Cryopreservation Protocol for BioPreservation Clients January, 1994 SUMMARY OF THE CRYOPRESERVATION PROTOCOL TO BE EMPLOYED BY BIOPRESERVATION, INC. (BPI) TO PREPARE BPI CLIENTS FOR CRYOPRESERVATION CAVEATS The following is presented as representative of the procedures and documentation to be used by BPI in preparing its clients patients for cryopreservation. It is acknowledged by contracting cryonics organizations that there may be variations in techniques used based on the patient's condition, availability of supplies, the surgeon's preference, etc. This notwithstanding, the general approach described below of cardiopulmonary resuscitation (where appropriate and permissible following pronouncement of legal death) followed by total body washout (TBW) with a tissue preservative solution, followed by transport to facilities where cryoprotective perfusion can be carried out to introduce multimolar concentrations of glycerol (up to 7.0 M) will be used. Significant variation from this protocol such as substitution or variation of cryoprotectant(s) and/or cooling protocol will be given to the contracting organization in writing before they are carried out, and their consent shall be obtained. The case history used to illustrate this document is based on that of a neuropatient who was cryopreserved by an Alcor/Cryovita team which included Mike Darwin and other BPI team members. The actual case history has been modified to remove dates and personal information, and to bring it into line with the advances in technique now employed by BPI. It is then presented as if it were the case history of a patient which the BPI team has taken from remote standby to the final cool-down phase. A neuro-cryopreservation was chosen because of the more complex surgical approach required. Whole-body patients are treated similarly with the differences being that the the quantities of perfusate prepared are larger (approx. 80 liters of 65% (v/v) glycerol and 40 liters of 5% (v/v) glycerol) and the patient's entire body is perfused. Following completion of cryoprotective perfusion the whole-body patient is decannulated, the chest wound is closed, and the patient is placed in heavy plastic bags and cooled to dry ice temperature in an isopropanol bath by controlled addition of dry ice. The protocols for transport, median sternotomy, and cryoprotective perfusion are generally the same for both whole-body and neuro- cryopreservation patients. MEDICAL HISTORY Where available to BPI, the patient's medical history and medical records is obtained, copied, and placed in the patient's record of care. A summary of the patient's medical history, particularly as it relates to the cryopreservation procedure, is prepared by BPI and entered into the patients file. DOCUMENTATION OF AGONAL COURSE AND LEGAL DEATH The patient's agonal (terminal) course commencing with the start of standby by BPI personnel is documented. The documentation includes an assessment of the level of consciousness, vital signs, perfusion status, and such other indicators of the patient's condition as may be appropriate and necessary to facilitate subsequent cryopreservation. The following is provided as an example of the standard of documentation of the patient's agonal course which BPI undertakes to provide. It is understood that the ability to achieve this level of documentation may be dependent upon factors outside of BPI's control such as cooperation from the patient, next-of-kin, medical staff, or others who have control over the patient and/or his or her medical records. Agonal Course At 16:00 on 9 June, the patient was obtunded (level of consciousness (LOC) = 3 on the Glasgow Coma Scale) and diaphoretic, with vital signs as follows: B.P. 50/38, pulse 140, and respirations 16. Capillary refill time was 2-3 seconds and the extremities were dusky and mottled with marked pedal cyanosis. Pupils were mid-position and were sluggishly responsive to light. Vital signs up to the time of deanimation are given in tabular and graphic form below: ============================================================================= Time Blood MAP Pulse Respirations Observations Pressure (Apical) 12:00 90/50 103 120 14 LOC 3, extremities mottled 13:00 88/48 101 136 16 Temp. 99.0 axially 14:00 78/46 89 140 16 Temp. 100.1 axially 15:00 68/44 76 128 14 poor capillary filling in nail beds with 2-3 second return 16:00 60/40 67 152 16 Temp. 101, shallow respirations 17:00 50/38 56 140 --- long periods of apnea 17:20 38/-- 52 140 --- extremities cool 17:47 0 0 0 0 deanimation =========================================================================== MAP = D + (S-D)/3 Cardiac arrest occurred 17:47 on 9 June and legal death was pronounced by Jane Roe, R.N., the attending Registry nurse at that time. (17:47 has been used as the arrest time wherever decimal hours post-arrest are calculated.) DOCUMENTATION OF INITIAL CARDIOPULMONARY SUPPORT BPI documents the efforts of its personnel to restore perfusion and to determine the adequacy of that perfusion. The attached copies of the Transport Data Collection and External Cooling Logs are used to gather this data. The patient is (where feasible) initially supported using a heart-lung resuscitator and medicated per the attached Emergency Instructions For Stabilization of Biopreservation's Cryopreservation Patients. The procedure used during transport and its documentation are approximately as illustrated here: Transport Immediately following the pronouncement of legal death the patient was moved from the bed in which she experienced cardiac arrest and was quickly carried to the portable ice bath (PIB) in an adjacent room. An esophageal gastric tube airway (EGTA) with an attached Fenem end-tidal CO2 detector was quickly placed to facilitate ventilation and a model M/1004 Michigan Instruments Thumper was installed. Cardiopulmonary support was initiated at 17:50 at a rate of 80 compressions per minute. Chest deflection was initially set at 2" on the Thumper and then re-set to 2.5" at 18:00 with sixteen ventilations per minute via EGTA at 30 cm of water airway pressure. There was an immediate return of agonal gasping upon the initiation of CPS accompanied by "pinking-up" of the patient with the skin on the thorax, abdomen, and face appearing ruddy and well perfused. This was in sharp contrast to the duskiness and cyanosis observed during the hours of antemortem shock which preceded cardiac arrest. An array of tubing driven by a submersible sump pump (15 GPM @ 10') and terminating in multiple lengths of perforated 1/2 ID rubber tubing was used to circulate ice-cold water in the PIB over the patient, particularly over the head and anatomical areas with large, high flow vessels near the surface of the skin: the groin, neck, and axilla. This device, owing to its appearance, is referred to as the Squid. Initial response to CPS was judged to be excellent as indicated by prompt return of agonal gasping, pinking-up of the skin, and end-tidal CO2 readings of 2-3%. A venous blood sample drawn at 18:25 via the PICC line was bright red, indicating good ventilation and perfusion. IV access was via an implanted PICC using a 16 g. Sorenson thin-wall dialysis needle to puncture the Port-A-Cath septum. Administration of transport medications began at 17:54 with IV push medications as follows: sodium pentobarbital 1092 mg, 2 gm deferoxamine at 17:55, 400 ug nimodipine at 17:54, 4368 mg sodium citrate at 17:55, 2912 mg Trolox at 18:19, 4,550 mg ascorbic acid (Cevalin) at 18:12, 15,288 IU sodium heparin at 17:55, 1 g methylprednisolone at 17:56, 109 mg chloropromazine at 17:55 and 2.55 mg metubine iodide at 17:56. Continuous IV infusion of 500 cc of 0.3 M tromethamine (THAM) and 500 cc of 20% mannitol in water were begun at 17:56 and 17:58, respectively. A venous blood sample drawn from the PICC at 17:50 disclosed the following: ========================================================================= pH 7.65 mOsm 358 HCT -- SGOT 780 IU/l SGPT 220 IU/l Total Bilirubin 1.4 mg/dl Direct Bilirubin 0.6 mg/dl Indirect Bilirubin 0.8 mg/dl BUN 47.0 mg/dl Creatinine 1.4 mg/dl Cholesterol 394 mg/dl Alkaline Phosphatase 662 IU/l Glucose 127 mg/dl Phosphorus 7.4 mg/dl Total Protein 6.0 g/dl Albumin 2.9 g/dl Globulin 3.1 g/dl Sodium 140 mEq/l Potassium 9.7 mEq/l Chloride 91 mEq/l gamma-GT 1385 IU/l Uric Acid 8.8 mg/dl Lactate Dehydrogenase 1507 IU/l ========================================================================= At 18:40 Squid assistance to external cooling was discontinued and the patient was loaded into a mortuary van at 18:45 for transport to Smith's Mortuary for femoral cutdown and Total Body washout (TBW). Mechanical cardiopulmonary support and infusion of THAM and mannitol were continued en route to the mortuary and the infusions were completed at approximately 18:17. Arrival at the mortuary occurred at 18:53. The patient's axillary temperature at the time of deanimation was 38.3*C. The first temperatures obtained during transport were recorded at 18:33 and were 24.0*C rectal and 24.8*C esophageal. Temperature was monitored with Cole Parmer, Co. Digi-Sense (Cole-Parmer Cat. 8528-20) thermocouple thermometer employing a copper-constantan vinyl coated TC probe to measure esophageal temperature (Cole-Parmer Instrument Co. Model # 8505-90). The patient's temperature descent during external cooling is presented graphically below with accompanying end-tidal CO2 readings. Times shown are decimal hours post-arrest. SQUID supported cooling was re-initiated after arrival at the mortuary at approximately 18:06. Thumper CPS was briefly interrupted between 18:48 and 18:49 when the H-oxygen cylinder supplying the unit became exhausted and was switched out for a fresh tank. Patient temperatures at the the time of this interruption were 11.2 *C esophageal and 13.1 *C rectal. At 20:10 a small quantity of the blood-tinged foam characteristic of pulmonary edema was noted in the patient's oropharynx. There was no evidence of erosion of the gastric mucosa, as evidenced by lack of gastric bleeding. TBW PERFUSATE COMPOSITION AND PREPARATION Perfusate preparation and composition is documented in detail. This includes a complete list of components used, the supplier and lot numbers, and the procedure used to filter sterilize the perfusate prior to administration (if applicable). Cryoprotectants used and their concentration in both the reciculating and concentrate perfusates are documented. The following serves to illustrate the general procedure employed and the level of documentation provided: Total Body Washout Perfusate Preparation The composition of the Viaspan total body washout flush solution is given in Table I. TABLE I - Viaspan Composition =========================================================================== Component g/l Lactobionic Acid (as Lactone) 50.00 Potassium Phosphate monobasic 3.40 Magnesium Sulfate heptahydrate 1.23 Raffinose pentahydrate 17.83 Adenosine 1.34 Allopurinol 0.136 Total Glutathione 0.922 Dexamethasone 0.16 Glucose 0.90 Insulin (U-100 Regular) 40 IU Heparin 2000 units/l Water for Injection q.s. Potassium Hydroxide q.s. Sodium Hydroxide adjust pH to 7.4 Measured Osmolality: 305 mOsm/kg Measured pH: 7.5 Note: Dexamethasone, insulin, glucose and heparin were added to the ViaSpan immediately before use. =========================================================================== PROCEDURE FOR TOTAL BODY WASHOUT The procedure employed to carried out total body washout in the field (if applicable) is documented with respect to the surgical approach, details of cannulation (cannulae size, vessels employed, complications) and general conduct of perfusion (tubing pack, filter, oxygenator, blood/perfusate flows, gas flows etc.). The attached Perfusion Data Collection Sheet is used to gather this data in an orderly fashion. While details of equipment/procedures may vary from patient to patient, the following serves to illustrate the procedure used and its level of documentation: Total Body Washout (TBW) Femoral Cannulation Femoral cut-down to connect the patient to the extracorporeal circuit for total body washout was begun at 19:25. The patient's esophageal temperature at that time was 13.3*C and the rectal temperature was 15.1*C. The patient's right groin was prepared for femoral cut-down by scrubbing/swabbing with povidone iodine solution (Betadine) and draping with sterile towels. The anatomical position of the right femoral artery and vein were located by reference to the pubic tubercle and the anterior superior iliac spine. An incision with a #10 scalpel blade was made at the midpoint between these two structures, beginning with the inguinal ligament and running parallel to the longitudinal axis of the leg for approximately 5 cm. The femoral artery and vein were dissected free and #2 silk ties placed on the proximal and distal exposures of both vessels. The distal ties were tied to achieve occlusion. There was a vigorous arterial pulse from the HLR, and the capillary blood appeared well-oxygenated. An arteriotomy was made with with a #11 scalpel blade. Upon opening the femoral artery, it was noted that the arterial blood appeared bright red. A USCI Type 1966, 18 Fr. arterial cannula was then introduced and secured with the proximal tie. A veinotomy was performed in the same fashion and a USCI type 1967, 26 Fr. venous cannula was advanced until the tip was well within the inferior vena cava and near the heart, and secured with the proximal tie. Venous blood appeared well saturated with oxygen. The arterial perfusion line was connected to the arterial cannula with a 3/8" straight connector with port, and the port fitted with a Cobe 3-way stopcock for evacuation of air. The venous return line was connected to the venous cannula with a 1/2" straight connector with port, and air was removed from the venous cannula and venous line with a 35 cc plastic syringe. Washout was carried out employing a Travenol roller pump, a a Sarns 16310 oxygenator, and a Pall Leukoguard-6 40u blood filter. Perfusion pressure was measured at the Leukoguard filter, anterior to the arterial cannula, employing an aneroid manometer with a sterile Gish ECPML flexible membrane pressure monitoring assembly to protect the aneroid from fluid contamination. A calibration curve of measured back-pressure versus measured flow was generated in advance to account for the pressure increase resulting from cannula-induced flow restriction. Temperatures were measured with a Cole-Parmer Digi-Sense type T thermocouple thermometer (catalog #8505-90). The extracorporeal circuit was primed with 3 liters of Viaspan washout solution, the composition of which is given in Table I. At the start of blood washout, chest compressions were discontinued and the mask of the Esophageal Gastric Tube Airway (EGTA) was removed (the obturator was left in place to guard against aspiration). Blood washout was begun at 20:10 in the preparation room of the mortuary, with a 10 liters of Viaspan at arterial pressures of between 60-80 mmHg, a flow rate of approximately 1600 cc/min. and an oxygen flow rate (to the oxygenator) of 5 liters per minute. The patient's temperatures at the start of washout were 9.5*C esophageal and 11.8*C rectal. Perfusion with the 10 liters of Viaspan was completed at 20:30. A venous effluent sample drawn from the venous line at 20:12 near the beginning of the first pass disclosed the following: =========================================================================== pH 7.56 mOsm 417 HCT -- SGOT 925 IU/l SGPT 182 IU/l Total Bilirubin 0.8 mg/dl Direct Bilirubin 0.4 mg/dl Indirect Bilirubin 0.4 mg/dl BUN 34.0 mg/dl Creatinine 1.1 mg/dl Cholesterol 95 mg/dl Alkaline Phosphatase 214 IU/l Glucose 109 mg/dl Phosphorus 6.3 mg/dl Total Protein 2.2 g/dl Albumin 1.0 g/dl Globulin 1.2 g/dl Sodium 101 mEq/l Potassium 11.2 mEq/l Chloride 79 mEq/l gamma-GT 488 IU/l Uric Acid 0.1 mg/dl Lactate Dehydrogenase 1382 IU/l ============================================================================= A venous effluent sample taken at 20:39, at the conclusion of ViaSpan flush revealed the following: ============================================================================= pH 7.70 mOsm 322 SGOT 27 IU/l SGPT 7 IU/l Total Bilirubin 0.0 mg/dl Direct Bilirubin 0.0 mg/dl Indirect Bilirubin 0.0 mg/dl BUN 0.7 mg/dl Creatinine 0.2 mg/dl Cholesterol 0 mg/dl Alkaline Phosphatase 2 IU/l Glucose 120 mg/dl Phosphorus 41.0 mg/dl Calcium 1.4 mg/dl Total Protein 0.3 g/dl Albumin 0.0 g/dl Globulin 0.3 g/dl Sodium 40 mEq/l Potassium 70.0 mEq/l Chloride 0.0 mEq/l gamma-GT 7 IU/l Uric Acid 0.0 mg/dl lactate Dehydrogenase 57 IU/l ============================================================================= TBW was concluded at 20:40, with an esophageal temperature of 4.60*C and a rectal temperature of 4.9*C. The patient was cleaned up on the mortuary prep table and transferred to a heavy-duty (8 mil) vinyl body bag. At this time (21:24) it was noted that there was no rigor present. The body bag containing the patient was then placed atop a bed of Zip-Loc bags containing crushed ice which had been laid down inside an insulated air transport box. The patient was then covered with additional bags of crushed water ice and the transport container was closed for air transport to the cryoprotective perfusion facility. (BPI's cryoprotective perfusion facility is located in Rancho Cucamonga, California.) Air Transport Air transport was by private, prop-driven aircraft which arrived at Ontario Airport at approximately 01:45 on 10 June. Air transport was uneventful. The patient was collected from the aircraft and transported to the cryoprotective perfusion facility by "ambulance", arriving at approximately 02:12. (BPI maintains a two fully equipped "ambulances" for the transport of patients.) Gross Assessment The patient's arrival temperatures were: 1.8*C esophageal and 3.8*C rectal. At 04:23 the patient was transferred from the air shipment container to the weighing surface of an Acme model SRD-2S in-bed where scale the patient's weight was determined to be 32.8 kg. The patient was then transferred to the operating table, the surface of which had been previously prepared with a cooling blanket placed atop 2"-thick eggcrate foam. After the patient was on the operating table, she was briefly examined. The exam disclosed a profoundly cachectic caucasian female in her late 60's. The thorax and limbs were skeletal in appearance, the abdomen was sunken. The eye exam disclosed bilaterally dilated pupils with corneal misting. The globes were somewhat retracted in their sockets. The buccal mucosa was whitish-yellow and apparently blood-free. The skin was pale, uniform in color, and apparently blood free. The skin under the HLR piston was bruised and erythematous in appearance. The distal half of the sternum exhibited the "caved in" appearance which is typical following extended HLR support. The patient was free of any signs of rigor mortis and there was no evidence of post-mortem lividity. CRYOPROTECTIVE PERFUSATE COMPOSITION AND PREPARATION Perfusate preparation and composition is documented in detail. This includes a complete list of components used, the supplier and lot numbers, and the procedure used to filter sterilize the perfusate prior to administration (if applicable). Cryoprotectants used and their concentration in both reciculating and concentrate perfusates are documented. The following serves to illustrate the general procedure employed and the level of documentation provided: Perfusate Preparation The composition of the perfusate employed to carry out cryoprotective perfusion is given in Table II. Dry chemical perfusate components were prepared from reagent or medical grade chemicals weighed out using an Ohaus Centogram model 311, and Ohaus Triple Beam 2610 g balances. Dry components were mixed with American Chemical Society (ACS) reagent grade glycerol and/or sterile water for injection USP, or sterile water for irrigation USP. Perfusates were sterilized by filtration into the recirculating and cryoprotective concentrate reservoirs through a Pall PP3802 0.20u pre-bypass filter. Perfusate was prepared in two batches with the following volumes and glycerol concentrations: Description Volume Glycerol%(w/v) Recirculating 20 liters 5% (w/v) Concentrate 40 liters 65% (w/v) TABLE II - Total Body Washout Perfusate ============================================================================= Component Molar Concentration (mM) g/l Mannitol 170 (MW 182.20) 30.97 Adenine HCl 0.94 (MW 180.6) 0.17 D-Ribose 0.94 (MW 150.2) 0.14 Sodium Bicarbonate 10.0 (MW 84.0) 0.84 Potassium Chloride 28.3 (MW 74.56) 2.11 Calcium Chloride 1.0 (MW 111) 0.28 ml 10% soln. Magnesium Chloride 1.0 (MW95.2) 1.0 ml 20% soln. HEPES (Na+ salt) 15.0 (MW 260.3) 3.90 Glutathione 3.0 (307.3) 0.92 Glucose 5.0 (180.2) 0.9 Hydroxyethyl Starch --- 50.0 Heparin --- 1000 units/l pH was 7.8 (measured). mOsm (base perfusate) was 398 (measured). Cryoprotective perfusate was prepared by dissolving the above components for 40 liters in sufficient water to yield 3 times the above concentrations, dividing the resulting solution into two equal parts, and diluting to the indicated component concentration (making to 20 liters) with water for injection/irrigation USP and glycerol in the appropriate proportions to make 20 liters of solution at the desired glycerol concentration. ============================================================================ SURGICAL PROCEDURES The nature, operative approach and course of all surgical procedures used is fully documented. Operative Data Collection Sheets for aortic root and right atrial cannulation/ligation of thoracic vessels for neuro-cryopreservation procedures, and for burr-hole trephination are attached. The following serves to illustrate the general procedures used and their level of documentation: Operative Procedures Pre-operative Prep The patient was prepared for a median sternotomy and cranial burr- hole by shaving the head and thorax and scrubbing/swabbing them with povidone iodine solution (Betadine). The sternal operative site was defined by draping with sterile towels and an adhesive operative drape (3M) was placed over the sternum. A cardiac drape was placed over the patient, "tented" on two IV poles at the head, and allowed to extend down over the feet and over the sides of the table by a minimum of 24". The top of the scalp was draped with a fenestrated adhesive drape over the right parietal lobe. Median Sternotomy/Vascular Access Median sternotomy was begun at 05:00 on 10 June with an incision over the midline of the sternum with a #10 scalpel blade. Fascia and connective tissue were cleared down to the sternum with an electrosurgical knife. A median sternotomy was then performed with a Stryker vibratory sternal saw. The edges of the sternotomy were padded with laparotomy sponges, a self-retaining retractor placed, and the sternotomy retracted open. Blunt and sharp dissection were used to expose the pericardium. The ascending aorta was freed from the pulmonary artery by blunt dissection with Metzenbaum scissors. An aortic cross-clamp was placed just above the aortic valve to exclude the coronary circulation. A second aortic cross-clamp was applied to the descending aorta just distal to the left subclavian artery in order to exclude any arterial circulation to the body. The left subclavian artery was identified and followed to locate the left vertebral and mammary arteries. Number 2 silk ties were placed on the mammary artery and on the subclavian, just distal to the vertebral, and secured to exclude these vessels. This directed flow to the left vertebral artery, supplying the brain, and excluded the brachial and thoracic wall circulation. The innominate artery was located and followed to identify the right subclavian artery. The right subclavian was followed to identify the right vertebral and mammary arteries. Silk ties were placed, as was done over the left side, to direct flow to the vertebral artery. A ventral midline pericardiotomy was made using Metzenbaum scissors. Four stay sutures of 3-0 silk were placed in the margins of the pericardiotomy. These sutures were tied to the sternal retractor, thereby reflecting the pericardium away and creating a pericardial "cradle", and exposing the heart and aorta for cannulation. A Sarns cardiotomy sucker was used to suction away the pericardial fluid. A 3-0 Ti-cron purse-string suture was placed in the aorta and a snare applied. An aortotomy was made with a #11 scalpel blade. A 22 Fr. aortic perfusion cannula was primed with normal saline and a clamp placed on the distal end. The cannula was then introduced into the aorta and snared in place with a hemostat. A Satinsky partial occlusion clamp was placed on the right atrium just below the apex. A purse-string suture of 2-0 Ti-cron was placed in the atrium and a snare tube applied. An atriotomy was made by removing the apex of the right atrium with Metzenbaum scissors. A tubing clamp was placed on the distal end of the 32 Fr. USCI type 1967 venous catheter, and it was advanced through the atriotomy (with concurrent release of the Satinsky clamp) into the right atrium to the superior vena cava. Umbilical tape was passed around the superior vena cava and tied below the cannula tip. In order to prevent contamination of the recirculating system with venous circulation from the extremities, silk ties were placed on the left and right innominate veins just distal to the left and right internal jugular veins. Venous return was collected from the cannula in the superior vena cava. A third small purse-string suture of 5-0 silk was was placed in the left lateral aspect of the ascending aorta and an aortotomy made with a #11 scalpel blade. A Cobe 3-way stopcock was fitted to an Aloe arterial pressure monitoring catheter, and the catheter was flushed with normal saline and introduced through the aortotomy into the ascending aorta. The catheter was secured in place by applying a snare to the 5-0 suture. The sterile perfusion tubing was then brought up to the surgical field and secured in a Travenol tubing holder towel-clamped to the drapes. The arterio-venous loop of the perfusion circuit was clamped and divided by cutting out the 1/2" - 3/8" adapter with Mayo scissors. A 1/2" connector with a Cobe 3-way stopcock was used to connect the 1/2" ID venous return line to the venous cannula. Air was cleared from the system with a 100 cc glass syringe. A Cobe 8 ft. pressure monitoring line was fitted to the arterial pressure catheter, flushed with normal saline, and handed off the field to be connected to a Trantec Model 800 pressure transducer and a Tektronix Model 414 monitor. Surgery to connect the patient to the perfusion circuit was completed at 07:40. Cranial Burr-Hole Surgery to open the cranial burr-hole was begun at 05:27. The vertex of the scalp approximately 3 cm to the right of midline over the right parietal lobe was incised with a #10 scalpel blade and an incision approximately 4 cm long was made down to the periosteum. A periosteal elevator was used to expose the bone approximately 3 cm to the right of the midline. A 10 mm hole was made with a neuro burr and drill. The dura mater was opened and trimmed away with iris scissors to expose approximately 6 to 8 mm of the cortical surface. The burr hole was opened at 05:50; the pial vessels, including a large pial vein directly under the burr hole were noted to be free of blood and the cortical surface appeared pearly white. The cortical surface was 1 mm below the cranial bone. Perfusion Circuit The extracorporeal circuit for cryoprotective perfusion consisted of two parts: a recirculating system to which the patient was connected, and a cryoprotectant addition system which was connected to the recirculating system. The recirculating system was a 20 liter reservoir sitting atop a magnetic stirring table, an arterial (recirculating) roller pump, a Sarns 16310 hollow fiber oxygenator/heat exchanger and a Pall EC1440 40 micron blood filter. The recirculating (mixing) reservoir was continuously stirred with a 2" teflon-coated magnetic stirring bar driven by a Thermolyne type 7200 magnetic stirrer. The cryoprotectant addition system consisted of a 60-liter polyethylene reservoir containing 65% (w/v) glycerol (see Table I) and a Drake-Willock model #7401 hemodialysis pump acting as a withdrawal pump which removed perfusate from the recirculating system, while simultaneously adding 65% (w/v) glycerol perfusate from the concentrate reservoir to to the recirculating reservoir. Arterial and venous samples for evaluation of chemistries and glycerol concentration were drawn at 15-minute intervals during cryoprotective perfusion. Arterial samples were drawn from a 3-way, 3 stopcock manifold interposed between the arterial filter and the filter vent line. Venous samples were drawn from a 8' Cobe monitoring line connected to the Cobe 3-way stopcock and attached to the venous return line approximately mid-way along its course to the recirculating reservoir. (The dead-space of the Cobe monitoring line was determined and this volume was drawn up and held in a syringe attached to the middle stopcock of the Cobe manifold before each sample was taken.) The perfusion circuit was prepared in advance of need and was sterilized with ethylene oxide using an appropriate protocol of post- sterilization out-gassing and aeration. Oxygen gas delivered to the oxygenator at a flow rate of 2 liters per minute was used throughout cryoprotective perfusion. CRYOPHYLAXIS Cryoprotective perfusion is carried out to a terminal venous concentration of glycerol as close to 7.5M as possible. Starting and terminal concentration of glycerol in the arterial and venous perfusate is documented as well the rate of glycerol introduction. The procedure of cryoprotective perfusion and its documentation are illustrated below: Cryoprotective Perfusion Closed-circuit perfusion of 5% (w/v) glycerol perfusate was begun at 08:01 at a flow rate of 500 cc/min., a sinus temperature of 5.2*C, an arterial temperature (perfusate) of 6.3*C and a mean arterial pressure (MAP) of 50 mmHg. At 08:05 arterial and venous pH and gases were as follows: arterial pH 7.58, arterial pO2 35, arterial pCO2 10.3, venous pH 7.46, venous pO2 48, and venous pCO2 20.8. The glycerol ramp was begun at 08:01 and at 08:10 at a flow rate of 160 cc/min. Pulsatile flow was initiated using a Sarns pulsatile roller pump at a rate of rate 60 pulses per minute. Pulse pressure was adjusted to 100/10 mmHg and the flow rate was gradually increased from 500 cc/min. to a peak of 850 cc/min. At the start of pulsatile perfusion until well into the glycerol ramp at approximately 09:00 the cortical surface was observed to pulsate; rising and falling with each pulse. At 08:20 glycerolization of the face and scalp was noted to be very uniform, with no patchy non-perfused areas noted. Circulation through the scalp and dura was judged to be excellent with drainage from the burr hole occurring at a rate of 150-200 cc/min. Cryoprotective Ramp The recirculating perfusate withdrawal/glycerol concentrate addition flow rate was set at 160 cc/min., yielding a 50 mM/min. rate of increase in arterial glycerol concentration. This resulted in an average arterial/venous difference in glycerol concentration of approximately 550 mM over the course of cryoprotective perfusion. The initial response to the start of the cryoprotective ramp was good, with cerebral cortical volume rapidly decreasing to 2-3 mm below the margin of the burr hole. The cortical surface continued to shrink away from the burr hole and terminated at 6-7 mm below the cranial bone with brain appearing waxy yellow and shrunken within the burr hole. Cryoprotective perfusion was concluded at 09:45 at an arterial flow rate of 488 cc/min., MAP of 110 mmHg, arterial temperature of 6.0*C, sinus temperature of 6.2*C, and recirculating withdrawal/CPA addition rate of 164 cc/min. The final venous cryoprotectant concentration was 7.5 M. The concentration of glycerol in the arterial and venous effluent, the arterial and esophageal temperatures, arterial pressure, arterial flow rate and arterial and venous pH and gases are shown graphically below: At 09:45, the silastic-coated tip of a 15' long, 30 gauge Kapton-wrapped copper-constantan (type T) thermocouple probe (Instrument Laboratory #53- 30-513) was threaded into the burr-hole and placed on the cortical surface. The burr-hole was then filled with bone wax and the scalp closed with surgical staples. The probe wire was anchored to the scalp with surgical staples. Brain surface temperature was measured at 6.7*C at 09:57. Cephalic Isolation Surgery for cephalic isolation was begun at 10:03 with a circumferential skin incision made at the base of the neck and extended anteriorly and posteriorly to just below the margins of the clavicle. The skin was dissected free from the underlying connective tissue up to the level of the 5th cervical vertebra to form skin flaps. The cervical musculature and other anatomical structures were then severed with an electric knife (Black and Decker) down to the junction of the 5th and 6th cervical vertebrae. A Stryker saw was then used to cut throough the vertebral column at approximately the level of the 5th cervical vertebrae, which freed the head from the body. The cervical skin and musculature were observed to be dark in color, evenly stiff, and somewhat waxy in texture, consistent with uniform glycerolization. The spinal cord was observed to somewhat shrunken within the vertebral foramen. Skin flaps were then closed over the stump of the neck using a skin stapler, after the edges of the flaps were first approximated using interrupted 2-0 Ti-cron suture. Cephalic isolation was completed at 10:14. COOLING TO -77*C Cooling to the temperature of solid carbon dioxide at atmospheric pressure (technically, -78.5*C, but -77*C is as close as we ever get) was carried out immediately following cryoprotective perfusion. The precise cooling protocol employed is dependent upon the terminal glycerol concentration achieved during cryoprotective perfusion. The procedure for cooling to -77*C and its documentation are illustrated below: Cooling to Dry Ice Temperature Temperature descent to -77*C was monitored with probes in the frontal sinus, the brain surface, and head surface (placed temporally) and an additional probe was used to monitor bath temperature. Bath, external, and sinus probes were Instrument Laboratories 53-20-507, "load type", 20 gauge, teflon-coated copper-constantan thermocouples. (The 53-20-507 TC probe placed in the frontal sinus was used to replace the clinical TC probe employed to monitor pharyngeal temperature during perfusion.) TC probes were anchored into place with surgical staples. The patient (cephalon) was placed inside two 2 mil polyethylene plastic bags and lowered into a 15 liter bath of 5 centistoke polydimethylsiloxane oil (Silcool) which had been pre-cooled to -40.2*C. The first temperature readings taken at 10:25 were: brain surface 3*C, sinus 5*C, temporal -5*C and bath -10*C. Cooling to -77*C was at a rate of approximately -7*C per hour to -44*C and 15*C per hour to -77*C The temporal to sinus temperature differential of approximately 26*C to -40*C and 18*C to -77*C. Cooling to -77*C was complete by 16:00 on 11 June. The patient's dry ice cooling curve is presented below. Times shown are in decimal hours post-arrest. Serum/Perfusate Electrolytes and Enzymes Laboratory evaluations of samples taken during cryoprotective perfusion are presented in full in both graphic and tabular form as an addendum to this document. CASE SUMMARY The findings from the case, including discussion of the patient's response to transport, TBW, and cryoprotective perfusion, are briefly summarized as illustrated below : Discussion Not surprisingly the patient was markedly dehydrated at the time of cardiac arrest as evidenced by a serum osmolality of 358 mOsm and a BUN of 47. SGOT, SGPT, LDH and GGT were all elevated at the time of cardiac arrest presumably as a result of both the malignancy and the antemortem agonal period with its associated period of lengthy and profound shock. However as can be seen from the data, ongoing release of tissue specific enzymes during TBW and subsequent cryoprotectant perfusion was minimal if it occurred at all. Most enzyme levels had declined to very low or undetectable levels at the conclusion of TBW and aside from a modest and expected rebound during the cold ischemic period of air transport, remained low throughout cryoprotectant perfusion. This is in sharp contrast to cases where antemortem ischemic injury was more profound or where adequate CPS during transport was unachieveable or not possible. The acute response of this patient to CPS was judged excellent as evidenced by the prompt return of agonal gasping, rapid drop in core temperature, and visible improvement of skin color: all signs indicative of good perfusion and ventilation. Subsequent direct visualization of the vigorous pulse in femoral artery on Thumper support during cutdown as well as the bright red condition of the capillary and venous blood indicate that this patient continued to perfuse and ventilate well up until the the time of TBW. Subsequent analysis of serum/perfusate chemistries bear out the likely cerebral viability of this patient (within the context of contemporary medical criteria) throughout transport.