X-Message-Number: 7684
Date: Thu, 13 Feb 1997 10:37:58 -0500 (EST)
From: Charles Platt <>
Subject: Cryopreservation report (final)
This is the last subsection of the second part of Mike Darwin's report of
the cryopreservation of CryoCare patient James Gallagher.
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The Cryopreservation of James Gallagher (part 2b)
by Mike Darwin
Initiation of Cryoprotective Perfusion at BPI
Cryoprotective perfusion was begun at BPI at 0834 at a
flow rate of 1.1 LPM, a MAP of 45 mmHg, an FiO2 of 9.2, sweep
gas flow rate of 4 LPM an esophageal temperature of 3.2 C, a
right brain surface temperature 1.8 C and left brain surface
temperature of 2.2.0 degrees C. Patient temperatures during
cryoprotective perfusion are presented graphically in figure
9.
(Figure 9 shows temperatures at the start of perfusion near
+2 degrees C and at the end of perfusion near +9 degrees C.)
A Sarns 16310 oxygenator-heat exchanger was used for
oxygenation and temperature control. Sweep gas FiO2 was
decreased to 2.4 at 0842. Cryoprotective perfusion was
initiated with 10% (v/v) glycerol in MHP-2 base perfusate.
This perfusate was recirculated for 10 minutes before
beginning the glycerolization ramp.
Increase of glycerol concentration over 10% (v/v) was
begun at 0840 by the addition of 200 cc/min of 60% (v/v)
glycerol to the recirculating system (patient loop) and the
removal of 170 cc /min of perfusate from the venous return
line to discard. Initial arterial and venous glycerol
concentrations were 0.2 M and 0.7 M respectively at 0846.
Arterial and venous glycerol concentration during the course
of cryoprotective perfusion are presented graphically in
figure 10.
Arterial and venous perfusate samples were drawn at 15
minute intervals during cryoprotective perfusion. The initial
two venous chemistry samples were of questionable value due
to technician error during collection (the arterial side of
the 3 gang stopcock was not completely shut off during venous
sample collection). The first venous sample (chemistry)
results are thus not reported. The first arterial and venous
(gases) perfusate samples were collected at 0839 and
disclosed the following:
Arterial Sample
Measured Normal
Values Range
glycerol (M) 2.8
pH 7.748 7.35 (mean)
pCO2 9.1 mmHg 45-55
pO2 324.2 mmHg 40-50
BUN 5.0 mg/dl 7-25
Creatinine 0.6 mg/dl 0.7-1.4
Sodium 54 mEq/l 135-146
Potassium 29.7 mEq/l 3.5-5.3
Chloride 53 mEq/l 95-108
Calcium 3.0 mg/dl 8.5-10.3
Phosphorus 6.7 mg/dl 2.4-4.5
Protein, Total 0.3 g/dl 6.0-8.5
Glucose 182 mg/dl 70-125
Bilirubin, Total 0.0 mg/dl 0.0-1.3
Alkaline Phosphatase 0.0 U/L 20-125
LDH, Total 113 U/L 0-250
GGT 0.0 U/L 0-65
AST 25 U/L 0-42
ALT 10 U/L 0-48
CPK 187 U/L
Venous Sample
glycerol (M) 2.8
pH 7.223 7.35 (mean)
pCO2 32.7 mmHg 45-55
pO2 99.1 mmHg 40-50
(Chemistries were not available on the first venous
sample.)
The next labs were drawn as follows:
Arterial (gases) 0855:
Measured Normal
Values Range
pH 7.462 7.35 (mean)
pCO2 18.7 mmHg 45-55
pO2 113.6 mmHg 40-50
Arterial (chemistries) 0904:
Sodium 52 mEq/l 135-146
Potassium 28.8 mEq/l 3.5-5.3
Chloride 53 mEq/l 95-108
Calcium 2.6 mg/dl 8.5-10.3
Phosphorus 2.6 mg/dl 2.4-4.5
Glucose 177 mg/dl 70-125
Alkaline Phosphatase 0.0 U/L 20-125
LDH, Total 63 U/L 0-250
GGT 0.0 U/L 0-65
AST 17 U/L 0-42
ALT 8.0 U/L 0-48
CPK 216 U/L
Venous (gases) 0905
pH 7.426 7.35 (mean)
pCO2 21.4 mmHg 45-55
pO2 375.5 mmHg 40-50
Venous (chemistries) 0904:
Sodium 58 mEq/l 135-146
Potassium 30.0 mEq/l 3.5-5.3
Chloride <50 mEq/l 95-108
Calcium <2.5 mg/dl 8.5-10.3
Phosphorus 2.9 mg/dl 2.4-4.5
Glucose 190 mg/dl 70-125
Alkaline Phosphatase 5.0 U/L 20-125
LDH, Total 64 U/L 0-250
GGT 0.0 U/L 0-65
AST 17 U/L 0-42
ALT 5.0 U/L 0-48
CPK 196 U/L
Data for arterial and venous perfusate gases, relevant
chemistries are presented graphically as figures 11 through
17. Graphic data for mean arterial perfusion pressure is
presented in figure 18.
Cryoprotective perfusion proceeded uneventfully. CVP
remained below 10 mmHg until 1000 at which time it was 11
mmHg at a MAP of 68, flow rate of 1.1 LPM and a glycerol
concentration of 5.0M arterial, and 4.2M venous.
(Figure 15 (mislabeled in the printed article) shows arterial
and venous CPK during cryoprotectant perfusion. CPK values
are shown graphically both corrected for dilution (using
dilution of BUN as a reference) and uncorrected. The
uncorrected CPK remains at or below 200 IU/L throughout
cryoprotectant perfusion. Correcting the CPK for dilution
indicates true levels were about 1600 IU/L at the start of
cryoprotective perfusion with a marked and steady decline to
approximately 500 IU/L near the end of perfusion.The final
arterial CPR value is anomalously high at approximately 1580
IU/L
The cerebral cortical surface was repeatedly examined
during cryoprotective perfusion using both flexible and rigid
fiberoptic endoscopes. A Storz Hopkins 26156B 30 degree angle
rigid endoscope was used for maximum resolution of the
cortical surface and could be extended through the burr holes
to view the cortical surface over a 5-6 cm area underlying
the burr hole once cerebral dehydration had become pronounced
(greater than 20%). The flexible scope is a 4 mm diameter 20
cm long custom "cerebroscope" manufactured by Trimedyne Corp.
of Santa Ana, CA. A Storz endoscope camera and Xenon light
were used as the cold light source and imager.
Resolution with the Storz rigid endoscope is at the
level of small arterioles and venules, and particles in the
range of 20 to 30 microns can be easily seen inside vessels.
As a consequence of altered tissue refractive index due to
glycerolization the cortical surface becomes translucent and
it is possible to look into the cerebral cortical surface to
a depth of approximately 3-5 millimeters by adjusting the
focal plane.
In this patient blood washout was judged to be
excellent. The cortical microvasculature was examined at
multiple locations in both brain hemispheres and only
occasionally were any aggregates of RBCs observed; the
frequency of RBC aggregates was comparable to that observed
in non-ischemic dogs undergoing cryoprotective perfusion
following induction of hypothermia and TBW under controlled
(and optimum) conditions.
Optical resolution limitations do not allow for such
detailed evaluation of the intravascular space using the
flexible fiberoptic cerebroscope, however the device does
allow gross evaluation of the cortical surface for non-
perfused areas as large pial vessels which are blood filled
are easily resolved with this instrument. Flexible fiberoptic
endoscopy of the surface of both cerebral hemispheres
disclosed no visible areas of failed perfusion as evidenced
by the absence of blood filled pial vessels. Because the
results of the endoscopic exam indicated uniform cerebral
perfusion, and because clinical observations did not indicate
any problems with cryoprotective perfusion (i.e., no edema,
acceptable MAP and flow rate) intravascular dye was not
administered to evaluate brain perfusion status in this
patient.
Near the end of cryoprotective perfusion an external
temperature probe was anchored with surgical staples to the
left temple. The esophageal probe was repositioned (guided by
fluoroscopy) in the left frontal sinus with the tip resting
on the bone abutting the forebrain.
The brain was noted to be moderately dehydrated at the
conclusion of cryoprotective perfusion with an estimated 30%
reduction in volume.
Terminal glycerol concentrations were 6.7M arterial and
5.45M venous at 1045. Perfusion was discontinued at 1050.
Cephalic Isolation
Surgery for cephalic isolation was begun at 1055. The
skin, cervical musculature, and spinal cord all exhibited
complete blood washout and typical signs of thorough and
uniform glycerolization (dehydration, waxy texture, ambering
of the skin and deepening of skeletal muscle color).
Closure of the burr holes was delayed until the
completion of cephalic isolation. The cranial vault was then
bilaterally suctioned of perfusate (burr hole drainage) and
the isolated head was turned calvarium down to facilitate
additional drainage of perfusate from the burr holes while
the stump was covered in gauze 4"x4" squares and stockinette
put in place. The head was then positioned calvarium up at
which time the burr holes were filled with bone wax (with the
thermocouple probes still in place) and the skin incisions
over the burr-holes were closed with staples. All probes were
further secured with surgical staples to the skin of the
patient's head.
Cooling to -79 Degrees Celsius
The stockinette was then unrolled to cover the entire
head with the temperature probes exiting from the crown of
the head through the stockinette. The stockinette was secured
to the thermocouple probe bundle and excess stockinette
trimmed. The patient (cephalon) was then placed in two 1 mil
polyethylene bags. The patient was then submerged in a 15
liter Silcool bath which had been pre-cooled to -39.8 degrees
C.
The first temperature readings after submersion in the
Silcool were right brain 5.3 C, left brain 6.cc, frontal
sinus, 3.7 C and skin surface -12 C.
The patient's cooling curve to dry ice temperature is
shown in figure 19.
(Figure 19 shows cooling to -80 C taking place over a period
of approximately 16.5 hours. The maximum surface to core
temperature difference was approximately 30 degrees C and
occurred during the first two hours of cooling. Surface to
core temperature differences shown for the remainder of
cooling are in the range of 5 to 10 degrees C.)
Postmortem Examination
A thorough postmortem examination was performed on the
non-cryopreserved remains of this patient. Examination of the
abdominal and thoracic viscera disclosed no infarcted areas
and apparently uniform distribution of cryoprotectant with
the exception of the left ventricular . On cross section of
the left ventricle it was noted that the endocardium had not
perfused and that epicardial glycerolization extended only 5-
7 mm into the ventricular wall. The transition from perfused
to un-perfused tissue was strikingly sharp. We believe this
selective failure of left ventricular endocardial perfusion
is a result of distention of the left ventricle under the
static pressure load of the retrograde aortic perfusion.
Distention of the left ventricle and presumed compromise
of endocardial blood flow are normally avoided in sustained
circulatory arrest cardiopulmonary bypass by the expedient of
venting the left ventricle through the cardiotomy reservoir.
Use of the closed chest approach to cryoprotective perfusion
prohibits this technique from being applied. While this is
likely of no significance in patients who have elected for
neuro-cryopreservation it may be a relative contraindication
to the use of this technique in whole-body cryopatients.
Certainly this finding (confirmed in canine cryoprotective
perfusion using a variety of CPAs) indicates that in whole
body patients undergoing open chest cardiopulmonary bypass
the left ventricle should be routinely vented to assure
adequate perfusion of the endocardium.
Samples of spinal cord, liver, kidney (renal cortex) and
cardiac muscle (left ventricle) were collected for subsequent
evaluation. One set of samples was cooled with the patient,
and is currently undergoing freeze-substitution at -80
degrees C so that transmission electron microscopy can be
performed to determine the ultrastructural integrity of the
tissue and the quantity and location of ice in the
cryopreserved state.
Samples of spinal cord, left ventricle, and renal cortex
were weighed to 0.01 g and then homogenized in known weights
of distilled water for determination of glycerol
concentration by osmometery. Glycerol concentration was
highest in the kidney and lowest in the left ventricle.
Results are given in the Table below.
Glycerol Concentration in Selected Tissues
Tissue Glycerol Concentration in Moles
Left Ventricle 4.25*
Spinal Cord 5.01
Renal Cortex 5.10
*Note that this sample included some visibly
nonglycerolized endocardium.
Postmortem examination disclosed widespread metastatic
adenocarcinoma of the bowel. Metastases were noted in the
liver, both kidneys, lungs, pancreas, mesentery, abdominal
and thoracic lymph nodes, and the mediastinum. The liver was
heavily invaded with tumor both macroscopically and
microscopically. Remarkably a number of the patient's
arteries were invaded with linear rod or wire-like metastases
(confirmed histologically) including the right femoral and
iliac arteries.
Also atypical was the presence of multiple cyst-like,
spherical metastases in the kidney, and widespread invasion
of the skin with multiple metastases ranging in size from 1
cm to 6 cm and also typically presenting as spherical, cyst-
like masses.
The patient had suffered unrelenting nausea with
occasional vomiting and was unable to take normal quantities
of food during the final months of his illness. Despite
aggressive treatment with a wide range of potent anti-emetic
(including marijuana) this remained an intractable problem
throughout the patient's illness. CT of the abdomen was
unremarkable save for the presence of hepatic and renal
masses, and the cause of the patient's nausea remained
undiagnosed during life.
Autopsy disclosed extensive carcinomatous invasion of
the stomach presenting the classic "leather bottle"
appearance with extension of the tumor from the cardiac
portion of the stomach into the mediastinum. The vagus nerve
was encased in tumor to a level above the bronchial hilus.
This is noteworthy in that the patient developed a moderate
bradycardia (HR of 50-60) during the last months of his
illness which was in sharp contrast to his previous high
resting heart rate of 80-90 when he had enjoyed good health.
We presume that vagal involvement with malignant disease was
responsible for this bradycardia as the few cardiac
metastases that were observed were epicardial and right
ventricular and did not appear to impact the cardiac
conduction system.
Another remarkable finding at autopsy in this patient
was the presence of bead-like coal-black nodules in the
mediastinum with many of the hilar lymph nodes exhibiting a
similar appearance. These lesions were strikingly pigmented
and yielded an oily black smear when cut on gauze. Subsequent
histopathological evaluation of these masses and of the lung
disclosed these lesions to be anthracosis. This finding is
remarkable in that the patient had no history of exposure to
coal dust or hydrocarbon pyrolysis products and the patient
had not smoked cigarettes (or cohabited with smokers) in over
a decade. The finding of anthracosis is consistent with the
histological finding of bilateral moderately advanced
emphysema in all lung samples submitted for pathological
evaluation. The etiology of the anthracosis and chronic
obstructive pulmonary disease remains unknown.
Discussion
We believe the care this patient received during the
premortem, agonal, and transport phases of his
cryopreservation represents the best achieved anywhere to
date. Mitigation of antemortem and postmortem shock-mediated
ischemia-reperfusion injury by premedication seems to have
played a critical role in protecting this patient's lungs and
brain from ischemic injury. The use of advanced methods of
CPR allowed for restoration and prolonged maintenance of
acceptable mean arterial pressure and optimum levels of blood
gases and CO2.
However, we believe further improvements to transport
can be made, particularly improved rates of cooling using
intracorporeal (intraperitoneal and intrapulmonary) methods
until extracorporeal circulation and cooling can be achieved.
It is now arguably possible to recover and stabilize
selected cryopatients who have been pronounced legally and
medically dead without the complication of cerebral ischemic
injury (i.e., to stabilize such patients at near 0 degrees C
with brains which are viable by _contemporary_ medical
criteria). However, we note with continuing frustration that
inflicting massive gross, histological, and ultrastructural
disruption as a result of cryoinjury is still unavoidable. We
suggest, in the strongest possible terms, that future
research efforts (and the expenditure of nearly all
discretionary money available to cryonics organizations) be
focused on improving the subzero aspects of human
cryopreservation (cryoprotection and cooling to long-term
storage temperature).
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End of report
References Available Upon Request
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