X-Message-Number: 2644
Date: 12 Mar 94 20:36:27 EST
From: Mike Darwin <>
Subject: SCI.CRYONICS BPI Tech Brief #5
CREATION AND ELIMINATION OF AIR EMBOLI DURING
PERFUSION OF HUMAN CRYOPRESERVATION PATIENTS
by Michael G. Darwin and Steven B. Harris, M.D.
INTRODUCTION
Permeable cryoprotectives used to achieve colligative
cryoprotection of human cryopreservation patients are
osmotically active. In order to minimize injury from the
perfusion of these agents in hyperosmolar concentrations it
is necessary to gradually increase their concentration
during the course of cryoprotective perfusion (1). This
allows time for the agent(s) to diffuse across the capillary
and cell membranes and gradually exchange with both
intracellular and interstitial water. Introduction of
cryoprotectant(s) at too rapid a rate causes cellular
dehydration resulting in direct injury to the cell membrane
as well as disruption of cell-cell connections.
Achieving a slow, controlled rate of cryoprotectant
introduction is most commonly achieved by the linear gradual
addition of perfusate containing concentrated
cryoprotectant(s) to a recirculating system (which
initially) contains perfusate which is isosmotic with, and
circulated through the vascular system of, the patient or
organ to be cryoprotected (2,3).
A schematic of such a system which has been employed
by the Alcor Foundation to achieve cryoprotective perfusion
is shown in Figure 1. The system consists of two reservoirs
and two pumps: One reservoir, the concentrate reservoir,
contains perfusate which consists of salts, sugars, colloid
and buffer dissolved in a concentrated solution of
cryoprotectant and water. Commonly this would be a solution
made up of perfusate components as shown in Table 1
dissolved in a 65% (v/v) solution of glycerol in water. The
second, or recirculating reservoir, contains the base
perfusate with either little or no added cryoprotectant.
The contents of the concentrate reservoir are then added
gradually to the recirculating reservoir while, at the same
time, an identical amount of perfusate is withdrawn and
discarded from the recirculating system.
One pump, the arterial pump, is used to pump perfusate
from the recirculating reservoir through the patient from
where it returns to the recirculating reservoir by gravity
drainage. The second pump serves to add perfusate
containing concentrated cryoprotectant to the recirculating
system (from the concentrate reservoir) while at the same
time withdrawing an identical amount of the recirculating
perfusate (usually drawn from the more dilute venous return)
and discarding it. In this way the concentration of
cryoprotectant is gradually increased in the recirculating
system (including the patient) until the desired terminal
concentration of agent is achieved, usually 4M to 6M
glycerol. A detailed mathematical analysis and computer
model of this system of cryoprotectant introduction has been
done by Perry (4).
*Illustration Not Shown.
FIGURE 1: Typical Cryoprotective Perfusion Circuit
1) Concentrate Reservoir
2) Recirculating Reservoir
9) Stopcock
10) Arterial Cannula
3) Arterial Pump
4) Pulsatile Flow Generator
11) Venous Cannula
12) Venous sample line
5) Oxygenator
6) Heat Exchanger
13) CPA add/withdrawl pump
14) Drain
7) Arterial Filter
8) Sample manifold
15) Cardiotomy sucrtion
16) Cardiotomy pump
17) Vent line to recirc.
res.
18) Magnetic stirrer
TABLE I
FORMULA FOR MHP-2 BASE
PERFUSATE
Component Molar Concentration
mM
Mannitol 170.00 (182.2)
Adenine HCl 0.94 (MW 180.6)
D-Ribose 0.94 (MW 150.2)
Sodium Bicarbonate 10.00 (MW 84.0)
Potassium Chloride 28.3 (MW 74.56)
Calcium Chloride 1.0 (MW 111)
Magnesium Chloride 1.0 (MW 95.2)
Sodium HEPES 30.0 (MW 260.3)
Glutathione (free acid) 3 .0 (MW 307.3)
Hydroxyethyl Starch ----
50.00 g/l
Glucose 5.0 (MW 180.2)
Heparin ----
1,000 IU/l
-------------------------------------------------
GENERATION OF AIR EMBOLI
The introduction of concentrated cryoprotectant
solution into the recirculating perfusate in such a way as
to minimize osmotic stress requires that the concentrate be
rapidly and completely mixed with the perfusate present in
the recirculating reservoir. This is especially important
since the concentrate has a higher specific gravity than the
recirculating perfusate. A consequence of this is that the
added concentrate solution will sink to the bottom of the
recirculating reservoir where, depending upon the mechanics
of the system, it will either remain as a static, unmixed
layer or, if the intake line of the arterial pump originates
at the bottom of the recirculating reservoir, (a desireable
place for it since this minimizes the chance of pumping air
to the patient) serve as a source of concentrated and
injuriously hyperosmotic cryoprotectant solution which will
be delivered undiluted to the patient.
The solution to this problem has been to vigorously
stir the contents of the recirculating reservoir so that
added cryoprotectant concentrate is quickly diluted and
mixed with the recirculating perfusate. Mixing is typically
achieved through the use of a teflon coated, magnetically
driven stirring bar identical to those used to mix solutions
both in the laboratory and in industry (See figure 2).
* Illustration not shown.
FIGURE 2: Typical magnetically driven laboratory stirrer
set-up.
A consequence of the stirring of the recirculating
reservoir by the rapidly spinning magnetic stir bar is the
generation of an air vortex in the recirculating perfusate.
While this vortex is very effective at both rapidly and
completely mixing the concentrate with the perfusate in the
recirculating reservoir, it is also very effective at
introducing air into the recirculating perfusate as well. At
rates of rotation fast enough to achieve good mixing, the
bottom of the vortex of air reaches the rapidly rotating
stir bar. Air is thus turbulently mixed into the perfusate
where it forms bubbles of widely varying size; the smallest
of which are very stable. As the concentration of
cryoprotectant rises, and the viscosity of the solution
correspondingly increases, air bubbles generated by stirring
in the recirculating reservoir become more and more stable
and begin to saturate the recirculating perfusate creating
large amounts of foam.
This phenomenon was first observed by the author
during a human cryoprotective perfusion carried out at the
Alcor Foundation in Riverside, California in August of 1991.
During that case the top of the acrylic housing of the
hollow fiber bundle of the Sarns 16310 oxygenator and the
top half of the housing of the Pall EC-1440 40 micron
extracorporeal filter were noted to contain large amounts of
foam. A careful examination of the arterial line leading
from the filter to the patient did not disclose the presence
of any visible bubbles, but the phenomenon was nevertheless
very troubling and constituted an unacceptable hazard.
Introduction of air into the arterial circulation
during cardiopulmonary bypass (perfusion) is a catastrophe
about which many articles have been written and about which
many pages of any textbook dealing with perfusion will be
dedicated (5). Introduction of air into the arterial
circulation leads to generation of emboli interrupting the
flow of blood or perfusate to the tissues. While
extracorporeal filters are reasonably effective at excluding
*limited amounts* of macroscopic air, they allow some
microbubbles to pass. Elimination of foam from the arterial
side of the extracorporeal circuit is thus of paramount
importance and constitutes a fundamental of safe and
responsible perfusion of cryopreservation patients.
Attempts to control the generation of foam as a result
of stirring the recirculating reservoir initially consisted
of keeping the perfusate level in the recirculating
reservoir high and keeping the r.p.m. of the stirrer bar to
the minimum required to achieve thorough mixing.
While this approach was moderately successful in
reducing the amount of foam generated during perfusion, it
was not completely effective. Furthermore, as the desired
terminal patient cryoprotectant concentration (i.e., the
desired terminal concentration of glycerol in the patient's
tissues) has risen from 4M where it was in 1985 (6) to the
currently recommended 6M (7) the problem of foam generation
secondary to stirring of the recirculating reservoir has
increased as a result of the increasing peak viscosity of
the recirculating perfusate; perfusate containing 6M
glycerol is far more viscous than perfusate containing 4M
glycerol. More viscous perfusate results in more stable
bubbles.
Recently Biopreservation began a series of experiments
wherein dogs are subjected to transport, blood washout and
cryoprotective perfusion to 6M glycerol employing a model
closely approximating that used to cryopreserve human
patients. During the course of the cryoprotective perfusion
of two of these animals generation of significant amounts of
foam was again observed despite efforts to minimize air
entrainment by the stir bar. While the Pall 40 micron
extracorporeal filter appeared to trap most of this foam,
microbubbles were detected ultrasonically using a Renal
Systems Sonalarm Foam Detector. Furthermore, at the
conclusion of cryoprotective perfusion a fine line of
microbubbles was observed in the arterial line leading from
the Pall filter to the femoral arterial cannula.
PREVENTING THE INTRODUCTION OF AIR
These observations lead to redoubled efforts to solve
the problem of microbubble/foam generation during stirring
of the recirculating perfusate.
The first attempt to solve the problem was made by
floating a polyethylene lid atop the liquid in the
recirculating reservoir to prevent generation of an air
vortex. This lid consisted of a circular 3.5 cm high dish
of polyethylene (closely resembling an inverted plastic tank
lid) of slightly smaller diameter than the recirculating
reservoir. The flat surface of the lid contained small
raised air cells to entrap air and allow the lid to stably
float. Unfortunately, the vigorous stirring required to mix
viscous multimolar solutions of glycerol created sufficient
turbulence at the top of the reservoir to result in tipping,
filling and sinking/tumbling of the floating lid.
Additionally, the air entrapped in the lid air cells was
also entrained into the solution generating foam as a
result. It thus became clear that elimination of the air
vortex would require a floating lid which could not be
tipped by turbulent fluid flow and which was carefully
designed to exclude all air/fluid contact.
A second generation floating lid was then developed
which consisted of a custom-fabricated hollow cylinder of
high density polypropylene 11 cm high by 33 cm wide (See
Figure 3)(manufactured by Custom Fab or Riverside, CA).
This cylinder was closed at both the top and the bottom.
The top surface of the lid was penetrated by a 5 cm screw-
cap port opening. This top port was sealed by an
unscrewable handle approximately 10 cm tall which could
removed to allow for the addition of sterile solution to the
lid so that the depth to which the lid sank in the
recirculating perfusate could be controlled by moderating
its buoyancy with added fluid ballast. This was found to be
of importance since allowing the lid to float too high
permitted air to be entrained beneath the lid as it wobbled
atop the turbulent column of recirculating perfusate. This
wobble could be greatly reduced both by partially sinking
the lid in the perfusate, and by weighting it with the same
fluid ballast use to partuially submerge it, thus
effectively increasing the amount of energy required to
disturb the lid.
*Illustration not shown.
FIGURE 3: Variably bouyant floating lid assembly.
While the use of this sealed, variably buoyant lid
(VBL) was very effective at preventing air from being
introduced if it was applied without interruption from the
start of perfusion, it could not eliminate air that was
accidentally introduced after perfusion began, such as
introduction of air into the venous return line or into the
cryoprotectant concentrate addition line. While these
sources of air are not likely to be routinely encountered,
they nevertheless exist as real possibilities. Further, once
air is introduced and converted into microbubbles, it is
stable and difficult to get rid of.
ELIMINATING FOAM
In order to deal with this problem a second series of
experiments was conducted using both 1% human albumin in
water and 4M glycerol with 2% human albumin in water in a
recirculating system identical to that employed in human
cryopreservations. Both the albumin-water and the glycerol-
albumin-water solutions were very effective at generating
large amounts of stable foam when stirred with a magnetic
stir bar at rates comparable to those employed to mix the
recirculating perfusate during human and canine
cryoprotective perfusions. While the use of the (VBL) was
very effective at preventing foam generation it did nothing
to deal with the problem once it occurred.
To solve the problem of foam generated in the
recirculating reservoir as a result of the inadvertent
introduction of air after the VBL was in place two changes
to the system were made. First, the VBL was treated with
Dow -Corning Antifoam-A (Dow-Corning, Midland, MI) so that
foam accumulating under it would be decomposed into large
bubbles which the centrifugal force of the rotating fluid
column might more easily push out from under the VBL.
Secondly, a Sarns 9438 Filtered Venous Reservoir was
inserted in-line between the recirculating reservoir and the
intake of the arterial pump. The Sarns 8438 reservoir has a
large .15 meter surface area 40 micron filter which is
underlaid with a layer of Antifoam A-treated coarse
debubbling material and overlaid with a layer of Antifoam-A-
treated fine debubbling material.
This reservoir is capable of being operated under
vacuum without leaking air (it is designed to function as a
cardiotomy reservoir as well) and consequently is suitable
for use on the negative pressure side of the perfusion
circuit. The perfusate level in this reservoir was adjusted
(and maintained) at the minimum level of 400 cc (reservoir
capacity is 4500 cc) by use of a Mityvac hand-held vacuum
pump manufactured by Neward Enterprises of Upland,
California.
The interposition of the Sarns 9438 reservoir between
the recirculating reservoir and the arterial pump was
effective at removing all foam and bubbles introduced as a
result of stirring the recirculating reservoir with or
without the use of the VBL. One added advantage to the use
of the Sarns reservoir is the extra filtering capacity which
is likely to be especially useful during perfusion of
patients with intravascular clotting and/or cold
agglutination where steady streams of particulate matter in
the patient's venous return are encountered which might load
the comparatively small surface area of the arterial filter.
This system, consisting of both the VBL and the Sarns
9438 Reservoir, as shown in Figure 4, was then used during
the cryoprotective perfusion of a dog to 6.5M glycerol. The
system was found to perform as effectively in a model
simulating almost exactly the conditions encountered during
cryoprotective perfusion of humans for long-term
cryopreservation.
*Illustration not shown.
FIGURE 4: Human cryoprotective perfusion circuit
incorporating VBL and Sarn 9438 reservoir.
On 6 March, 1994 this system was employed clinically
for the first time during the cryoprotective perfusion of
American Cryonics Society patient ACS 9577. The system
functioned flawlessly and allowed perfusion of the patient
to a terminal concentration of 6.76M glycerol without the
introduction of any air into the arterial perfusate.
Perfusate coming from the recirculating reservoir to the
Sarns 9438 reservoir and from the Sarns reservoir to the
oxygenator and extracorporeal filter (Pall EC-1440) was
observed to be free of air. The VBL was sunk approximately
2 cm into the recirculating solution by the addition of
approximately 2L of Dianeal 5% dextrose containing
peritoneal dialysis solution to the lid as ballast. (Dianeal
was chosen simply because it was cheap and available; we had
a large overstock of it on hand.)
CONCLUSIONS
An undesirable side effect of vigorous stirring of the
recirculating perfusate reservoir during human
cryoprotective perfusions is the introduction of air into
the recirculating perfusate resulting in the generation of
foam. Elimination of this air is achieved by a two-step
process: prevention of air entrainment by elimination of the
air vortex in the recirculating reservoir through the use of
a variably buoyant lid, and defoaming and filtration of the
recirculating perfusate by interposition of a Sarns 8438
Venous Filtration reservoir between the recirculating
reservoir and the intake of the arterial pump. This system
has been shown to be effective at eliminating foam
generation during both experimental and clinical
cryoprotective perfusion.
1) Levin, RL. A generalized method for the minimization of
cellular osmotic stresses and strains during the
introduction and removal of permeable cryoprotective agents.
J. Biomech. Eng. 1982;104:81-86.
2) Darwin, MG, Leaf, JD, and Hixon, HL. Case report:
neuropreservation of Alcor patient A-1068. Cryonics
1986;7:17-32.
3) Jaconsen, IA and Pegg, DE . Cryoprotection of rabbit
kidneys with glycerol. in Organ Preservation II, eds. DE
Pegg and IA Jacobsen, New York: Churchill Livingstone. 1979.
4)Perry RM. Mathematical analysis of recirculating perfusion
systems with apoplication to cryonic suspension. Cryonics
1988;9:24-37.
5) Reed, CC, Kurusz, M, and Lawrence, JR. EA. Safety And
Techniques In Perfusion. Stafford, TX: Quali-Med, Inc. 1988.
6) Darwin, MG, Leaf, JD, and Hixon, HL, ibid.
7) Fahy, GM, personal communication.
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