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.


     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 

(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 

     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 
     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 

     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 

     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. 


     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). 


End of report

References Available Upon Request 

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