Postmortem Examination of Three Cryonic Suspension Patients
From Cryonics, September 1984
Postmortem Results: Some Perspectives
By Mike Darwin
[Below] we present a technical paper documenting observations made on the bodies of frozen-thawed suspension patients who were converted to neuropreservation. First, we should point out that long-term care for all three of these patients is continuing and will continue as long as we’re around. Second, we should point out that while the discovery of fractures in the bodies of all three patients is not good news, it is the kind of “bad” news we’ve been prepared for all along.
While the fracturing problem is a serious one (many vital organs were disrupted by one or more fractures) it is not overwhelming in the sense of the bodies being heavily or minutely fractured. While the fractures we observed would certainly disrupt gross functioning, they have little, if any, impact on the stability and integrity of the fine structure and thus the information content of the tissues. A good analogy to this kind of injury would be to envision a phonograph record which someone has scored with a screwdriver. The fidelity of the recording would appear to be hopelessly compromised if it was placed on a standard stereo and played. Yet, the same record with advanced Dolby (TM) noise removal would be virtually indistinguishable from the original. The information is there; the breaks and glitches simply need to be removed.
What these results DO point up is that it is time for those (hopefully few) of us who have been thinking of cryonic suspension in terms of simple viability and traditional surgical repair techniques to wake up and face reality. Contemporary freezing techniques are disruptive and they will require the development of very sophisticated repair techniques. Undoubtedly we will discover damage on the molecular level which is equivalent to or even worse than the fracturing problem we have encountered on a gross level. Repairing such injury will require that we be able to move atoms around almost on a one by one level. It will require that we have tools and engineering capability on a molecular level and that we be able to build molecular repair and fabrication machines–basically our own version of enzymes.
While we may not be able today to design such tools, it is possible for us to set the parameters for repair and to know what such tools will have to do, as well as what their physical and design requirements will have to be. Just because we don’t yet have the capability to build something doesn’t mean we don’t know if it can or cannot in fact be built. In this respect we are in much the same position as Leonardo da Vinci in the 15th Century. Leonardo could envision many, many mechanical devices (indeed he envisioned ALL of them now known but two!) but the levels of engineering and materials science available in his time were just not good enough to build them. Leonardo lived in a time when ball bearings could be thought of but the existing craftsmen were unable to cast and machine metals to that quality and degree of precision. Indeed, it was to be hundreds of years before metallurgy evolved to the point that metals hard enough to withstand the loads required were even developed!
We are in much the same position today. We can see quite clearly the kinds of repair techniques that will generally be required to recover patients from suspension, and yet they are beyond our grasp today! But not tomorrow! That is the beauty of cryonics: we, unlike da Vinci, can afford to wait. It behooves all of us to realize that subtle and powerful techniques will have to be developed to recover those of us frozen with existing techniques. This is one reason why so many of us have elected for neuropreservation as opposed to whole body storage: the kind of repair technology which will likely have to be available to repair our brains will make regrowth of a new body a trivial exercise by comparison. Indeed, new bodies are grown every day in nature right now, while freeze-injured and cryofractured brains are not repaired in nature at all. We urge everyone who is whole-body to rethink the basis of their choice. If the basis of choice is concern over a severed spinal cord or emotional concerns not related to the hard facts, we urge you to reconsider.
On the other hand, we should point out that we intend to try and solve the fracturing problem. At this point it is hard to know how easy this will be to do, or even if we can do it in a fashion consistent with existing resources and realities. Rest assured we are going to be looking for solutions and we have some reasonable amount of optimism that we will find them!
In the meantime, keep the issue of fracturing in perspective. While it isn’t the most pleasant thing to discover, it’s far from the end of the world and in fact just confirms what we were reasonably sure was happening all along: existing freezing techniques cause injury on gross and microscopic levels.
On a more optimistic note, histological studies done on tissues from one of these patients demonstrated excellent cellular preservation on a light microscopy level. Cell to cell relationships were well preserved and other visible structures appeared intact as well. This work gives us much reason for optimism.
A word or two needs to be said about why it took well over a decade to find out such a problem was happening! It cannot be emphasized too much that many cryonicists don’t take the need to do research seriously. They are content to sit on their duffs and let “our friends in the future” fix us up. Well, friendship is a two-way street. Our “friends” of the future had better have some debt of gratitude to owe us or they’re not likely to have our reanimation as a high priority. Research work successfully completed now helps everyone and is the foundation upon which future capabilities will rest. Technical problems are solved a little bit at a time. If we allow ourselves to be demoralized by the magnitude of the task required, or worse if we sit back and wait for others to solve problems we wouldn’t tackle ourselves, we’ve failed before we’ve begun. By and large no one is going to do the kind of work necessary to perfect cryonics except cryonicists. In a very real sense the amount of injury YOU suffer and the degree of recovery YOU experience IF you are revived depend upon your actions NOW. The fracturing problem is just one example of the kinds of problems which need to be and CAN be identified early and solved early.
Finally, a deep debt of gratitude needs to be acknowledged to the husband of one of the suspension patients whose body we examined. All of us owe him our thanks for his generosity in allowing such an examination. That kind of courage and depth of commitment are rare. All of us will benefit from his generosity. Reflecting on the stupidity and short- sightedness of other relatives in similar situations, it is especially apparent that this cryonicist made a major contribution. Our sincere thanks to him, and to the patients themselves, two of whom I know were strongly motivated to participate because of what could be learned by their doing so. Our thanks to all of you.
POSTMORTEM EXAMINATION OF THREE CRYONIC SUSPENSION PATIENTS
by Michael Federowicz, Hugh Hixon and Jerry Leaf
INTRODUCTION
Since the first person was placed into cryonic suspension in l967, well over 30 intact (whole-body) humans have been cooled to liquid nitrogen temperature (1). At the time of this writing, only 3 whole-body patients remain in liquid nitrogen storage (2). Despite the fact that the majority of patients treated by cryonic suspension have been removed from liquid nitrogen storage and interred conventionally, to our knowledge, no previous efforts have been made to subject these individuals to a postmortem examination in order to determine the efficacy of the cryonic suspension protocol used to prepare them.
Postmortem examination of cryonic suspension patients being removed from long-term storage could serve to answer many important questions and could facilitate quality control just as it does in medical practice (3). Key questions of concern to cryonicists are:
1) How effective are various approaches to perfusion in achieving blood washout and cryoprotective agent (CPA) equilibration?
2) How is CPA distributed during “optimum” perfusion vs. post-ischemic or “suboptimum” perfusions?
3) What are the gross and microscopic effects of CPA perfusion and freezing and cooling to liquid nitrogen temperatures?
4) What is the impact of transport or other handling techniques on the gross integrity of the body?
While answers to many of these questions may be approximated by the use of small animal models (4), many problems and complications introduced by the advanced age of many suspension patients and the large mass of adult humans cannot be easily answered in this fashion.
Due largely to the unfortunate circumstances which usually surround removal of patients from cryonic suspension (5), it has not been possible until very recently to subject patients being removed from cryonic suspension to autopsy. During November and December of l983 the authors were afforded the opportunity to conduct a postmortem examination on the remains of three whole-body cryonic suspension patients who had been converted to neuropreservation.
PATIENT HISTORIES
On November 2, 1983 two whole-body cryonic suspension patients who had been maintained in liquid nitrogen storage for 5 and 9 years respectively, were removed from storage and converted to neuropreservation. PATIENT 1 (P1) was a male, caucasian, 65 years of age who suffered a lethal cerebrovascular accident in 1974. The patient was not near a cryonics facility when he deanimated and it was necessary for a team to be flown a considerable distance to perform perfusion, resulting in a delay of almost 24 hours after declaration of legal death. Perfusion was complicated by a number of technical difficulties and equipment failures as well as by severe edema, secondary to the use of a colloid-free DMSO-based perfusate (6). Vascular access for perfusion was via the left common carotid artery and was achieved by a local mortician who assisted with the case. Perfusion was open circuit and drainage was via both the left and right internal jugular veins. Perfusion consisted of initial blood washout with 32 liters of bicarbonate-buffered Ringer’s solution and cryoprotective perfusion (also open circuit) with 32 liters of a glycerophosphate-based perfusate containing 15% DMSO (v/v). Standard embalming equipment (Porta-Boy embalming pump) was used for perfusate delivery. A total of 64 liters of perfusate was employed. Effluent samples collected at the time of perfusion were lost late in 1983 when they exploded (as a consequence of rapid boiloff of liquid nitrogen which had leaked into the vials) during a transfer of the patient from one storage dewar to another.
The protocol for cooling the patient to dry ice temperature consisted of wrapping in a thin insulating blanket and packing in crushed dry ice. The patient was prepared for cooling to liquid nitrogen temperature by being wrapped in multiple layers of fiberglass cloth, polyethylene, and aluminum foil. Following wrapping, the patient was placed on a heavy gauge aluminum stretcher and cooled to liquid nitrogen temperature by placement in a cryogenic dewar which was gradually filled with liquid nitrogen over a period of 5 days (Figure 1).
Figure 1. First Cooldown of P1. Thermocouple probe placed externally on head. Vertical marks on horizontal axis indicate midnight. “Start” indicates beginning of cooldown.
PATIENT 2 (P2) was a female caucasian 68 years of age who was pronounced legally dead in 1978 in a location remote from cryonics facilities. Deanimation was sudden and reportedly due to an acute myocardial infarction. There was approximately a 72-hour delay between the time of death and the time perfusion was begun (all of this delay period was in the presence of either air or ice cooling). Vascular access for perfusion was via the aortic root and the right atrium following median sternotomy (7). Perfusion was accomplished utilizing a heart-lung machine with appropriate 40 micron in- line filters and a Bentley Q-100 “bubbler” oxygenator. The perfusate was a glycerophosphate type with PVP-40 as the colloid. Perfusate was filter- sterilized by passing through a 0.2 micron filter. The patient was exposed to three open circuit passes of perfusate consisting of 10 liters of 5% DMSO (v/v), 10 liters of 10% DMSO (v/v) and 40 liters of 15% DMSO (v/v). Following completion of perfusion the patient was cooled in an isopropyl alcohol bath at an average rate of of 2.27 degrees centigrade per hour. Following cooling to dry ice temperature, the patient was removed from the cooling bath, wrapped in two layers of 1/4″ closed cell nitric rubber (ensofoam) and placed in a mylar bag. Via a lifting block attached to the feet the patient was then lowered into a cryogenic dewar and cooled to liquid nitrogen temperature very rapidly by partial submersion. The temperature descent curve to -196 degrees centigrade are not available on this patient as a result of loss of records by the contract company which prepared this patient for long-term storage.
In 1982 P1 was removed from liquid nitrogen storage due to inadequate maintenance funds, placed in a sleeping bag and immediately transferred to a dry ice storage chest which had been filled with dry ice previously cooled to near liquid nitrogen temperature. The patient was stored on dry ice for 19 days and was then transferred to a cryogenic dewar and again gradually cooled to liquid nitrogen temperature over a period of 8 days at a rate of 0.9 degrees centigrade per hour (Fig. 2).
Figure 2. Second Cooldown of P1. Thermocouple probes placed externally on head and ankle. Vertical marks on horizontal axis indicate midnight. “Start” indicates beginning of cooldown.
Both patients were maintained in the storage dewar in a head-down position in order to minimize any thermal cycling of the central nervous system which might result from fluctuating liquid nitrogen levels. The distal 1 to 1.5 meters (i.e.-knees to feet) of the patients bodies were alternately exposed to vapor and liquid nitrogen as a result of normal boil-off/fill cycles. Both patients were maintained in the same cryogenic dewar during the last year of their liquid nitrogen storage. Prior to this both patients had been maintained in separate storage dewars and both had been transferred on two occasions owing to logistic needs or vacuum failure of the storage vessels.
PATIENT 3 (P3) was a 36-year-old caucasian female who deanimated suddenly in 1980 following numerous chronic illnesses including profound immune deficiency, multiple opportunistic infections, adenocarcinoma of the throat, therapeutic radiation overdose, idiopathic liver disease, and an unclassified central nervous system myelopathy. Since deanimation was sudden and remote from cryonics facilities there was approximately a 24-hour delay before the start of perfusion during which time thorough external cooling with ice was carried out.
Perfusion consisted of blood washout and extracorporeal circulation with a heart-lung machine, employing the aortic root and right atrium for vascular access (8). Perfusion was closed-circuit, employing a glycerophosphate-based perfusate using PVP-40 as the colloid glycerol as the cryoprotective agent. The concentration of glycerol was gradually increased in the recirculating system until the venous concentration reached 2.85M. Following perfusion the patient was cooled to dry ice temperature at a rate of approximately 2 degrees centigrade per hour by submersion in an isopropanol bath and gradual addition of dry ice.
Five days following cooling to dry ice temperature the patient was placed in a urethane (open cell) foam insulated aluminum cassette, positioned in a storage dewar and gradually cooled over a two-week period to liquid nitrogen temperature (Figure 3).
Figure 3. Cooldown of P3 Thermocouple probes at head and ankle placed externally on skin. Esophageal and rectal probes placed internally. Vertical marks on horizontal axis indicate midnight. “Start” indicates beginning of cooldown, with LN2 liquid level below head of patient in upside down position. “Add LN2” indicates addition of more liquid.
REMOVAL PROTOCOL
P1 and P2 were removed from the storage dewar using a high-reach forklift and lowered to the ground on their supporting stretchers in a supine position. The head and upper torso of each patient was unwrapped and conversion to neuropreservation was achieved using a high speed electric chain saw while the patient’s head was lavaged with liquid nitrogen. A block of tissue approximately 12 by 16 cm. was cut from the decapitation site on the body and banked with the heads as a reference sample. Following conversion to neuropreservation the bodies were wrapped in several layers of 4 mil plastic tarpaulin and allowed to rewarm for 21 hours on a concrete floor. All insulating wrappings (sleeping bags, foil-fiberglass, Ensofoam) were allowed to remain in place. After 21 hours of rewarming, the insulating materials were removed, the patients were examined externally, samples were taken, and surface and “core” temperature measurements were made. After 21 hours, “core” temperature was -47 degrees centigrade and surface temperature was -46. (Core temperature was approximated by inserting the thermistor probe into deep cuts made as a consequence of sample taking.) At the time of unwrapping, numerous samples were removed by chiseling with a precooled steel chisel and were thawed in fixative at 22 degrees centigrade for possible light/electron microscopy at a later date.
Samples of tissue were also allowed to thaw out at room temperature and fluid collected from them for examination by light microscopy.
P3 had been placed into long-term liquid nitrogen storage in an aluminum cassette which was lined and heavily packed in open cell urethane foam to act as a thermal barrier. Inside the cassette P3 was wrapped in a 1/2-inch thick layer of Ensofoam and two ethylene vinyl acetate (EVA) bags.
The cassette containing the patient was removed from the storage dewar using an overhead crane and lowered to the ground with the patient in a supine position within the cassette. The cassette was then opened and sufficient urethane foam was cleared away to facilitate removal of the patient. The patient was removed to an isolation tent with specially constructed supports, where a rapid conversion to neuropreservation was done using a high speed electric chain saw.
After conversion to neuropreservation, the body of P3 (still enclosed in a 1/2-inch Ensofoam wrap and EVA bags) was transferred to an insulated rewarming chest for attempted controlled rewarming to 0 degrees centigrade. This was done in an effort to minimize the possible effects of large thermal gradients during warming.
The rewarming box consisted of a cavity 24″ in width and 74″ in length. The container was insulated with 2″ of Styrofoam and 2″ of urethane foam on the bottom and sides. The body was placed in the box, which had been precooled with dry ice and liquid nitrogen, on two thicknesses of R-19 fiberglass insulation with a wooden support block across the shoulders and buttocks to prevent crushing of the insulation. Following placement of the body in the rewarming box, the body was covered over with three layers of R-19 fiberglass insulation and the box was closed.
The temperature of P3 was monitored via copper-constantan thermocouple probes placed rectally, on the external abdomen and on the ankle. Another probe was placed midway in the covering R-19 insulation to monitor the box temperature. P3 was allowed to gradually rewarm over 19 days following removal from liquid nitrogen storage. The course of rewarming is shown in Figure 4.
Figure 4. Rewarming of P3. Thermocouple probes for ankle and abdomen were placed on the skin. Rectal probe was internal. Box probe was next to inside wall of box. Vertical marks on horizontal axis indicate midnight. Vertical (temperature) scale changes at -80°C. Arrow at -145°C indicates addition of LN2.
POSTMORTEM EXAMINATION
During examination of P1 following unwrapping, it was noted that full thickness fractures of the skin had occurred. These fractures were most unusual in appearance and can best be described as resembling the type of cracking observed in deteriorating coatings, such as is seen in paint peeling away from a wall. The skin adjoining the fracture fissure was somewhat raised from the underlying fat and gave the appearance of having “peeled away” slightly. Another striking feature about these fractures was their symmetry. Fractures were observed in either identical or nearly identical locations bilaterally. (Figure 5) Fractures were observed in the skin along the inner aspect of each upper arm and lower arm, in the skin of the hand just below the wrist in the fleshy area between thumb and forefinger, on the inner aspects of the upper thigh and on the upper aspect of the feet. Many of these fractures were 2 to 3 inches in length. External examination of P2 revealed only one similar fracture, which measured only a few millimeters in length and was at the edge of what appeared to be a diabetic ulcer. Several other areas on the lower extremities which looked slightly “crazed” were noted, but no gross fractures were observed with separation from subcutaneous fat comparable to the ones observed in the remains of P1. At the time of initial examination we attributed the presence of visible fractures on P1 to be due to an episode of thermal cycling (up to dry ice temperature and then down to liquid nitrogen temperature again) which had occurred a year before when funding for this patient became exhausted and the contract company caring for him removed him from liquid nitrogen storage.
External Fracture Locations: Figure 5 (P1), Figure 6 (P2), and Figure 7 (P3).
Following thawing, numerous large fractures were observed on the surface of P2’s remains (Figure 6) and additional fractures were noted on P1’s remains. These fractures were also often bilaterally symmetrical. In some instances these fractures penetrated more than 4 cm into the subcutaneous fat, usually ending at the body wall or at the next underlying tissue plane (such as the fascia covering the musculature) (Plate 1). Both patients’ remains exhibited massive cutaneous fractures over the pubis (Plate 2(A)). In the remains of P1 a fracture separated a large flap of skin and subcutaneous fat over the pubis and in P2 these fractures penetrated the mons and labia majora to the underlying musculature.
Plate 1. Arrows A and B indicate massive fractures of abdominal skin and subcutaneous fat observed in P2. These fractures penetrated to the body wall. A thorough external examination at -40 degrees centigrade failed to disclose these fractures.
Plate 2. Arrow A indicates fracture of skin and subcutaneous fat observed in P1. The fracture penetrated almost to the body wall. This fracture as well most of the others observed in P1 were clearly visible at -40 degrees centigrade. Arrow B points to areas of lividity which outlined the course of veins in the legs. Much of this network of lividity is not visible in the printed photograph owing to loss of fidelity during printing. The discoloration of the left hand and forearm is lividity apparently secondary to a venous infiltration probably sustained during the patient’s hospitalization.
External examination of P1 following thawing revealed much more extensive surface fracturing than was visible at first examination in the subzero state. In the limbs of P1, particularly in the lower limbs, a tracery of veins was etched in bright red on the skin surface, apparently as a result of the release of hemoglobin into the tissue adjacent to the vessels (Plate 2(B)). The skin of P1 also exhibited an overall pink cast which was absent from P2 and P3.
Internal examination of these patients’ remains disclosed multiple fractures in almost every organ system with only the livers being completely spared and the kidneys only slightly affected. In the remains of P2 fracturing was so severe that numerous organs were often completely, or almost completely transected by fractures. The pulmonary artery had fractured and completely separated from the heart (Plate 3). A large fracture had almost completely severed the aorta. Numerous small fractures were observed in other great vessels of the chest (Plate 4). The lungs (Plate 5) and spleen were at one point in each organ virtually severed by fractures. The intestine, mesentery, mesenteric fat (Plate 6), peritoneum, abdominal wall, and pancreas were riddled with fractures too numerous to count. P1 was similarly fractured, although not as extensively.
Plate 3. All of the great vessels in P2 were seriously fractured adjacent to the heart. Arrows indicate where the pulmonary artery and aorta were almost completely severed by fractures. Numerous small longitudinal fractures were noted elsewhere in the great vessels such as in the descending aorta and inferior vena cava.
Plate 4. Arrows indicate small circumferential fractures observed in numerous locations in both arteries and veins. This photo is of the pulmonary artery of P2.
Plate 5. Large fracture almost completely severing the right lung of P3. The arrow point out the small band of tissue which uniting the nearly severed tip of the lower right lobe with the rest of the lung.
Plate 6. Fractures in the mesentery and mesenteric fat adjacent to the ileum. The ileum was also fractured in numerous locations.
In sharp contrast to P1, P2 exhibited excellent blood washout with small blood-filled vessels being exceptional rather than the rule, as was the case with P1. The circulatory system of P1 was filled with blood or perfusate containing large amounts of blood. The partially frozen contents of the pulmonary artery and aorta were collected from both patients, thawed and subjected to osmometry. Additionally, a fluid sample from the femoral artery of P2 and a sample of interstitial fluid draining from a right thigh muscle fracture were also subjected to osmometry. Osmolalities for these body fluids are shown in Table I. These osmolalities must be regarded as only approximate at best due to the fact that the samples were collected in the partially frozen state. Nevertheless, the data does at least provide a basis for comparison of the two perfusion techniques employed and unequivocally supports the use of great vessel perfusion utilizing large quantities of perfusate if the open circuit approach is used.
Table I. BODY FLUID OSMOLALITIES
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Patient Collection mM Me2SO
Site
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P1 Pulmonary Artery 20
" Aorta 40
" Pericardial Fluid 0
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P2 Pulmonary Artery 900
" Aorta 760
" Femoral Artery 800
" R. Adductor Muscle 250
(fluid drainage)
Other findings of interest were that both patients apparently suffered from pathologies which contributed to or caused their deaths which were not listed on the death certificates. In addition to having suffered a cerebrovascular accident P1 was also found to have suffered a large, “recent” infarct of the posterior wall of the left ventricle. The heart was also hypertrophied secondary to chronic heart failure. P2 apparently died as a result of a massive intra-abdominal hemorrhage, not of an acute myocardial infarction as listed on the death certificate. Examination of the liver of P2 revealed it to be severely fibrosed and shrunken, having a knobby, scarred appearance.
The autopsy results of P1 and P2 prompted us to attempt to remove too rapid rewarming as a possible cause of fracturing in P3. To this end a mathematical model of rewarming was generated by Art Quaife of Trans Time (9) which indicated that the rewarming scheme which was eventually employed with P3 would yield a rate of temperature rise between -196 and -120 degrees centigrade in the range of 2 to 3 degrees centigrade per hour. It was also hoped that the surface-to-core temperature gradient could be held to a maximum of 10 degrees centigrade. As can be seen in Figure 4 neither of these objectives was met. However, rewarming was still much slower than in P1 and P2. Results of the postmortem examination lead us to believe that rewarming is not the primary event responsible for massive internal fracturing observed in these patients.
When the rectal temperature of P3 reached 0 degrees centigrade the body was removed from the rewarming box and subjected to a thorough external examination. The initial examination gave us considerable cause for optimism concerning the prospect for minimal internal fracturing. P3 had only a few external fractures: one of the skin on the dorsum of each hand approximately 3 cm in length (Plate 7); one between the thumb and forefinger of the right hand; and bilateral fractures of the skin and subcutaneous fat in the brachial area (Figure 7). The rest of the body was free of fractures. Careful external examination also failed to disclose the livid tracery of veins under the skin which was observed on P1.
Plate 7. Cutaneous fractures of the hands and wrist of P3. These fractures penetrated only a millimeter or two below the skin surface.
Of particular interest was the fact that no fractures were observed adjacent to the decapitation wound. This seems especially remarkable considering the amount of mechanical and thermal stress introduced by application of a chain saw at liquid nitrogen temperature. The absence of fractures associated with sawing at liquid nitrogen temperature confirms for humans what had been found in earlier, unpublished animal work conducted by the ALCOR Foundation at Cryovita Laboratories in Fullerton, California.
Examination of the internal organs of P3 revealed fractures present in almost every organ. The spinal cord, aorta, thoracic inferior vena cava, pulmonary artery, myocardium (Plate 8), right lung, liver, pericardium, stomach, ileum, colon, mesentery, spleen, skeletal muscle, and pancreas, were all seriously fractured. In some organs, such as the spleen, lung, liver (Plate 9) and great vessels, fractures penetrated the organ to the point of completely or almost completely severing them. In other instances the fractures were confined to the capsule, serous coat, or first two or three millimeters of the organ (Plate 10). The only organ which was consistently spared gross fracturing in all three patients was the kidney: in P1 and P3 the kidneys were free of visible fractures and in P2 the left kidney suffered only one small fracture of the cortex (Plate 11). The kidneys of P1 were free of any fractures and were somewhat fibrotic and shrunken showing evidence of renal disease.
Plate 8. Two small fractures 2 to 3 mm wide in the wall of the left ventricle. The myocardium was riddled with fractures of this kind.
Plate 9. The liver was separated by a large, sinuous fracture which penetrated the full thickness of the anterior lobe. Several other lobes of the liver were similarly disrupted by fractures.
Plate 10. The arrow indicates a fracture of the serous coat of the lung in P3.
Plate 11. Fracture of the renal cortex (arrow) observed in P2. The fracture did not quite penetrate the cortex to the medulla. Note the excellent blood washout as evidenced by the solid white appearance of the renal cortex.
A length of spinal cord approximately 20 cm in length was exposed in P3 and was collected for later evaluation by light and electron microscopy. When the dura was opened it was noted that the cord had fractured into three pieces, each approximately 6 cm long. These fractures completely transected the cord giving the impression of a broken glass rod.
During the course of the autopsy on P3 two sets of tissue samples were collected from the left ventricle, lung, liver, kidney, spleen, diaphragm, spinal cord, skin and fat. One sample of each set was fixed in Karnovsky’s solution (10) and the other sample of the set was fixed in buffered formalin (11) for later evaluation by light and electron microscopy. Fluid was also collected from the femoral artery, hepatic artery, abdomen and pericardium and subjected to light microscopy and osmometry. Light microscopy revealed numerous intact red cells in the femoral artery and pericardial fluid with few ghosts and little debris present. Many of these cells were noted to be agglutinated upon initial examination, and progressive clumping and agglutination of the red cells was noted with continued exposure to ambient temperature on the stage of the microscope. Osmometric determinations are shown in Table II.
Table II. BODY FLUID OSMOLALITIES,P3
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Sample Source Glycerol M
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Abdominal fluid .729
Pericardial fluid 1.367
Femoral Artery 1.967
Hepatic Artery B .690
The abdominal and thoracic viscera of P3 showed numerous areas where blood washout was incomplete. The distal two-thirds of the right kidney appeared very dark and infarcted and the upper pole of the left kidney also appeared to have been poorly perfused (Plate 12). There were numerous areas in both lungs which were atelectic and unperfused. The liver, mesentery and gastrointestinal tract appeared well perfused and free of any obvious blood. The spleen was very bloody on sectioning and demonstrated many intact red cells on microscopic examination.
Plate 12. The right kidney of P3 showed very poor blood washout with one pole of the kidney being very dark and the other 2/3rds of the organ showing extensive red mottling. The flap of membranous tissue at the top of the photo is the renal capsule which has been removed to facilitate examination of the cortex.
In 1980, when cryoprotective perfusion of this patient was carried out by the authors, a large, firm abdominal mass was noted both on external examination and during median sternotomy. At that time we elected not to perform a laparotomy to investigate the nature of the mass because of both time constraints and concern over our ability to close the abdomen should edema develop. This mass, which was severely compressing the diaphragm was found to be the massively hypertrophied anterior lobe of the liver. The liver was sectioned and it was demonstrated that the lobe was indeed hepatic parenchyma and not tumor or cystic material. Histological examination of liver sections taken from this lobe later demonstrated severe liver disease with massive areas of necrosis interspersed with small islands of apparently histologically normal tissue. The other lobes of the liver were similarly enlarged, and the liver was noted to have a dusky brownish-yellow color rather different from that normally seen following blood washout.
Prior to the discovery of massive internal fracturing in P3 we had hoped to reperfuse the remains with a high molecular weight dye solution with a glycerol concentration comparable to what the glycerol concentration in the patient’s venous return had been at the conclusion of cryoprotective perfusion. We hoped to determine if reperfusion was possible and if any small fractures were present in organs which might not be readily apparent on gross inspection. Due to the disruption of the great vessels and cracks in the abdominal and thoracic viscera, complete reperfusion of the remains was not possible. Instead, we elected to reperfuse the left arm by cannulating the axial artery and vein. Unfortunately, the dye used (2,000,000 molecular weight blue dextran) did not impart enough stain to adequately determine distribution of perfusate into the limb. Consequently we continuously added methylene blue to the perfusate as it was delivered. Distribution of the dye was quite striking. Dye penetration was first noted in the hand and a few small patches of skin on the brachial and dorsal aspects of the arm. Perfusate began leaking from the brachial fracture almost immediately (Plate 13).
Plate 13. A 12 g angiocath has been positioned in the axial artery to allow for reperfusion of the left arm of P3. Drainage was via the axial vein which was opened at the point of the incision. The arrow indicates a fracture which is leaking perfusate containing dye. The skin of the arm is mottled with dye.
Within 1 minute following the start of perfusion massive edema was noted in the limb. Little venous return was noted at any time during the perfusion. Due to edema, perfusion was stopped after 5 minutes, 21 seconds and the limb was dissected in order to evaluate dye distribution.
There was very poor distribution of dye in the skin and subcutaneous fat with the exception of the hand and distal portion of the forearm, which showed moderate staining with patchy distribution. The deep musculature of the upper arm showed staining with dye with very even distribution (Plate 14). Virtually all of the extensor and flexor muscles of the forearm perfused very poorly if at all. In the case of the brachioradialis and the flexor pollicis, perfusion was confined to an area approximately 2 cm in diameter in the center of the muscle.
Plate 14. Deeply dye-stained brachial muscle of the upper left arm following reperfusion. Note the mottled appearance of the subcutaneous fat which failed to perfuse well with dye.
During dissection of the arm immediately following perfusion several highly pressurized blebs of perfusate were encountered, apparently the result of fractured vessels within the limb.
DISCUSSION
The most unexpected finding as a result of these autopsies is the discovery of serious fracturing in all of the suspension patients. While the cause of these fractures has yet to be established, two explanations may be put forth. The first, and most obvious cause would be shell deformation effects resulting from expansion of water in the core of the body following phase change on the outside of the body (12). Unfortunately, this fails to explain the presence of many internal fractures in tissues which should have frozen more or less simultaneously and at high enough temperatures to have accommodated any expansion of ice. A more likely explanation is that fracturing occurred long after freezing was complete and the patient was being cooled to below dry ice temperature. As cooling proceeds below the glass transition phase of water (TG), different organs and tissues within the patient’s body will begin to contract at different rates. However, because the system is now in a solid state, these materials, bonded to each other by ice/cryoprotective agent mixtures, will be unable to contract independently. A logical consequence of this would be the development of tremendous mechanical stress in tissues contracting at differential rates. As a result of the inflexibility and low tensile strength of frozen tissues at or below TG, fracturing occurs.
If the latter explanation is indeed the correct one, then cooling to very low temperatures in the absence of serious fracturing may be extremely difficult in large biomasses. Perhaps the solution to this problem may be to anneal the patient for a prolonged period of time at or near TG prior to completing the descent to -196 degrees centigrade. Alternatively, it may be determined to be both safe and feasible to pursue storage at higher temperatures, perhaps in the region of TG, where there will be no available liquid water/cryoprotective agent to allow for appreciable propagation of chemical reactions.
It should also be noted that in the case of P3 there were surface to core temperature gradients of 20 degrees centigrade or greater during descent from -100 to -196 degrees centigrade (Figure 4). Head to foot gradients during cooling below – 100 degrees centigrade were consistently in the range of 40 to 60 degrees centigrade. It should be emphasized that these gradients were probably not as extreme in P3 as in P1 and P2 due to the fact that P3 was surrounded by multiple layers of insulation and a metal cassette. Large head to foot gradients, as well as surface to core gradients should be carefully examined as a possible cause of fracturing. It may be that simply by eliminating large temperature gradients during cooling much of the fracturing observed might be avoided. Clearly, much additional research is needed in this area.
One puzzling observation for which we have no explanation as yet is why none of these patients experienced any fracturing on the dorsum of the body. The entire back side of the bodies was completely free from fractures, including fatty areas such as the buttocks which would seem prime candidates for fracturing. Additional areas which were consistently free of fractures were the palms of the hands, soles of the feet, and genitals in the male patient. A cursory examination of the patient’s severed heads under liquid nitrogen revealed no sign of fracturing (such as was observed in the subzero state in the case of P1), however it seems unlikely that the head would occupy a privileged position in this respect. Since these patients have been converted to neuropreservation and their care continues it was not possible to examine the brain and head for post- thaw fracturing.
The first two patients were cooled to liquid nitrogen temperature while strapped to heavy aluminum stretchers which completely covered the dorsum of the body. P3, who experienced far less external fracturing than P1 and P2, was enclosed in an insulating container which, as shown in Figure 3, resulted in a much slower rate of temperature descent. It remains a possibility that the stretchers may have acted in some way to mitigate fracturing on the dorsum of P1 and P2.
On gross external and internal observation, all of the remains appeared well preserved post-thaw. The skeletal muscles in both P1 and P2 had a softened, somewhat “mushy” feel which was not present prior to freezing and which was noted only to a slight extent in P3. The remains of all three patients exhibited considerably less rigor post thaw than was observed at the conclusion of perfusion. The change in tissue texture and reduction in rigor suggest the possibility of autolytic degradation of skeletal muscle. The texture and appearance of the heart and the other abdominal and thoracic viscera were for the most part unremarkable. The pancreas of P3 appeared edematous with separation of the parenchyma into rosette-like islands imbedded in a clear, gelatinous matrix. This type of edema has been observed during ischemic glycerol perfusion of animals (4) and during failed total body washout experiments. In contrast to the experimental situations where such edema has been observed, it appeared confined to the pancreas in P2. All three patients exhibited some degree of pulmonary edema.
It was difficult to find free fluid available for sampling in any of the peripheral vessels of any of the three patients. Cut tissues exhibited very little weeping of fluid and even with the application of pressure, oozing was negligible. This observation suggests absorption of extracellular fluid by the tissues. Transcellular water in the form of intestinal chyme was noted in all three patients. The bladder of P3 was noted to contain a small amount of urine which was still partially frozen at the time of autopsy suggesting absence of cryoprotective equilibration. The spinal canal of P3 contained only a small amount of very viscous fluid (too small for sampling) and the cord was shrunken to approximately 1/2 of normal diameter. The dura of P3 had a dark, abnormal reddish cast and areas adjacent to fractures in the cord were similarly discolored. The virtual absence of significant amounts of CPA in P1 are very disappointing in view of the amount of perfusate used. It seems likely that failure to introduce meaningful amounts of CPA in this patient are a result of both the postmortem delay and the mode of vascular access used: the internal carotid artery. The absence of adequate blood washout and significant levels of cryoprotective agent point out the inadequacy of embalming equipment and techniques in human cryoprotective perfusions.
Both P2 and P3 showed good blood washout when contrasted with P1. However, in both of these patients numerous infarcted and nonperfused areas were noted. In P2 the CPA concentration was, not surprisingly, very low, no doubt as a consequence of the low volume of DMSO perfusate which could be delivered prior to the development of obstructive edema. A greater disappointment is the low levels of glycerol observed in the body fluids from P3. Despite four hours of closed circuit perfusion, the highest concentration of glycerol observed was still under 2M. These preliminary indications of poor CPA distribution should serve to again point out the devastating effects of long postmortem time delays to perfusion in the absence of adequate cardiopulmonary support. It is unfortunate, but not surprising that these results with humans bear out work conducted earlier with animals subjected to long ischemic times prior to glycerol perfusion (4). In the future, greater efforts should be made, where possible, to provide good postmortem cardiopulmonary and metabolic support in order to avoid the complications of inadequate perfusion and CPA distribution observed in all three of these patients.
Our thanks to Trans Time, Inc. and the Bay Area Cryonics Society, Inc. for their co-operation and support. Special thanks to Art Quaife of Trans Time for his thermal modeling efforts and to John Day and the rest of the BACS-Trans Time crew for their outstanding organizational efforts.
REFERENCES
1) Personal communication with Robert C.W. Ettinger.
2) Personal communication with Cryovita Laboratories, Inc., Trans Time, Inc., Cryonics Society of South Florida and the Cryonics Institute.
3) The Autopsy in Clinical Quality Control, Scottolini A.D. and Weinstein S.R., “Journal of the American Medical Association,” 250, pp. 1192-1194 (1983).
4) “Cryoprotective Perfusion and Freezing of the Ischemic and Nonischemic Cat,” Federowicz M.G. and Leaf J.D., Cryonics, issue 30, p.14, 1983.
5) “Berkowitz Removed From Suspension: Lawsuit Settled,” Cryonics, December 1983 p.1.
6) “Suspension Records of R.M. Sr. 3/27/76,” Unpublished, ALCOR Archives.
7) “Case Study: K.V.M. Suspension,” Leaf J.D., Cryonics, August 1981, pp. 8-18.
8) “Case Report: Two Consecutive Suspensions, a Comparative Study in Experimental Suspended Animation,” Leaf J.D., In press.
9) “Heat Flow in the Cryonic Suspension of Humans: Survey of the General Theory,” Quaife A., Manuscript in preparation.
10) Modified from the original (Journal of Cell Biology 27:137A, 1965): 10g paraformaldehyde, 100 ml 25% glutaraldehyde, 8.39 g sodium chloride, 1.42 g disodium phosphate, .5 ml 22.2 calcium chloride, glycerol concentration adjusted to 3.0M, pH adjusted to 7.4, final volume 1 liter.
11) General Zoological Techniques, Weesner F.M., p. 30, Williams and Wilkins Co. Baltimore, 1965 (4% formalin in 3M glycerol/water, acid neutralized with calcium carbonate).
12) “The Ductility of Mammalian Tissue in Dependance on the Deformation Temperature,” Thom, F. et al, Cryo-Letters, 4, 341 (1983) pp. 341-348.