of Hetastarch (HES).
Example 2
[0078] Hamster Revived after 1 hour of Ice-cold
Blood-substitution.
[0079] A 41 g female hamster (Mesocricetus auratus), approximately
1 month old, was injected i.m. with 0.04 ml of Vetalar, a 100 mg/ml
solution of the anesthetic ketamine. The animal was packed in
crushed ice and chilled until its rectal temperature was 10.degree.
C. The animal was removed from the crushed ice and placed ventral
side up on a custom-designed stage positioned so that specific
portions of the animal could be observed through a
stereo-microscope during surgery. Its limbs were secured, and the
animal was instrumented with EKG leads and a rectal telethermometer
probe.
[0080] An incision-was made in the right groin region, and the
right femoral vein, and then the right femoral artery, were
cannulated using specially designed micro-cannulas filled with
solution A. After cannulation, 0.02 ml of heparin (1000 U/ml) in
solution A was injected into the animal through the venous cannula,
which was then capped.
[0081] After the right femoral arterial cannulation, the cannula
was connected to a luer-tipped segment of sterile plastic tubing
which was connected to a stopcock mounted on the surgical stage.
The stopcock was connected to another tubing segment which was in
turn connected to a wider, thicker, and more compliant tubing
segment passed through the head of a roller pump. The end of this
wider tubing segment contained a tube for drawing up fluid from a
reservoir. This tube for drawing up fluid from a reservoir termed a
"pick-up" herein was fashioned from the luer end of an 18 gauge
hypodermic needle. This "pick-up" was covered with blood filter
material which was secured by a small rubber "O" ring. The
"pick-up" was inserted into a reservoir of solution A contained by
a centrifuge tube immersed in crushed ice. 0.06 ml of 1M KCl was
added to the solution (15 ml), yielding a molar concentration of
about 4 mM KCl. The line was closed using the stopcock to prevent
back-bleeding into the arterial cannula.
[0082] The hamster was surrounded with crushed ice, and chilled to
4.degree. C. Then 0.2 ml of 1M KCl was injected into the stopcock,
which was opened to allow the injected solution to flow into the
line connecting to the arterial cannula, and from there, into the
animal's femoral artery. The hamster's heart arrested. The animal
was allowed to cool further, and was perfused through the arterial
cannula with 8 ml of solution A 4 mM KCl. Effluent, containing most
of the hamster's blood, was collected from the venous cannula.
After the hematocrit dropped below 5, the roller pump was turned
off for 67 minutes.
[0083] The hamster was then perfused through the arterial cannula
with 8 ml of solution A without KCl, followed by 8 ml of
heparinized blood taken from other hamsters by cardiac puncture. An
equal amount of effluent was collected from the venous cannula.
After the hematocrit exceeded 40%, perfusion with whole blood was
ended, and the cannulas removed.
[0084] The hamster was warmed with a desk lamp, until it became
reactive to stimuli. The cannulas were removed, open blood vessels
ligated, and incisions closed. Further rewarming continued. The
animal fully recovered, and continued to live for weeks following
the experiment.
Example 3
[0085] Cardiac Preservation After Sub-zero Storage.
[0086] A fasted (overnight) female hamster, 40 grams, was injected,
i.m., with 0.02 ml of Ketamine anesthetic (100 mg/ml). The hamster
was immersed in crushed ice until its body temperature lowered to
+14.degree. C. It was then placed on a surgical stage and
instrumented with EKG leads and a rectal temperature probe. The
carotid artery and jugular vein were exposed surgically while the
animals body temperature was maintained between 10-14.degree. C.
and cannulas were inserted into the artery and vein. The arterial
cannula was attached to tubing connected to a peristaltic pump. The
tubing was filled with solution A, containing in addition 20 mM
KCl. The venous cannula was capped until the animal's body
temperature was lowered to SIC using crushed ice and a
temperature-controlled stage set at -1.0.degree. C.
[0087] The animal stopped breathing on its own when its body
temperature fell below 10.degree. C. Respiration with 100% O.sub.2
was initiated. At 5.degree. C., the venous cannula cap was removed
and 3.5 ml of solution A was pumped into the artery at a flow rate
of about 0.3 ml/minute. Afterwards, 4.5 ml of a cryoprotective
solution composed of solution A and in addition 4 mM KCl, 1.0M
glucose, 4% propanediol (i.e. 1.8 g glucose +0.4 g propanediol per
10 ml solution) was infused. During perfusion, the venous effluent
was collected. The animal's temperature was lowered gradually to
0.degree. C. during perfusion. Respiration was discontinued 5
minutes following the onset of perfusion. At this time, more than
30% of the subject's blood volume had been removed. The heart
continued beating until it eventually stopped. Following perfusion
with the cryoprotective solution described in the preceding
paragraph, the animal was placed in a sub-0.degree. C. NaCl slush
(0.6M) solution which was placed in a freezer overnight.
[0088] The freezer temperature was kept at an average of -5.degree.
C. Fifteen minutes after the animal was placed in the freezer, its
rectal temperature lowered from 0 to -1.0.degree. C. The animal's
rectal temperature 12 hours later was -2.5.degree. C. The animal
was then warmed to a temperature of about 2.5.degree. C. in a
Quasar commercial kitchen microwave oven using 7 second pulses with
the setting on warm. The pulses were generated 1 minute apart.
Eighteen pulses were needed to thaw the animal.
[0089] The animal was again placed on the surgical stage and
instrumented with EKG leads and a rectal telethermometer probe.
Three and one half ml of solution A was perfused into the carotid
artery at a flow rate of approximately 0.2 ml/min. The animal's
body temperature was maintained below 5.degree. C. The hamster was
then perfused with whole blood, and gradually warmed.
[0090] After 2 ml of blood had been infused, and the animal's
temperature had climbed to 13.degree. C., rhythmic EKG signals were
detected. With continued perfusion and warming, the amplitude of
the signals became greater, and they increased in frequency. After
5.5 ml of blood had been infused, and the animal's temperature had
reached 25.degree. C., the chest of the animal was opened and its
heart was observed to beat continuously.
Example 4
[0091] Synthetic Solution Substitutes for Blood in a Hyperbaric
Chamber.
[0092] A 40 g hamster, previously fasted overnight, was injected
with 0.03 ml Ketamine (100 mg/ml) i.m. The hamster was placed in
crushed ice, until its body temperature fell below 15.degree. C.
The hamster was removed from crushed ice, and placed ventral side
up on a temperature-controlled stage positioned for microsurgery
below a stereo-microscope. The hamster's temperature was maintained
between 12-15.degree. C.
[0093] Following an incision in the right groin area, the right
femoral vein and artery were exposed. The femoral vein was
cannulated, 0.1 ml of heparin (1000 u/ml) was injected, and the
cannula was capped to prevent bleeding.
[0094] The right femoral artery was then cannulated, and the
cannula was briefly attached to tubing filled with solution A. The
tubing was threaded through the head of a peristaltic pump. A small
volume of the solution (approximately 0.3 ml) was infused to keep
the arterial cannula void of blood. Both the venous and arterial
cannulas were secured to the animal with surgical suture.
[0095] The arterial cannula was capped and the animal was moved
onto the stage in a hyperbaric oxygen (HBO) chamber. A temperature
probe was inserted into the rectum.
[0096] The arterial cannula was attached to tubing which passed
through a peristaltic pump and into a reservoir. The tubing and
reservoir were filled with solution A containing 4 mM KCl.
[0097] The cap was removed from the venous cannula, and the HBO
chamber was closed and pressurized. The peristaltic pump was turned
on, and the animal perfused with solution, which replaced most of
its blood. This blood was allowed to drain from the animal as a
venous effluent. The final chamber pressure was 1.5 atm over
ambient pressure, which was kept constant. The flow rate of
solution into the animal was about 0.3 ml/min. The hamster was
maintained between 14-16.degree. C. using the
temperature-controlled stage on which the hamster was positioned in
the HBO chamber.
[0098] Cardiac activity and breathing were maintained throughout
this period during the perfusion. After 15 ml of solution A
containing in addition 4 mM KCl was perfused into the hamster
replacing the blood, the chamber was gradually depressurized.
[0099] The chamber was then opened, and a hematocrit sample was
taken. The hematocrit was 5%. The venous and arterial cannulas were
capped and the chamber closed and pressurized to 1.5 atm over
ambient pressure.
[0100] The animal continued to breathe on its own in the chamber
for 4 hours after the removal of its blood. After this time, the
chamber was depressurized gradually. Concomitantly, the animal was
cooled to 12.degree. C. The chamber was opened, and the animal was
moved to another surgical stage. Ice was placed on the animal, and
whole blood was perfused into the animal at a flow rate of 0.2
ml/min, as solution was allowed to drain as venous effluent.
[0101] After 1 ml of blood was infused, the ice was removed. The
hamster's body temperature was at 4.degree. C. The animal was then
permitted to warm gradually as the hematocrit was raised by
continuous blood infusion.
[0102] Artificial respiration was initiated after 1 ml of blood was
put back in. The animal's heart never stopped beating rhythmically.
At 21.degree. C., the animal was breathing steadily on its own.
Artificial respiration was discontinued and warming and blood
infusion continued until the animal's temperature reached
25.degree. C. The hematocrit was measured to be 40%. Perfusion was
discontinued, the cannulas removed, blood vessels ligated and
surgical incisions closed.
[0103] One hour following the procedure, the animal was very active
and alert. Four hours after the experiment, the animal was eating
and drinking. At 24 hours after the completion of the
above-described procedure, it appeared completely normal with
respect to posture and behavior, and continued to live for weeks
after the experiment.
Example 5
[0104] Ice-cold Blood Substitution of a Hamster.
[0105] A 46 g hamster, approx. 1 month old, was injected i.m. with
0.02 ml Vetalar, a 100 mg/ml solution of ketamine. The animal was
surrounded by crushed ice until its rectal temperature was about
12.degree. C. The animal was then removed from the crushed ice and
placed ventral side up on an operating stage designed to keep the
animal cold, which is under a stereo-microscope. Its limbs were
secured, and the animal was instrumented with EKC leads and a
rectal telethermometer probe.
[0106] An incision was made in the right groin region. A cannula
was placed in the right femoral vein, and 0.02 ml of heparin
solution (250 U/ml) was injected into the animal through the
cannula which was then capped. Then the right femoral artery was
cannulated. The cannula was connected to a luer-tipped segment of
plastic tubing, and the tubing was passed through a peristaltic
roller pump and into a reservoir containing solution A containing
0.05 M glucose. At the end of the tubing was inserted an 18 G
hypodermic needle to which a mesh blood filter material was secured
at the hub by a rubber "n" ring. The pump was turned on, and fluid
in the reservoir was pumped through the tubing into the femoral
artery of the animal. When the animal's temperature fell below
9.degree. C., ventilation (at 20 breaths/minute) was initiated
using 100% oxygen. The animal was cooled further to a rectal
temperature of 4.degree. C., and 0.1 ml of 0.2M KCl was injected
into the 24 G angiocath which was inserted in the femoral vein.
This injection arrested the heart, and EKG signals ceased. The pump
was turned on, and solution A was perfused into the artery at
approximately 0.2 ml/min while venous effluent was collected.
During the perfusion the animal's temperature dropped to near
1.degree. C. After 4 ml of solution was perfused into the animal,
the pump was turned off and the animal was kept surrounded by
crushed ice in circulatory arrest for 2 hours. Then the animal was
perfused with approximately 7 ml of whole blood (which was
collected from other hamster blood donors) while the animal was
gradually warmed using a desklamp. During the perfusion venous
effluent was collected. The same volume pumped into the artery is
collected as venous effluent. At 10.degree. C., after the animal
remained in cardiac arrest for 3 hours and 11 minutes, heart beats
were first observed upon monitoring EKG signals. Ventilation (6
breaths/minute) of the animal was then initiated using loot oxygen.
As the animal was further warmed and heart beats became stronger
and faster, this rate was increased to about is breaths/minute.
When the animal's temperature was above 28.degree. C. the animal
began to breathe on its own and became responsive. Perfusion was
discontinued (the hematocrit reading 44%) and cannulas were removed
and surgical wounds closed. This hamster remained alive in
apparently normal health for many weeks after the experiment.
Example 6
[0107] Recovery of Heart Beat in an Ice-cold Hamster.
[0108] A fasted (overnight) female hamster, 45 grams, was injected
i.m. with 0.03 ml ketamine anesthetic (100 mg/ml). The hamster was
immersed in crushed ice until its body temperature lowered to about
14.degree. C. The animal was then placed on a surgical platform and
instrumented with EKG leads and a rectal temperature probe. The
carotid artery and jugular vein were exposed surgically using a
stereo microscope. The animal's body temperature was maintained
between 10-14.degree. C. Cannulas were inserted into the carotid
artery and jugular vein. The arterial cannula was connected to
tubing which passed through a peristaltic pump into a reservoir
containing cryoprotective solution composed of solution A
containing, in addition, 11 mM KCl, 1.0M glucose and 4%
propanediol. The venous cannula was initially capped until the
animal's body temperature was lowered to 5.degree. C. using crushed
ice and a temperature regulated platform set near -1.0.degree.
C.
[0109] The animal stopped breathing on its own as the body
temperature fell below 10.degree. C. At this time the animal was
ventilated at about 15 breaths per minute with 100% oxygen. When
the animal's temperature fell to 5.degree. C., the venous cap was
removed and the pump was turned on at a flow rate of about 0.20
ml/minute. The animal's heart stopped beating 21 minutes later, and
ventilation was discontinued 5 minutes after the onset of
perfusion. During the perfusion blood was collected as venous
effluent. Approximately 4 ml of the cryoprotective solution A was
infused into the animal. Then the animal was surrounded by a
salt-ice slurry whose temperature was -2.0.degree. C. The container
that held the slurry and animal was placed inside a temperature
bath set at -5.0.degree. C. The animal's rectal temperature
gradually lowered to -3.4.degree. C. in the morning (18 hours after
the animal was put in the cooling bath). The container was removed
from the cooling bath. The slurry was frozen solid. It was melted
using ice-cold water. Upon removing the "slurry" the animal felt
frozen. The animal was then placed in a kitchen microwave oven. The
oven was set on warm for 7 seconds. The animal was exposed to about
20, 7 second heating cycles over a 20 minute period. This thawed
the animal and raised its rectal temperature to about 2.degree.
C.
[0110] The animal was again. placed on the surgical platform, and
the animal was infused into the carotid artery with solution A. The
cryoprotective solution was collected as venous effluent. About 3
ml of solution A was perfused into the animal at a flow rate of
0.15 ml/minute. Blood which was collected from hamster blood donors
was then perfused in at the same flow rate. After 2 ml of blood was
perfused into the artery of the hamster, the hamster was warmed
slowly using a desk lamp. As blood perfusion and warming continued,
the animals temperature rose above 15.degree. C. and strong
rhythmic EKG signals were recorded. Upon surgical thoracotomy
actual heartbeats could be observed.
Example 7
[0111] Synthetic Solutions as a substitute for Blood in a
Hyperbaric Oxygen Chamber.
[0112] A 43 gram female hamster (fasted overnight) was injected,
i.m., with 0.02 ml of ketamine (100 mg/ml). The hamster was placed
in crushed ice until its body temperature fell to about 14.degree.
C. The hamster was then placed ventral side up on a
temperature-controlled stage positioned for microsurgery below a
stereo-microscope. The hamster's temperature was maintained between
12-15.degree. C. Following an incision in the right groin area, the
right femoral vein and artery were exposed. The femoral vein was
cannulated, 0.1 ml of heparin (250 u/ml) was injected, and the
cannula was capped to prevent bleeding. The right femoral artery
was then cannulated, and the cannula was attached to tubing passed
through a peristaltic pump and into a reservoir filled with
solution A. A small volume of the solution (i.e. 0.2 ml) was
infused to keep the arterial line void of blood. Both the venous
and arterial cannulas are secured to the animal. The arterial
cannula was capped, and the animal was transferred onto the
temperature-regulated stage of a hyperbaric oxygen (HBO) chamber.
The animal's temperature measured rectally was maintained between
13-18.degree. C. The purpose of maintaining the hamster in that
temperature range was to keep the animal's activity low while
ensuring the animal was breathing on its own and reflexively
responsive to stimuli.
[0113] The arterial cannula was connected to tubing that passed
outside the chamber through a peristaltic pump and into a reservoir
(inside the chamber) which contained solution A and 2.5 mM KCl. The
cap was removed from the venous cannula, and the pump was turned on
at a flow rate of about 0.2 ml/min. As the solution was perfused
into the animal, venous effluent (blood) was collected. The chamber
was quickly closed and gradually pressurized to 20-24 psi (100%
oxygen). After about 1 hour of perfusion under pressure the chamber
was gradually depressurized over a period of about 1 hour. Then
perfusion was discontinued. A total of about 13 ml of solution was
perfused into the animal. The cannulas were capped after a sample
of venous effluent was taken to determine the hematocrit. The
animal was placed again on a surgical platform, and the cannulas
were pulled out and wounds tied. The animal showed some very
minimal reflex activity during this time although the animal had
little blood and was breathing room air. The animal was quickly
placed in a box inside the chamber which was pressurized gradually
to about 20 psi. In the chamber was placed food and water for the
hamster. A heat lamp was used to warm the chamber and the animal.
The pressure in the chamber was gradually lowered (over a 1 hour
period) to s psi. The animal's activity increased over the one hour
period until it became quite active. The animal was maintained in
the chamber for about 16 hours at 5 psi. The pressure was then
gradually lowered to 0.5 psi (100% oxygen) and maintained at that
pressure 24 hours. Then the animal was taken out of the chamber and
was placed in a normal cage. The animal continued to appear
completely normal many weeks following the experiment.
Example 8
[0114] Use of Solution A Auaimented with Potassium Chloride to
Blood Substitute Primates
[0115] In this example an 8 kg. juvenile male baboon of the species
Papio anubis was injected i.m. with 60 mg of ketamine. A 22
gauge.times.11/4 in. catheter was inserted in the right cephalic
vein, and 3 ml of 2.5% pentothal was injected i.v. The animal was
then fitted with an endotracheal tube, placed on a surgical table,
and ventilated with a 0.7-2.5% mixture of Flether in 100% O.sub.2,
titrated to the animal's activity. The eyes were coated with
lacrylube for protection.
[0116] The ventilator was set at 18 breaths per minute (bpm), its
stroke volume was 240 ml, and the inspiratory/expiratory ratio was
37%. Airway pressure was maintained at approximately 10 mm Hg, and
the volume delivered with each respiration was checked by examining
the airway pressure trace on a CRT or strip-chart recorder. Airway
pressure was monitored on-line by computer.
[0117] The animal was shaved, and Ringer's lactate drip was
initiated i.v. at a flow rate of 1-3 ml/minute with the rate
titrated to the animal's arterial blood pressure. Terramycin was
administered.
[0118] The extracorporeal circuit consisted of a blood oxygenator,
blood reservoir and pump and was constructed with a secondary
in-line heat exchanger added as close to the animal as possible. It
was further equipped with an external ice water reservoir. The
ice-water reservoir had a pump to supply the oxygenator's built-in
heat exchanger, as well as the secondary heat exchanger with
circulating ice water. All tubing in contact with blood or blood
substitute was sterile. The oxygenator reservoir and circuit was
filled with 2 liters of solution A.
[0119] KCl (4 ml of 2.0 M) was added to the 2 liters of solution A
in an oxygenator reservoir and bypass circuit, yielding a KCl
concentration of 4 mM. A 5F NIH catheter for monitoring arterial
pressure was introduced into the left brachial artery. To it was
attached a 3-way stop-cock (to allow arterial blood sampling every
10-60 minutes throughout the entire procedure). Blood gases, pH, K+
and hematocrit were measured in each sample, and in some cases,
electrolytes, and enzymes as well. The catheter was attached to a
pressure transducer. The transducer was connected to a computer to
monitor central arterial pressure (CAP). Other temperature and
pressure parameters were also measured on-line by the same
computer.
[0120] A 6 F NIH catheter was inserted into a distal branch of the
left brachial vein to allow computerized monitoring of central
venous pressure (CVP). A thoracotomy was performed, and a 6 F
coronary catheter was inserted into the left atrium to monitor left
atrial pressure.
[0121] A 10 F arterial cannula was placed in the left femoral
artery and a 16F venous cannula was placed in the left femoral
vein. Methyl prednisolone (80 mg) was introduced i.v. An esophageal
tube was inserted, and 3 ml of Maalox was administered. The
esophageal tube was fitted with a thermistor probe for recording
deep esophageal temperature.
[0122] Due to the extensive surgical procedures, the baboon spent
about five hours on anesthetic. After the EKG leads were in place,
the animal was put in a netted sling and lowered into an insulated
ice chest. It was then immersed in crushed ice. After 1 hour and 6
minutes of chilling in crushed ice, body temperature sank to
23.degree. C. Nipride (25 mg sodium nitroprusside in 500 ml of 5k
aqueous dextrose) infusion was begun at a rate of 6 ml/hr. The
animal was placed on bypass 17 minutes later, when the temperature
had declined to 21.degree. C.
[0123] At that time, 200 ml of whole blood were removed from the
baboon as venous effluent. The clamps were released which isolated
the monkey's circulation from the bypass circuit, and 2 liters of
solution A, to which were added 2 ml of 2M KCl (final concentration
2 mM KCL), were allowed to blood-substitute the animal. Following
this, its heart was arrested by the i.v. administration of 15 ml of
2M KCl.
[0124] A blood--blood-substitute mixture was continuously removed
as a venous effluent until 4 liters of solution A (to which 22 ml
of 2M KCl had been added) replaced the circulating solution. After
50 minutes of chilled blood substitution, the primate's temperature
had declined to 3.degree. C. Flow through the animal appeared good,
and there was little tendency for the pulmonary arterial wedge
pressure to elevate along with perfusion of the femoral artery. The
cause of this increased flow, and relatively rapid pace of
temperature decline, may be related to the use of nitroprusside,
and also the relatively sparing use of anesthetics during chilling,
which resulted in the animal being somewhat more active as it was
cooled.
[0125] Following blood-substitution, the animal was placed on
circulatory standstill for one hour and 40 minutes. At the end of
the standstill period, 2 liters of ice-cold solution A was added to
the circuit, replacing 2 liters removed as venous effluent. The
minimum body temperature recorded was 2.8.degree. C. Rewarming was
then begun. After 13 minutes of warming, the animal's body
temperature reached 10.degree. C., and 800 ml of a 1:3 mixture of
blood and blood-substitute, followed by 450 ml of a 1:1 mixture,
and finally, approximately 1 liter of whole blood was added to the
circuit, replacing solution A.
[0126] Immediately after blood was introduced into the animal,
heartbeat was detected. Over the next hour and 22 minutes, 40 ml of
NaHCO.sub.3, were introduced i.v. Mechanical ventilation was begun,
and a dopamine drip (200 mg in 250 ml) was administered at 30
ml/hr. CaCl.sub.2 (50 mg) was also injected i.v. Approximately one
hour later, when the body temperature climbed to near normal, the
animal was taken off bypass and placed on a whole blood drip. The
animal's blood gases and blood pressures stabilized in the normal
range.
[0127] One hour later, the cannulas were removed. Since the animal
had been catheterized following a thoracotomy, it was decided that
the long term post surgical management of the animal would not be
attempted, due to the behavioral problems of restraining an untamed
baboon while treating potential chest infections. When ventilation
was discontinued after another hour, the animal displayed agonal
movements and went into cardiac arrest. As the monkey's blood
pressures and blood gases had stabilized, it is clear that the
animal had the potential to survive after being blood-substituted
below 10.degree. C. (deep esophageal temperature) for 2 hours and
30 minutes.
Example 9
[0128] Use of Solution A Without Augmentation in Blood Substitution
of Primates
[0129] In this example an 8 kg juvenile male baboon of the species
Papio anubis was chilled and blood-substituted below 10.degree. C.
for 1 hour and 22 minutes. Prior to chilling and blood replacement,
a 4 F 60 cm Swan-Ganz arrow wedge catheter was placed in the
pulmonary artery via the right femoral vein. This permitted
measurement of the pulmonary arterial wedge pressure without
performing a thoracotomy.
[0130] Keeping the animal anesthetically light, and using
nitroprusside when the temperature fell to 28.degree. C., improved
flow through the bypass circuit. Although the entire procedure went
smoothly, an i.v. injection of 50 mg calcium chloride after
citrated blood was introduced during warming caused massive clot
formation and termination of the experiment. At that time there was
no heparin in the cardiovascular system.
[0131] Procedure.
[0132] The baboon was injected i.m. with 70 mg of ketamine. A 22
gauge.times.11/4 in. catheter was inserted in the left cephalic
vein, and 3 ml of 2.5% pentothal was injected i.v. The ape was then
fitted with an endotracheal tube and moved to the x-ray room. It
was placed on an x-ray table, and ventilated with a 1% mixture of
isofluorane (Flether) in 100% O.sub.2, and a 4 F 60 cm arrow wedge
catheter was implanted in the pulmonary artery through the right
femoral vein.
[0133] The ventilator was set at 20 bpm, its stroke volume was 200
ml, and the inspiratory/expiratory ratio was 37%. Airway pressure
was maintained at approximately 10 mm Hg, and the volume delivered
with each respiration was checked by examining the airway pressure
trace on a CRT or strip-chart recorder. Airway pressure was
monitored on-line by computer.
[0134] The animal was shaved, and a 1-3 ml/minute Ringer's lactate
drip was initiated i.v., with its rate titrated to the animal's
arterial blood pressure.
[0135] The extracorporeal circuit was as described in the previous
Example. The oxygenator reservoir and circuit was filled with 2
liters of solution A.
[0136] A 20 gauge hydromere catheter was placed in the right
femoral vein to allow computerized monitoring of central venous
pressure (CVP). A 3-way stopcock was placed in-line to allow
sampling. A 20 gauge hydromere catheter for monitoring arterial
pressure was introduced into the right brachial artery. To it was
attached a 3-way stop-cock (to allow arterial blood sampling every
10-60 minutes throughout the entire procedure). Blood gases, pH, K+
and hematocrit were measured in each sample, and in some cases,
electrolytes, and enzymes as well. The catheter was attached to a
pressure transducer. The transducer was connected to a computer to
monitor central arterial pressure (CAP). Other temperature and
pressure parameters were also measured on-line by the same
computer.
[0137] A 14 F venous cannula was placed in the left femoral vein,
and a 10 F arterial cannula was placed in the left femoral artery.
After the venous cannula was implanted, 2.6 ml of heparin was
injected i.v. An esophageal tube was inserted, and 3 ml of Maalox
was administered. The esophageal tube was fitted with a thermistor
probe for recording deep esophageal temperature. Methyl
prednisolone (80 mg) was introduced i.v. The eyes were coated with
lacrylube for protection. As the animal was anesthetically light, 1
ml of pentothal was administered i.v.
[0138] The EKG leads were in place, the animal was put in a netted
sling and lowered into an insulated ice chest. It was then immersed
in crushed ice. After 29 minutes of chilling in crushed ice, body
temperature sank to 28.degree. C. The animal was kept
anesthetically light, Flether being turned off as the temperature
dropped below 30.degree. C. Nipride (sodium nitroprusside--25 mg in
500 ml of 5% aqueous dextrose) infusion was begun at a rate of 20
ml/hr and then increased to 40 ml/hr. Over the next 20 minutes, the
Nipride drip was turned on and off sporadically, as the blood
pressure and temperature fell. It was finally turned off when the
animal was placed on bypass 27 minutes later and the temperature
had declined to 23.degree. C. At that time, the clamps were
released which isolated the ape's circulation from the bypass
circuit, 2 liters of solution A were allowed to blood-substitute
the animal, and whole and diluted blood were removed as venous
effluent, and saved for revival. Following this, its heart was
arrested by the i.v. administration of 10 ml of 2M KCl.
[0139] A blood--blood-substitute mixture was continuously removed
as a venous effluent until 4 liters of solution A replaced the
circulating solution. After 39 minutes of chilled blood
substitution, the primate's temperature had declined below
4.degree. C. Flow through the animal was rapid. The pressure in the
pulmonary circulation, which was readily measured, indicated that
the circulation was good, and that the wedge pressure catheter was
well placed.
[0140] After 50 minutes of blood-substitution below 10.degree. C.,
the minimum body temperature recorded was 2.9.degree. C. Rewarming
was then begun, and after 28 minutes of warming, the animal's body
temperature reached 10.degree. C., and 750 ml of whole blood were
added to the circuit, replacing solution A.
[0141] Heartbeat was detected 8 minutes after blood was re-infused
into the animal. Over the next 30 minutes while the animal warmed,
10 ml of NaHCO.sub.3, were introduced i.v. and CaCl.sub.2 (50 mg)
was also injected i.v., as was 80 mg of methyl prednisolone. Within
a few minutes of adding the CaCl.sub.2, massive clot formation was
evident. It was thought that the blood, which was anti-coagulated
with citrate, clotted as a result of adding CaCl.sub.2. The
experiment was then discontinued.
[0142] In this experiment, the rate of flow of blood substitute
through the animal and bypass circuit appeared high, while the left
atrial pressure remained acceptably low. The factors which were
thought to contribute to this result were the use of nitroprusside,
and the maintenance of a light anesthetic state during the cooling
process. 1-2 ml of heparin will be added to the blood prior to its
re-introduction into the animal. It is believed that heparinizing
the re-introduced blood will eliminate the massive clotting which
caused an unexpected end to this experiment.
Example 10
[0143] Ice-cold Blood Substitution of a Dog with Solution HLB.
[0144] Place a 25-30 Kg dog on partial cardiopulmonary bypass.
Surface and core cool the dog to near the ice point (1-3.degree.
C.). Replace the dog's blood with solution HLB hypothermic blood
substitute, described in Example 1. Retain the blood for
transfusion during rewarming. Reduce the animal's body temperature
to near the ice point (below 4.degree. C.) and then rewarm. Replace
the blood substitute with blood with warming and revive the
animal.
[0145] Preparation.
[0146] Catheterize the dog by means of the right radial vein,
injected iv with pentothal, then fit with an endotracheal tube and
ventilate with isofluorane (or Flether) in 100% O.sub.2. Initiate a
Ringer's lactate drip at a rate titrated to the dog's arterial
blood pressure (approx. 40 ml/hr iv). Place the dog on a cooling
blanket cooled with recirculating ice water. Catheterize the right
carotid artery to allow for blood pressure (CAP) monitoring, and
add a 3-way stopcock in-line to allow arterial blood sampling every
10-60 min. throughout the entire procedure. Insert a foley catheter
for urine collection and measure the urine volume throughout the
procedure. Implant a 2 lumen, 7 F, Swan Ganz wedge catheter via the
right jugular vein or right femoral vein, which is fed through the
right heart into the pulmonary artery. Use the distal port to
measure pulmonary wedge pressure (PAW), the proximal port is used
for central venous pressure (CVP). (If necessary CVP may be
measured with a catheter inserted in one of the brachial veins.)
Isolate the left femoral artery and vein and prepare for
cannulation. Heparinize the animal (approx. 5,000 u). Insert a
Biomedicus venous return cannula (15-19 F) in the femoral vein and
a Biomedicus arterial cannula (12-15 F) in the femoral artery.
Measure the activated clotting time (ACT) every 45 min. (until
blood substitution) and adjust the heparin such that it remains
greater than 400 sec. Attach a thermocouple approx. midway to an
esophageal tube and insert the unit so that the tube enters the
stomach. A second thermocouple is placed rectally. Attach ECG
leads. Add Solu-Delta-Cortef (Upjohn, veterinary prednisolone Na
succinate), 80 mg by iv injection. Coat the eyes with Terrimycin
(or Lacrylube), and add DiGel (or Maalox, 20 ml) through the
esophageal tube.
[0147] Measurements.
[0148] Measure arterial blood gasses, pH and hematocrit in every
blood sample, and in some cases electrolytes, enzymes and other
chemistries. Monitor esophageal and rectal temperature as well as
the arterial inflow and venous return blood temperatures. Monitor
CAP, PAW, CVP, ECG, and airway pressure. Temperatures should be
displayed digitally and stored as a function of time in a
computerized data acquisition system. The pressures and ECG should
be displayed as real time waveforms or as numerical data and stored
by the computer.
[0149] Bypass Circuit Components.
[0150] The circuit features a Biomedicus centrifugal blood pump and
flow meter, a Terumo hollow fiber membrane oxygenator with built-in
heat exchanger, Shiley hard shell venous reservoir with filter and
a secondary heat exchanger with integral bubble trap
(Electromedics) located as close to the animal as possible. A drain
segment is located near the inlet of the venous reservoir and
terminates with a check valve. This allows rapid and efficient
blood/blood-substitute exchanges. There is an A-V shunt segment
that allows circulation when not on bypass.
[0151] The venous reservoir can be filled from either the 1 liter
separatory funnel through the "quick prime" port or from dual
infusion bags through one of the cardiotomy ports. The arterial
line from the oxygenator to the arterial cannula and the A-V shunt
are constructed from 1/4" tubing; the venous return, drain and
pump-head lines are 3/8". In those segments where severe bending
can occur, heavy-wall tubing is used or the tube is braced with
"spiral wrap."
[0152] The patient loop is double wrapped and the entire circuit
(sans the factory sterilized reservoir, secondary heat exchanger
and oxygenator) is ethylene oxide gas sterilized as six basic
sections (pump-head, flow meter section, central bypass loop,
funnel, infusion line, and gas filter line).
[0153] Bypass Circuit Support.
[0154] Ice water, pumped from one of two 10 gal. insulated
reservoirs, is used to cool the oxygenator and secondary heat
exchangers. The other reservoir supplies the cooling blanket. At
the onset of surgery, ice water is circulated through the cooling
blanket. At the onset of bypass, room temp. water is circulated
through the circuit heat exchangers.
[0155] Temperature is slowly decreased by adding ice to the
reservoir, in quantities sufficient to maintain a 7-10.degree. C.
difference between the esophageal and blood stream temperatures.
After blood substitution (i.e. to a hematocrit of less than about
4%) full ice water flow is commenced.
[0156] Upon rewarming, ice is removed from the reservoir and the
heater is activated. The temperature of the warming stream is
limited to a maximum of 10.degree. C. greater than the venous
return temperature, by manual adjustment of the heater
thermostat.
[0157] The oxygenator is supplied with sterile, filtered 100%
O.sub.2.
[0158] Blood Substitution.
[0159] The circuit is primed with 2 liters of solution L (Example
1), and recirculated through the A-V shunt to ensure
temperature-gas equilibrium. The cannulas are attached to the
arterial and venous lines of the bypass circuit, and the lines
remain clamped. The cooling blanket is wrapped around the patient
who is surface cooled until a deep esophageal temperature of
35.degree. C. is reached.
[0160] The clamps are removed, and bypass is commenced with the
solution L-diluted blood stream at room temperature (approx.
25.degree. C.). At the onset of cooling, gaseous anesthesia is
discontinued, and the dog is managed with 2.5% pentothal.
[0161] The blood stream is gradually cooled until the animal has an
esophageal temperature of 20.degree. C., at which time blood is
removed by clamping the venous return at the reservoir inlet and
draining from the drain segment while L solution is infused. During
this exchange, an additional 2 liters of L solution is added to the
venous reservoir and when the level of L solution drops to 250 ml,
approximately 6 liters of HLB is added stepwise until all of the
blood is removed (HCT less than 2%, visual observation).
Approximately 4 liters of blood/blood-substitute mixtures collected
in sterile bottles and retained for reinfusion. The very dilute
blood mixture (about 51/2 liters) is discarded.
[0162] After 4 liters have been exchanged (i.e. after the addition
of 2 liters of solution L and 2 liters of solution HLB), 20 meq KCl
will be injected via a stopcock on the secondary heat exchanger, to
arrest the heart. During the exchange, the inflow is adjusted such
that the PAW is kept below 5 mm Hg and the rate of efflux equals
the rate of influx, i.e. as close to isovolemia as possible. At the
end of the exchange the final reservoir level will be about 500 ml,
the PAW below 5 mm Hg and the CVP less than 5 mm Hg. Flow will be
adjusted such that isovolemia will be maintained (constant
reservoir level and the above pressure levels, i.e. PAW <5 mm Hg
and CVP <5 mm Hg).
[0163] When almost all of the blood is removed (HCT less than 4%,
visual observation), the cooling stream can be reduced to ice water
temperature (filling the reservoir with ice), and the dog rapidly
cooled to its minimum temperature. If the HCT is observed to rise
at any time during cold perfusion, the blood mixture can be removed
by exchanging with 2 to 4 liters of solution HLB by the method
described above.
[0164] During the entire procedure, arterial blood samples are
taken and blood gasses, pH, HCT, and in some cases electrolytes,
and other blood chemistries monitored.
[0165] After about 1-2 hours of blood substituted cooling, the
dog's temperature will be about 1-4.degree. C., and rewarming will
begin. The dog will be rewarmed, by removing the ice from the
supply reservoir and warming its contents with the heater which in
turn warms the blankets. When the esophageal temp reaches
15.degree. C., 4 liters of solution L with 25 g mannitol will be
exchanged with the solution HLB followed by the 4 liters of
collected blood mixture. The effluent will be discarded.
[0166] The animal will be warmed gently, blood stream temperature
differential less than 10.degree. C. and never above 40.degree. C.
The heart will spontaneously begin to beat. When the animal's
temperatures (esophageal and rectal) reach about 35.degree. C.,
physiological parameters are stabilized, and it can support itself,
it can be weaned from the extracorporeal circuit.
Example 11
[0167] Reviving an Ice-cold Blood-substituted Dog.
[0168] A 26.8 kg male dog was anesthetized with nembutal and
intubated. It was moved to the operating room, ventilated, and
catheterized with venous, Foley, arterial, and Swan-Ganz catheters,
and after i.v. heparin, its right femoral artery and vein were
cannulated. An esophageal tube was inserted and antacid
administered. Temperature sensors were placed in the esophagus and
the rectum. Methyl prednisolone was injected i.v.
[0169] The animal was wrapped in a cooling blanket, and surface
cooling initiated. The animal's cannulas were connected to a bypass
circuit, which consisted of a vortex blood pump, an oxygenator with
a built-in heat exchanger, a secondary in-line heat exchanger, and
a funnel for the rapid administration of blood and blood
substitute. Whole blood (225 ml) was removed from the dog and saved
for rewarming. Blood volume was quickly replaced with HLB solution.
The bypass circuit containing 1.05 liters of HLB solution was
opened to the animal, and core cooling began.
[0170] Thirty three liters of blood substitute were exchanged. By
the time the ice-point was approached, the hematocrit was far below
1%. The animal's deep esophageal temperature was below 10.degree.
C. for 4 hours and 5 minutes, with a minimum recorded temperature
of 0.70.degree. C. (Table 2).
[0171] Following the hypothermic period, the animal was warmed.
When body temperature climbed past 10.degree. C., venous effluent
and whole blood previously collected, as well as donor blood, was
returned to the circuit; hematocrit increased with increasing
temperature. Lidocaine and bicarbonate were administered, the heart
defribillated, an ventilation begun. When blood pressure and body
temperatures approached normal, the animal was weaned from bypass,
and protamine and Lasix injected. Several hours after warm-up, the
animal was conscious and responsive. The animal remained alive and
well after the procedure.
Example 12
[0172] Reviving an Ice-cold Blood-substituted Baboon.
[0173] A 24 kg male baboon of the species Papio annubis was
anesthetized first with ketamine and acepromazine i.m., then with
i.v. pentothal. It was then immobilized with pancuronium bromide.
It was intubated, ventilated, and catheterized with venous, Foley,
and arterial catheters. The animal was wrapped in a cooling
blanket, and surface cooling initiated. After i.v. heparin was
administered, the baboon's right femoral artery and bilateral
femoral veins were cannulated. Temperature sensors were placed in
the esophagus, rectum and brain. The animal was instrumented for
EKG, somatosensory evoked potentials (SSEPs) and EEG. Dexamethazone
was injected i.v.
[0174] The animal's cannulas were connected to a bypass circuit,
which consisted of a vortex blood pump, an oxygenator with a
built-in heat exchanger, and a funnel for the rapid administration
of blood and blood substitute. Whole blood (300 ml) was removed
from the baboon and saved for rewarming. The volume was quickly
replaced with 300 ml of physiological saline solution. The bypass
circuit, containing 2 liters of Plasmalyte (commercially available
electrolyte solution), was opened to the animal and core cooling
begun.
[0175] After the deep esophageal temperature declined below
13.degree. C., another 2 liters of Plasmalyte containing 12.5 g of
mannitol, was added to the circuit, replacing the mixture of blood
and Plasmalyte which previously filled the circuit. This diluted
blood was saved for use during warming. Immediately afterwards, 10
liters of HLB solution were added, replacing the Plasmalyte. By the
time the ice-point was reached, the hematocrit was far below 1%.
When the animal reached brain temperature of 3.4.degree. C. and
deep esophageal temperature of 2.8.degree. C., the blood pump was
stopped and the animal was maintained under a condition of
circulatory arrest (standstill) for 45 minutes. After this period,
circulation was resumed.
[0176] Following the hypothermic period, 4.2 liters of HLB solution
were added to the bypass circuit, and the animal warmed. When body
temperature reached 15.degree. C., 2 liters of Plasmalyte were
added to the circuit to replace the HLB solution. Mannitol (6.25
g/l) was added to the Plasmalyte in the circuit. Additionally,
venous effluent and whole blood previously collected, as well as
donor blood cells and fresh-frozen plasma, were returned to the
circuit; the animal's hematocrit increased with increasing body
temperature. Another 12.5 g of mannitol were added to the circuit.
When the esophageal and rectal temperatures approached normal, the
heart fibrillated during warming and began beating. Ventilation was
begun. When blood pressure and body temperatures approached normal,
the animal was injected with protamine i.v., weaned from bypass,
its cannulas and catheters removed, and its incisions closed.
[0177] The animal's deep esophageal temperature had been below
15.degree. C. for 3 hours, and below 10.degree. C. for 2 hours 17
minutes, with a minimum recorded temperature of 2.8.degree.C.
(Table 3). The following morning, the animal was able to sit erect
in its cage and pick up and eat pieces of banana, as well as drink
apple juice. It remained alive and well until sacrificed more than
one week later for histological evaluation.
1TABLE 2 REVIVAL OF AN ICE-COLD BLOOD-SUBSTITUTED DOG. MAP HR PAW
CVP Flow TIME SOLUTION TE .degree. C. TR.degree. C. mmHg bpm mmHg
mmHg L/min pH PCO.sub.2 PO.sub.2 Na K Hct 11:57 am 36.1 12:21 pm
225 ml 35.2 129 133 12 3 HLB in & 225 ml blood out @ 12:19 pm
12:39 pm 32.6 34.8 134 141 12 3 12:40 pm 7.41 34.7 581 151 3.1 37
1:35 pm @ 1:36 pm 32.2 32.9 141 132 12 5 1.7 on bypass w/1.05 L HLB
1:40 pm 29.9 31.5 115 128 10 3 1.7 1:43 pm 26.7 29.7 105 122 8 3
1.8 1:46 pm 7.36 37.1 719 143 2.6 24 1:50 pm 5 L HLB 21.9 24.8 66
77 7 2 0.9 0 1:58 pm 4 L HLB 18.5 20.1 19 1.1 2:00 pm 4 L HLB 14.9
18.8 28 1.0 7.48 9.2 812 155 2.5 0 2:02 pm 7.50 8.8 999+ 165 3.6 0
2:04 pm 10.4 16.9 37 1.5 2:05 pm 9.9 16.2 37 2:08 pm 4 L HLB 8.6
15.3 37 1.5 2:14 pm 7.50 11.6 999+ 159 4.2 2:16 pm 2 L HLB 5.7 12.3
27 1.5 2:20 pm 7.50 13.7 999+ 151 5.1 2:22 pm 3.7 10.4 36 1.4 2:25
pm 3.3 9.8 35 1.6 2:27 pm 2.9 9.1 36 1 1.4 2:33 pm 2.1 7.4 37 1.4
2:44 pm 2 L HLB 7.54 11.6 999+ 150 4.6 2:47 pm 1.4 4.8 36 3 1 1.3
2:50 pm 1.2 4.3 37 3 1 1.3 2:52 pm 1.2 4.2 37 3 1 1.3 -- 2:59 pm 2
L HLB 1.1 3.4 21 0.6 3:55 pm 0.9 2.3 22 0.4 4:00 pm 7.63 9.6 999+
150 5.4 4:22 pm 3 L HLB 1.1 2.1 20 0.3 5:00 pm 2 L HLB 0.8 1.6 18
0.4 5:30 pm 3 L HLB 0.8 5:50 pm 7.48 11.0 999+ 150 5.7 5:56 pm 1.8
1.8 19 0.4 6:04 pm 4.7 2.8 27 1.0 6:06 pm 2 L HLB 6.6 3.3 27 1.1 0
6:08 pm 2 L Half 9.7 4.1 30 1.1 20 blood 6:09 pm 9.9 4.2 6:11 pm
10.7 5.3 31 18 7 1.0 6:12 pm 7.30 28.0 902 151 4.6 26 6:15 pm 13.8
6.7 30 24 13 2 1.1 6:25 pm 20.2 10.7 38 6 1 1.4 7.28 27.2 716 154
5.0 27 6:34 pm 1 L blood 6:39 pm 7.34 38.9 670 158 3.2 26 6:42 pm
29.2 18.1 60 143 15 2 1.7 6:48 pm 7.37 28.9 587 154 2.9 27 6:57 pm
32.8 32.2 132 161 8 0 1.6 7:00 pm 7.33 27.3 496 150 2.7 Te:
Esophageal Temperature; Tr: Rectal Temperature; MAP: Mean Arterial
Pressure; HR: Heart Rate; PAW: Pulmonary Arterial Wedge pressure;
CVP: Central Venous Pressure
[0178]
2TABLE 3 REVIVAL OF ICE--COLD BLOOD--SUBSTITUTED BABOON. MAP HR ICP
Flow Plas- TIME TE.degree. C. TR.degree. C. TB.degree. C. mmHg bpm
mmHg L/min pH PCO.sub.2 PO.sub.2 HLB malyte* Blood Hct 1:23 pm 1.6
L + (on .apprxeq.18 12.5 g bypass) mannit ol 1:27 pm 31.3 32.8 33.2
60 83 9 2.2 1:30 pm 28.5 31.1 32.5 60 67 9 2.1 1:32 pm 23.4 29.0
30.9 50 45 8 2.2 1:35 pm 19.3 26.6 28.0 50 27 9 2.1 1:37 pm 18.0
25.5 26.5 50 24 11 2.2 1:38 pm 17.6 24.7 25.6 50 23 9 2.0 1:40 pm
16.8 23.7 24.3 50 25 8 2.0 1:44 pm 18.1 22.8 23.1 50 22 10 2.0 1:46
pm 18.0 22.2 22.3 50 8 2.1 0.3 L 1:50 pm 0.1 L 1:55 pm 12.2 19.3
18.2 50 4 7 L 0 2:02 pm 11.7 18.0 16.1 50 15 1.2 7.40 27 530 2 L
2:05 pm 12.7 17.5 15.1 40 9 1.0 3 L 2:10 pm 11.3 16.9 14.1 40 9 1.3
2:14 pm 10.5 16.2 13.3 50 11 1.3 7.34 17.1 578.6 2:21 pm 9.6 15.0
11.9 50 11 1.3 2:25 pm 8.8 14.3 11.0 50 10 1.3 2:30 pm 7.9 13.4 9.9
50 10 1.3 7.37 21.2 782 2:40 pm 6.4 11.7 8.0 50 9 1.3 2:49 pm 5.3
10.4 6.7 55 10 1.2 2:54 pm 5.4 9.8 6.3 50 7 1.2 3:18 pm 3.9 8.6 4.6
50 7 1.0 3:29 pm 3.2 7.8 3.8 50 8 1.0 3:32 pm 3.0 7.6 3.6 55 8 1.0
3:35 pm 2.9 7.4 3.5 50 7 1.0 3:37 pm 2.8 7.3 3.4 3 4:22 pm 3.7 10.1
4.8 1 4:24 pm 4.3 10.2 4.9 45 6 1.7 4:27 pm 6.5 10.4 6.4 55 8 1.0
2.2 L 4:32 pm 8.3 10.5 7.7 60 8 1.1 3L 4:34 pm 9.0 10.6 8.5 65 10
1.0 4:36 pm 9.4 10.8 9.0 65 7 1.0 4:38 pm 9.9 10.9 9.4 60 6 1.0
4:39 pm 10.0 10.9 9.6 60 7 1.0 4:45 pm 11.4 11.4 11.2 75 10 1.0
4:47 pm 11.9 11.6 11.9 80 9 1.0 4:51 pm 13.2 12.2 13.5 85 7 0.9
4:53 pm 14.1 12.6 14.6 85 slow 7 0.8 7.37 14 762 4:55 pm 14.6 15.2
15.9 90 slow 6 2 L 0 4:59 pm 0.3 L {fraction (1/10)} blood 5:01 pm
2 L 1/4 blood/ 0.3 L plasma 5:05 pm 18.0 15.3 18.1 55 7.33 22 224 2
5:16 pm +12.5 g mannit ol 5:20 pm 24.6 20.0 24.5 44 fib 12 2.1 5:24
pm 0.3 L plasma 5:25 pm 25.0 20.9 25.2 44 fib 13 2.0 7.30 25.3 593
5:36 pm 0.4 L 12 blood + 12.5 g mannit ol 5:37 pm 26.7 22.4 28.7 45
fib 12 2.0 5:43 pm 0.3 L 0.3 L blood 5:55 pm 32.0 24.8 32.8 45 fib
10 2.2 5:57 pm 32.2 25.3 32.9 45 fib 8 2.2 6:10 pm 35.3 28.8 36.6
55 beat 11 6:13 pm 36.3 30.3 36.8 ? 6:23 pm 37.3 33.7 36.2 60 7 7
1.3 7.34 28.2 435 17 6:34 pm 7.39 31.9 322 20 6:36 pm 0.3 L plasma
Te: Esophageal Temperature; Tr: Rectal Temperature; Tb: Brain
Temperature; MAP: Mean Arterial Pressure; HR: Heart Rate; ICP:
Intra-Cranial Pressure
[0179] The invention described above and claimed herein below
embodies novel solutions that may be useful in a number of
procedures. Those ordinarily skilled in the art may be capable in
light of the teaching of the S specification and claims to make
certain additions or modifications to the invention without
departing from the essence of the invention disclosed.
* * * * *