Date: 25 Jan 97 23:08:59 EST
From: Mike Darwin <>
Subject: Hemostasis in Cryopatients

The following is a BioPreservation, Inc. (BPI) Technical Briefing on the use of
cyanoacrylate to secure hemostasis in human cryopreservation patients.

Securing Hemostasis in the Human Cryopreservation Patient
By Mike Darwin, Steven Harris, M.D. and Sandra Russell, B.S.


Since the first human was cryopreserved over 30 years ago, one drug and one
alone has been retained in the protocols of all cryonics organizations: the
anticoagulant heparin.  There are sound reasons for this.  The
immune-inflammatory cascade (IIC) is activated by multi-system organ failure
(MSOF), trauma, ischemia, hypoxia and agonal shock, some or all of which are
experienced by human cryopreservation patients during their terminal course.
Activation of the IIC leads to a pro-coagulant state and may actually result in
activation of the clotting cascade before cardiac arrest occurs.  This may occur
either as disseminated intravascular coagulation (DIC) or more localized
clotting in the large vessels of the limbs (both arterial and venous) and in low
flow areas such as the atrial appendages.  Following  cardiac arrest, trickle
(during CPR) or absent blood flow results in platelet activation, platelet
adhesion and widespread clotting.  Post mortem, and more recently, agonal
intravascular clotting are recognized as very significant barriers to blood
washout and adequate distribution of cryoprotectant(s) during human
cryopreservation procedures.

For this reason heparin is among the first drugs given upon re-perfusion
following cardiac arrest.  In order to be effective in the setting of activation
of the IIC, acidosis, and quite often the clotting cascade itself,  it is
necessary to administer doses of heparin which completely inhibit hemostasis
(i.e., 400 IU/kg).

Surgical operations, particularly complex and invasive ones such as
cardiopulmonary bypass (CPB), are not carried out on fully anticoagulated
patients.  Indeed, the persistence of reduced hemostatic capability following
CPB is a significant source of morbidity and mortality and every effort is made
to normalize hemostasis by neutralization of heparin, utilization of platelet
conserving drugs during bypass (such as the protease inhibitor aprotinin), and
repletion of platelets via their selective administration.  

In cryopreserving patients, it is necessary to completely inhibit the clotting
cascade as soon as possible which invariably means _before_ invasive procedures
such as femoral cutdown and/or median sternotomy are performed.  If CPB is to be
initiated in the field via femoral-femoral perfusion, then meticulous and time
consuming surgery must be carried out using a radio frequency (RF)
knife/cautery, and/or a fair amount of bleeding must be accepted into the wound
site.  Bleeding slows and complicates surgery.  Further, even if the cautery is
used to maximize closure of microscopic vessels and Weck clips or ligatures are
used to divide or seal the visible vessels, a substantial amount of bleeding
continues from the transected capillaries of the cut surfaces of the wound.

Defining the Problem

If suction is available, this bleeding is mostly an annoyance to the skilled
operator and usually does not seriously interfere with cannulation.  However,
once blood washout is completed, if a long period of extracorporeally supported
ground transport is necessary (which has not infrequently been the case in the
past) then loss of significant amounts of the circulating volume of the
asanguineous perfusate may occur.  This is costly ($200 per liter) and may lead
to the termination of asanguineous perfusion before the patient reaches the
cryoprotectant perfusion facility.  Further, in patients with blood-borne
pathogens (principally HIV), the leakage of large amounts of blood/perfusate
into the circulating water of the portable ice bath (PIB) significantly
increases the risk of infection of transport team members.

If a median sternotomy is performed to facilitate cryoprotectant perfusion,
oozing and leakage of perfusate may be profound.  While cardiotomy suction can
be used to recover, filter and return most of the perfusate lost in this way to
the extracorporeal circuit, its use can be problematic in the presence of
profound pulmonary edema because the lungs fill the thorax and prevent its use
as a sump for collecting wound drainage.  Similarly, other operative sites such
as the incised skin and bone of the cranial burr holes also become significant
sources of perfusate loss.  This is both costly and inconvenient.

In the context of transport and cryoprotectant perfusion, the absence of
hemostasis has resulted in increased cost, increased risk to personnel, and at
best constitutes a major annoyance.  This burden has been an acceptable one in
the framework of the techniques formerly used to effect human cryopreservation. 

However, with the advent of new low viscosity cryophylactic agents and the
continuation of cooling to ultra-low subzero temperatures using agents with very
low viscosities and surface tensions, the need to achieve "hemostasis" (i.e., to
prevent leakage of circulating perfusate from the vascular system) has become
far more critical.

Over the past few years BioPreservation in conjunction with 21st Century
Medicine has increasingly focused on cryopreservation modalities which do not
conclude with introduction of cryoprotectant at +4 to +8 degrees C, but rather
continue with perfusion using a variety of agents (currently under patent) to
temperatures as low as -100 degrees C.  Massive leakage of perfusate from cut
surfaces complicates real-time determination of flow rate through the core
organs of the patient and creates many logistic problems in terms of recovery
and reprocessing of lost circulating fluid.  While the problems of recovery and
reprocessing of large volumes of circulating fluid are solvable both in
principle and in practice, the _best_ solution would be to minimize or stop the
leakage in the first place.

Meticulous use of the cautery and of hemostatic metal clips (we employ the Weck
vascular clip system) is of considerable help.  But it is far from adequate.
Further, when agents with viscosities of  0.5 centistokes or less are perfused,
even a few open paths out of the circulatory system become significant sources
of loss and "short circuiting" of fluid flow.

Solving the Problem

An effort was thus made to secure hemostasis by the application of a variety of
agents to the wounds during the surgical procedure.   Avitene (micro crystalline
collagen), topical thrombin and a number of agents were evaluated with little
success.  These agents had in common several of the same limitations and
disadvantages, chief amongst them was the need to apply them during the early
part of transport when whole blood was still present in the circulatory system.
Thrombin acts by activating the clotting cascade, even in the presence of
heparin.  Avitene acts by creating a sticky gel which acts to mechanically plug
small vessels (and also activates the clotting cascade if unheparinized blood is
present).  Unfortunately, median sternotomy and craniotomy are typically
performed following blood washout.  Additionally, movement of the patient and
manipulation of the wounds disrupts the superficial and relatively fragile
clots/gel that are formed as a result of the application of conventional
clinical hemostatic agents.

What was needed was a hemostatic agent that: 

*acts very rapidly (time course of seconds)
*acts in the cold (i.e., 0-4 degrees C)
*has a favorable spreading coefficient so that it reaches into crevices and
"troughs" in the cut tissue surface
*is not dissolved by water or the cryoprotectant drugs employed to treat the
*acts in the presence of water, and preferably is water-catalyzed to a non
water-soluble end product
*is acceptably non-toxic
*is affordable

This would seem a tall order, but the solution we have found was surprisingly
simple, effective and affordable: N-butyl cyanoacrylate, more commonly known as
"Super Glue" (TM) or Krazy Glue (TM).  Cyanoacrylate is available for soft
tissue applications in veterinary surgery and is marketed under the name of
VetBond (TM).  However, we have been unable to find any material difference in
the toxicological profile or impact on wound healing between VetBond and its
commercially available industrial counterpart Super Glue (TM).  Both achieve
wound closure in survival animals with minimal local irritation and good
outcome; with healing and lack of inflammation and infection as endpoint
criteria.  Four grams of Super Glue can be purchased for $0.89 versus $12.00 for
one 2 gram tube of VetBond.  Additionally, the dispensing system used for the
industrial/commercial product is in our opinion superior to the one used for the
veterinary surgical product (more control over dispensing, less fouling of
resealed tubes, etc.). A review of the literature shows that we were not alone
in reaching this conclusion (1,2).

We have used commercially available cyanoacrylate to achieve hemostasis over
large wound surfaces in the dog while perfusing low viscosity agents with high
spreading coefficients.  We have found the performance of this material to be
remarkable, even when applied _during_ active perfusion (and leakage) as opposed
to immediately after surgery but before perfusion commences. To our surprise we
have found that the material bonds very well to wet tissue surfaces and rapidly
creates a dry wound.  

We have also discovered that cannulae and other _plastic_ appliances can be
readily cleaned of the material by flexion and mild scrubbing.  The wound
surface can also be debrided of the material if it has been applied thickly
enough so that if a large tissue surface has been completely covered in a layer
of cyanoacrylate (which would prevent wound healing on closure) the material can
be peeled off the wound once normal hemostatic mechanisms (i.e., blood
reperfusion) have been restored with surprisingly little trauma.  Small wounds
such as jugular cut-downs can be closed with cyanoacrylate alone without an
adverse effect on healing.

The human cryopatient in some ways can be viewed as an "acute" laboratory
preparation.  An "acute" preparation in biomedical research refers to an animal
model wherein the animal will not survive.  While the objective in human
cryopreservation is most assuredly the recovery and survival of the subject, the
constraints are more relaxed.  For instance, we knowingly inflict currently
irreversible injury on cryopatients in anticipation that it will be reversible
in the future. 

Similarly, a break in sterile technique during cryoprotective perfusion is
unlikely to have any impact, present or future, on the survival of cryopatients
treated with today's techniques (in the context of the anticipated requisite
technology to recover them in the future).  Having made this observation, it is
also prudent to point out that it is wise to sharply constrain the burden put on
"tomorrow's medicine" by today's cryopreservationists.

Cyanoacrylate is not completely innocuous material.  While it has been used for
large-scale repair and hemostasis of wounds to vital parenchymatous organs in
humans (especially stellate fractures of, and lacerations of the liver) it does
have some local toxicity (3) and it has not been approved by the U.S. Food and
Drug Administration for clinical (human) use in soft tissue hemostasis or wound
closure.  However, in the context of cryopatient care today, the very
considerable benefits seem to outweigh the risks.  Cyanoacrylate rapidly and
reliably secures hemostasis of cut tissues even during the perfusion of
cryoprotective agents with good "solventing" capability for most conventional
adhesives.  It is also effective at minimizing leakage from aortotomies in cases
where atherosclerotic or very friable vascular tissue is all that is available
for achieving vascular access.  Dacron (although _not_ teflon) pledgets
saturated with cyanoacrylate can also be used as effective vascular patches for
the relatively low pressure venous side of the circulatory system.  We note that
the commercial product Krazy Glue has been used in desperate clinical situations
during thoracic surgery on humans (2).


We have found the intelligent application of cyanoacrylate to surgical wounds in
the heparinized or asanguineous animal to be a very efficacious hemostatic and
vascular repair agent.  We suggest that the use of cyanoacrylate glue in the
human cryopreservation patient will be similarly advantageous and will allow
conservation of perfusate, less risk of exposure of staff to etiologic agents,
and more effective cryoprotective perfusion and subzero cooling.


1) Mathews SC,  Tissue bonding: the bacteriological properties of a
commercially-available cyanoacrylate adhesive. British Journal of Biomedical
Science, 1993;50:17-20.

2) Robicsek F, Rielly JP, Marroum MC, The use of cyanoacrylate adhesive (Krazy
Glue) in cardiac surgery. Journal of Cardiac Surgery, 1994;9:353-6

3) Papatheofanis FJ, Barmada R,  Increased superoxide anion production in
polymorphonuclear leukocytes on exposure to isobutyl-2-cyanoacrylate.
Biomaterials, 1992;13:403-7.

Selected Bibliography:

Toyoda H, et al., Estimation of the usefulness of
N-butyl-2-cyanoacrylate-lipidiol mixture in transcatheter arterial embolization
for urgent control of life-threatening massive bleeding from gastric duodenal
ulcer.  Journal of Gastroenterology and Hepatology, 1996;11:252-8.

Vanholder R, et al. Cyanoacrylate tissue adhesive for closing skin wounds: a
double blind randomized comparison with sutures.  Biomaterials, 1993;14:737-42.

DeMeritt JS, et. al., Outcome analysis of preoperative embolization with N-butyl
cyanoacrylate in cerebral arteriovenous malformations.  American Journal of
Neuroradiology, 1995;16:1801-7.

Mike Darwin, BioPreservation, Inc.
10743 Civic Center Drive                     TEL: (909)987-3883
Rancho Cucamonga, CA 91730                   FAX: (909)987-7253