X-Message-Number: 2874
Date: 09 Jul 94 03:02:55 EDT
From: Mike Darwin <>
Subject: CRYONICS Case Report (3/3)
-- Continued --
Total Body Washout (TBW)
TBW was begun at 17:28 using 8 liters of Viaspan at a flow
rate of 630 cc/min, MAP of 50 mmHg, oxygen flow of 4 LPM,
arterial temperature of 8.7 C, esophageal temperature of 6.1 C
and a rectal temperature of 16.8 C. The composition of the
Viaspan with additives to it are given in Table I. TBW was
concluded at 17:49 at a flow rate of 630 ml/min, MAP of 50 mmHg
and an esophageal and rectal temperature of 4.8 C and 15.2 C
respectively. Oxygen flow-rate to the oxygenator was reduced to
2 LPM at the conclusion of TBW.
Table V: Composition of Viaspan
Component g/l
Lactobionic Acid 50.0
Potassium Phosphate, monobasic 03.40
Magnesium Sulfate, heptahydrate 01.23
Raffinose, pentahydrate 17.83
Adenosine 01.34
Allopurinol 0.136
Glutathione 0.922
Dexamethasone* 0.16
Glucose* 0.90
Insulin (U-100, Regular, Human)* 40 IU
Heparin* 2000 U/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
*Denotes component added immediately before use.
Recirculation of Viaspan was continued until 20:04 at which
time extracorporeal support was discontinued. At the conclusion
of bypass the patient's esophageal temperature was 1.8 C and the
rectal temperature was 6.2 C. The lines to the femoral cannula
were clamped and cut approximately 10 cm from where they were
connected to each cannula. A 1/2" -3/8" connector was used to
connect the stubs of the arterial and venous lines together
(carefully avoiding the introduction of air) effectively cross-
connecting the arterial and venous lines. The occluding clamps
were then removed and the wound (with cannula in-place) was
packed with sterile 4" x 4" gauze and covered with a 3M Steri-
Drape adhesive dressing.
The patient was then cleaned up in the PIB, transferred from
the PIB into a heavy-duty vinyl body bag, and the head rapidly
repacked in ice using 1-gallon Zip-Loc bags filled with shaved
ice. The body bag was closed and the patient was carried in the
body bag (using the body bag as a stretcher) down the steep
flight of steps from his condominium to the PIB on the Mobile
Advanced Life Support System in the parking lot. (The MALSS PIB
had been previously prepared by laying a layer of Zip-Loc bags
filled with shaved ice in the bottom of it) Once the patient was
in place in the PIB of the MALSS the body bag was opened and the
patient was thoroughly packed in ice from head to foot using Zip-
Loc bags filled with shaved ice. The PIB cover was placed and
the patient was wheeled into the BPI ambulance for ground
transport from Sunnyvale, CA where cardiac arrest had occurred to
BPI's cryoprotective perfusion facilities in Rancho Cucamonga,
CA.
The patient arrived at the facility in Rancho Cucamonga at
06:30 with an esophageal temperature of 1.0 C and a rectal
temperature of 0.6 C.
Gross Assessment
The patient was positioned on the operating table at
approximately 09:50. After the patient was positioned on the
operating table, he was briefly examined. Initial assessment of
the patient revealed a markedly dehydrated (globes deeply
retracted) cachectic male with complete rigor (except in the left
leg). The eyes were opened and the eye exam disclosed
bilaterally dilated pupils with corneal misting. The skin was
pale, bloodless in appearance, uniform in color, of markedly
decreased turgor, and free of lividity in dependent areas. The
skin/sternum under the Thumper piston was deeply indented and
bruised in appearance as is typical in patients subjected to
prolonged Thumper support.
Perfusate Preparation
The composition of the perfusate employed to carry out
cryoprotective perfusion is given in Table VII. Dry chemical
perfusate components were prepared from reagent or medical grade
chemicals using an Ohaus Model TS400D electronic balance
(previously weighed out and stored under dessicated conditions).
Dry components were mixed with American Chemical Society (ACS)
reagent grade glycerol and sterile water for injection USP,
and/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:
Table VI: Perfusate Volumes and Glycerol Concentrations
Description Volume Glycerol%(v/v)
Recirculating 40 liters 5%
Concenrtrate 60 liters 65%
TABLE VII
Mannitol-HEPES-2 (MHP-2) Perfusate Composition
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
Analysis of the perfusate with the Nova Stat-5 Blood
Gas/Electrolyte Analysis System disclosed the following:
pH = 8.202
Na+ = 72 mM/l
K+ = 24.1 mM/l
Cl- = 83 mM/l
Ca++ = 0.40 mM/l
Glucose = 77 mg/dl
mOsm/kg = 400
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 muslin towels.
A fenestrated 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 left frontal lobe.
Median Sternotomy/Vascular Access
Median sternotomy was begun at 12:04 on 02-06-1994 with an
incision over the midline of the sternum with an electrosurgical
knife. Fascia and connective tissue were cleared down to the
sternum with the electrosurgical knife. A median sternotomy was
then performed with a Stryker oscillating sternal saw. The edges
of the sternotomy were padded with laparotomy sponges, a self-
retaining retractor placed, and the sternotomy retracted open.
The left pleural space was opened and blunt and sharp dissection
were used to expose the pericardium. A ventral midline
pericardiotomy was made using Metzenbaum scissors. A Sarns
cardiotomy sucker was used to suction away the pericardial fluid
to discard.
Braided polyester purse string sutures (2-O Ti-cron) were
placed in the right atrial appendage and two were placed in the
aortic arch. A 5 Fr. Argyle polyethylene cannula was placed in
one of the purse-string sutures to monitor arterial pressure. A
24 Fr. x 40 cm USCI type 1967 Venous cannula was placed in the
right atrial appendage and passed into the right atrium. The
caudal vena cava was ligated with #1 silk. The descending
thoracic aorta at the level of the 4th rib was isolated and
doubly ligated with umbilical tape. A 16 Fr. Sarns 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 anchored in place with the purse string suture.
All cannulae were secured with # 1 silk suture to each line.
Industrial/automotive stainless steel hose-clamps were used
as tourniquets and were placed on each of the patient's arms at
the level of the axilla to achieve non-surgical vascular
isolation of the upper appendages.
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 4-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 sterile 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 13:29.
Cranial Burr-Hole
Surgery to open the cranial burr-hole was begun at 12:12.
The vertex of the scalp approximately 3 cm to the right of
midline over the right frontal 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 parietal bone approximately 3 cm to the right of the midline.
A 10 mm hole was made with a DePuy pneumatic perforator. The
burr-hole was then increased in diameter using a Hudson brace and
10 mm neuro burr. The dura mater was left closed and the
cortical surface was palpated through the dura and determined to
be about 3 mm below the inner aspect of the cranial vault The
dura appeared blood free.
Figure 4: Burr-hole location.
Opening of the dura was deferred until 14:43 in order to
attempt to determine the source of the copious drainage normally
observed coming from the burr hole during cryoprotective
perfusion of patients. Drainage from the scalp and bone wounds
was determined to be 10-15 cc/min. Approximately 30 minutes
after the start of perfusion the dura was again depressed and
found to be moderately tense. Either cerebral edema or fluid
accumulation in the subdural space had begun to occur. Seventy-
three minutes into cryoprotective perfusion the dura was breached
by perforating it with a 16 g needle and a copious, moderately
high pressure flow of clear fluid was observed to issue from the
needle. At this point the needle was withdrawn and the puncture
in the dura was widened with a #11 scalpel blade to approximately
1 mm in diameter. A copious and pressured flow of fluid was
observed streaming from the puncture. A 16 g blunt needle was
then passed through the opening in the dura and a sample of the
fluid collected in a 3 cc syringe for analysis of glycerol
concentration and gases and electrolytes. The results of that
analysis demonstrated a close correlation in composition between
venous perfusate and subdural effluent as contrasted with the
composition of arterial perfusate.
Table VIII: Composition of Subdural Fluid Compared with Venous
Perfusate (14:45)
Test Subdural Fluid/Venous Perfusate/Arterial Perfusate
pH 7.509 7.518 7.576
pO2 58 66 109.1
pCO2 18.1 17.4 3.9
Refractive Index 28.2 28.4 30.8
Following evaluation of the subdural fluid the dura was
elevated with a dura hook and trimmed to the margins of the burr
hole allowing for inspection of the cortical surface and free
drainage of fluid from the subdural space.
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 60 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% (v/v) glycerol (see Table
II) and a Drake-Willock model #7401 hemodialysis pump acting as a
cryoprotective addition pump which added 65% (v/v) glycerol
perfusate from the concentrate reservoir to the recirculating
reservoir. A Travenol 5M1153 roller pump was used to remove
perfusate from the recirculating system via the venous return
line and discard it to the drain. (This system is illustrated in
Figures 5 below.)
Figure 5: Cryoprotective perfusion circuit incorporating VBL and
Sarns 9438 reservoir.
A floating lid was used atop the recirculating perfusate
column to prevent air from being entrained in the perfusate by
the stirrer bar. A Sarns filtered (40 micron) hard-shell venous
reservoir was interposed between the recirculating reservoir and
the arterial pump to serve as an air trap/defoamer in the event
foam was generated in the recirculating reservoir and also as an
arterial prefilter for particulates (agglutinated RBCs, etc.) .
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, 4-stopcock manifold interposed between the arterial
filter and the filter vent line. Venous samples were also drawn
from the stopcock assembly which was connected by an 8' Cobe
monitoring line 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 a volume in excess
of the dead-space 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. A blend of
oxygen and helium gases was delivered to the oxygenator at a flow
rate of 2-liters per minute (this sweep gas flow rate was
maintained throughout cryoprotective perfusion). FiO2 to the
oxygenator was maintained between 18-25% throughout perfusion
Cryoprotective Perfusion
Open-circuit perfusion of 5% (v/v) glycerol perfusate was
begun at 13:30 at a flow rate of 1100 cc/min., an esophageal
temperature of 2.7C, an arterial temperature (perfusate) of
12.0 C and a mean arterial pressure (MAP) of 61 mmHg.
Approximately 15 liters was used to flush the patient's
circulatory system of Viaspan and residual blood and accumulated
metabolic byproducts from the period of cold ischemia during
transport before the start of cryoprotective perfusion. A
significant amount of cold agglutination was noted during this
initial flush (typical "grains of sand" clumps of red cells
which disappeared on collection and warming), but there were no
signs of clotted material. Additionally, it should be noted that
whole blood samples drawn on this patient prior to and during
transport (anticoagulated with EDTA) also showed marked cold
agglutination upon refrigeration to 2-4 C (Agglutination Score =
9, after Marsh, RL, Transfusion 12;5: 352-353, 1972). At 13:30
arterial pH and gases were as follows: pH 7.772, pO2 202.1, pCO2
1.4 and venous pH and gases were pH 7.328, pO2 21.8, pCO2 4.5 at
an FiO2 of 23%. The glycerol ramp was begun at the start of
closed circuit perfusion at 13:45 at an addition/withdrawal rate
of 200 cc/min. Closed circuit cryoprotective perfusion was begun
at 13:45 with 20 liters of 5% (v/v) glycerol perfusate in the
recirculating reservoir and 40 liters of 65% (v/v) glycerol
perfusate in the concentrate reservoir.
At 14:42 glycerolization of the face and scalp was noted to
be patchy with many non-perfused areas noted (possibly due to
cold agglutination in the skin). The skin on the neck and upper
thorax was observed to be glycerolizing somewhat more evenly and
appeared to have fewer non-perfused areas. The left ear and
left face appeared to be glycerolizing better than the right ear
and face. It was noted that the right side of the patient's head
was the dependent side (the patient's head was turned to the
right) during the last few hours of life. In particular a large
(20-30 cm) area of scalp near the occiput of the head on the
right side was noted to be cherry red and nonperfusing --
apparently an infarcted area as a result of a compression of the
skin (and resultant ischemia) during the hours of shock and
immobility which characterized the patient's agonal course.
Cryoprotective Ramp
After the burr hole was opened at 14:43 withdrawal of
perfusate from the recirculating system was temporarily
discontinued as leakage from the burrhole was in excess of the
desired withdrawal rate (leakage was 150 cc/min). After
evaluating the recirculating reservoir level for 30 minutes the
withdrawal pump was again started at a rate of 110 cc/min. The
rate of increase in arterial glycerol concentration during most
of the first third of perfusion was 46 mM/min.. This
differential had declined to 25 mM by the second third of
perfusion and was approximately 29 mM for the last third of
cryoprotective perfusion.. This resulted in an average
arterial/venous difference in glycerol concentration of
approximately 36 mM over the course of cryoprotective perfusion.
Glycerol concentration increase in both the arterial and venous
perfusate are given in graphic form at the end of this report and
in tabular form below:
Time (Arterial M) (Venous Glycerol M)
1352 1.13 0.95
1400 1.43 1.18
1418 1.71 1.62
1430 3.18 2.73
1445 3.84 3.40
1500 4.41 3.90
1515 4.94 4.38
1530 5.33 4.90
1545 5.84 5.28
1600 6.08 5.69
1621 6.38 6.09
1635 7.14 6.76
The initial response to the start of the cryoprotective ramp
was poor in the skin but good in the brain, with cerebral
cortical volume rapidly decreasing to 2-3 mm below the inner
aspect of the cranial vault. The pial surface was observed to be
blood free over the visible area of the right hemisphere (an area
of about 20-30 mm in all directions from the burr hole; the
limits of illumination of the cortical surface with a pen-type
flashlight). The cortical surface continued to shrink away from
the burr hole to a low of approximately 7 mm below the inner
aspect of the cranial vault. At approximately 15:23 brain volume
was noticed to have increased slightly with the cortical surface
at 3-4 mm below the cranial vault. By 16:21 the cortical surface
was at the level of the burr hole opening or perhaps .5 mm into
the burr hole. At 16:41 The cortical surface was approximately
+1 mm into the burr hole at the conclusion of cryoprotective
perfusion. Immediately after the termination of perfusion (i.e.,
after the arterial pump was turned off) the cortical surface
receded to 1.0 mm below the inner aspect of the cranial vault.
At 16:22 the rate of concentrate addition to the
recirculating system was increased from 200 cc/min to 400 cc/min
and the withdrawal pump rate was increased from 115 cc/min to 220
cc/min. Perfusate levels in the concentrate and recirculating
reservoir were 7.5L and 17L, respectively. Arterial flow rate
at that time was 675 cc/min, MAP was 81 mmHg, arterial
temperature was 14.0 C and esophageal temperature was 9.7 C.
Blood gases, pH and electrolytes during perfusion are
appended to the end of this case history.
Cryoprotective perfusion was concluded at 16:35 at an
arterial flow rate of 500 cc/min., MAP of 81 mmHg, arterial
temperature of 11.6 C, esophageal temperature of 9.2 C,
recirculating system addition/withdrawal rate was 400 /220
cc/min. Final venous gases/electrolytes were not measurable
due to the high viscosity of the perfusate (the Nova Stat-5
Profile is unable to process such viscous samples). The last
venous gases/electrolytes for which data wereobtainable was drawn
at 16:00 and were as follows: pH 7.543, pO2 28.2 mmHg, pCO2 4.4
mmHg, Na+ 104 mM, K+ 71.1 mM, Cl- 77 mM, Ca++ 0.34 mM, glucose 81
mg/dl. Laboratoy analysis of arterial and venous samples drawn
during cryoprotective perfusion are presented in graphic form at
the end of this report.
Reservoir levels at the conclusion of cryoprotective
perfusion were: concentrate 1-2 L, and recirculating 7 L. The
final venous glycerol concentration was 6.76 M.
Burr-Hole Closure
At 16:43, the silastic-coated tip of a 15' long, 30 gauge
Capton-clad 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.
Evaluation of cerebral cortex temperature was delayed until the
burr-hole had been closed, at which time it was discovered that
the thermocouple was not functional. The burr hole was reopened
(staples and bone wax removed) and a new (functional)
temperature probe was placed. The burr hole was closed as before
and the probe wire was anchored to the scalp with surgical
staples. Brain surface temperature was measured at 9.5 C at
17:00, esophageal temperature was 7.0 C.
Between 17:00 and 17:12, 20 gauge, teflon clad, copper-
constantan TC probes were placed nasopharyngeally and on the skin
of the right temple. Both probes were anchored to the patient by
stapling the probe wires to the skin with surgical staples.
Cephalic Isolation
Surgery for cephalic isolation was begun at approximately
17:12 by making a circumferential incision at the base of the
neck at just above the level of the clavicle using a Black and
Decker Slim-Grip (Catalog # EK200, Type 1) Electric Carving
Knife. The Electric Knife was used to incise the cervical skin,
musculature, and other structures down to approximately the 5th
or 6th cervical vertebrae. The vertebral column was then cut
with a Satterlee amputation saw, freeing the head from the body.
The cervical musculature (in contrast to the facial skin)
was observed to be uniformly perfused and glycerolized as
indicated by its stiff and waxy texture and dark (maroon) color.
The spinal cord was observed to be blood free, slightly shrunken
within the vertebral foramen, and otherwise giving an appearance
consistent with good glycerolization. The cervical vessels were
observed to be blood free and the cervical tissues at cross-
section appeared to be free of infarcts and evenly glycerolized.
The cephalic cervical stump was covered with sterile gauze 4" x 4" s
which were in turn covered and held in place with a sterile
Stockinet. Cephalic isolation was completed at 17:20
Cooling to Dry Ice Temperature
Temperature descent to -79 C was monitored with probes in/on
the pharynx, brain surface and head surface (placed temporally).
An additional probe was used to monitor bath temperature. Bath,
external, and nasopharyngeal probes were Instrument Laboratories
53-20-507, "load type", 20 gauge, Teflon-coated copper-constantan
thermocouples.
The patient (cephalon) was placed inside two 1.4 mil
polyethylene plastic bags and lowered into a 20 liter bath of 5
centistoke polydimethylsiloxane oil (Silcool) which had been pre-
cooled to -22 C with dry ice. The first temperature readings
taken at 17:24 were: brain surface 7.0 C, temporal 8.7 C,
nasopharyngeal 7.5 C and Silcool bath - 21.4 C. Controlled rate
cooling was achieved by the manual addition of dry ice to the
Silcool bath.
Cooling to -77 C was at a rate of approximately 5 C per hour
to -40 C, and approximately 4.5 C per hour from -40 C to -77 C.
Cooling to - 77 C was complete by 10:45 on 02-07-1994. Patient
cooling data to -79 C are presented tabularly and graphically as
an addendum to this document.
Cooling was achieved by addition of dry ice to the bath per
the following protocol:
1) Hold bath temperature at -40 C until nasopharyngeal
temperature reaches - 35 C.
When nasopharyngeal temperature reaches -35 C decrease bath
temperature to -50 C.
3) Hold bath temperature at -15 C below nasopharyngeal
temperature to maintain a bath-to-core temperature differential
of 15 C.
4) When an nasopharyngeal temperature of -65 C is reached add
sufficient dry ice to take the bath temperature down to -79 C and
hold it there.
Once cooling to -79 C was complete the patient was lifted
out of the Silcool bath, the outer, Silcool wetted plastic bag
was stripped off and the patient was placed within a polyester
cloth bag which was in turn placed inside a standard aluminum
neurocan which had been precooled to -90 and packed with dacron
wool. Temperature probes were left externalized to the neurocan
for subsequent cooling to -196 C.
Serum/Perfusate Electrolytes and Enzymes
Laboratory evaluations of samples taken during
cryoprotective perfusion are presented in full in tabular and
graphic form as an addendum to this document. All samples were
analyzed with a Nova Stat Profile 5 (in-house) or by SmithKline
Beecham Clinical Laboratories of VanNuys, California using an
Olympus AU5061 Clinical Chemistry Analyzer.
Final venous chemistries were as follows:
Na+ 46 mM/L
K+ 28.4 mM/L
Cl- 16 mM/L
Ca++ 0.9 mg/dl
Glucose 74 mg/dl
Urea Nitrogen 7 mg/dl
Creatinine 0.2 mg/dl
Phosphorus 2.2 mg/dl
Protein, Total 1.5 g/dl
Albumin 0.5 g/dl
Globulin, Total 1.0 g/dl
A/G Ratio 0.5
Bilirubin, Total 0.1 mg/dl
Alkaline Phosphatase 3 U/L
Lactate Dehydrogenase 211 U/L
GGT 2 U/L
AST 72 U/L
ALT 18 U/L
Uric Acid 0.3 mg/dl
Triglycerides 1121 mg/dl
Cholesterol, Total 1 mg/dl
Michael Darwin, Cryopreservation Team Leader Date
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