Cryonics, July 2014 (some data updated September 2016)
By Aschwin de Wolf
Two of the most important variables in cryonics patient care are time and temperature. These two concepts are clearly related. If the time between pronouncement of legal death and the start of cryoprotection is minimized we can place the patient in long-term cryostasis without incurring unnecessary cold ischemic injury. Not surprisingly, it has occurred to a number of people in the cryonics field that the quality of care could be improved if we eliminate the prolonged cold ischemic time that is typically associated with remote cryonics cases (i.e. cases outside of the Scottsdale area). In this article I will outline potential protocols and challenges concerning field cryoprotection. Field cryoprotection is the replacement of blood and tissue water by solutions of cryoprotective agents (CPAs) near the location of legal death, followed by prompt cooling to dry ice (-79°C) or lower temperatures at the same remote location. If a temperature cold enough to achieve a solid state is attained (approximately -130°C), the procedure could be called field cryopreservation. For Alcor members, these procedures have historically been done only in Alcor’s Scottsdale, Arizona, facility.
RATIONALE OF FIELD CRYOPROTECTION
To understand the rationale and challenges associated with the idea of field cryoprotection it is useful to briefly describe the current procedure for remote cases. Currently, when a member is considered terminal and close to legal death Alcor deploys a standby team to the bedside of the patient. For cases outside of Arizona in the continental United States, the team will typically be from Suspended Animation, Inc., and include a surgeon and clinical perfusionist. Upon pronouncement of legal death the team starts (mechanical) chest compressions and rapid cooling and administers a series of medications to mitigate ischemia. If qualified surgeons are part of the team, an additional procedure is to perform a field washout in which blood is replaced by a cold (but not freezing) organ preservation solution. The three most important objectives of the washout are to (a) increase the cooling rate (b) remove the blood and risk of coagulation and cold agglutination and (c) protect the patient against cold ischemia by introducing an organ preservation solution. The patient is then shipped on water ice to the cryonics facility for cryoprotective perfusion and long-term care.
As is clear from this suggestion, between the end of blood washout and the start of cryoprotective perfusion the patient is basically experiencing a prolonged period of cold ischemia (lack of oxygen), the duration of which is dependent on the airline schedule and distance to Alcor. While experimental evidence at a number of cryonics-associated research labs indicates that remote blood washout is superior to leaving the blood in the patient, it should be evident that prolonged cold ischemia is not beneficial to the patient and could be completely eliminated when there is a smooth transition from stabilization to cryoprotectant perfusion. For example, blood substitution with a static organ preservation solution can keep the brain viable (able to spontaneously resume function upon reperfusion with blood) for about 6 hours in the most optimistic projections.
Proposed benefits of field cryoprotection include:
TERMINOLOGY, HISTORICAL BACKGROUND, AND RESEARCH
In the context of this article field cryoprotection is defined as the procedure of conducting cryoprotective perfusion with a vitrification agent at a location remote from the cryonics facility followed by transport of the patient on dry ice for further cryogenic cooldown and long term care at the cryonics facility.
It is important to stress here that this procedure does not entail field vitrification. Field vitrification would not just require remote cryoprotective perfusion but also cryogenic cooling on-site and shipping at around -130 degrees Celsius (below the glass transition temperature of the vitrification agent) or -196 degrees Celsius (liquid nitrogen temperature). While it is not impossible to ship the patient at such temperatures it would introduce a number of non-trivial technological and logistical challenges. This would also likely offset any cost reductions associated with conducting cryoprotective perfusion in the field. As will be discussed below, in field cryoprotection the patient is cooled below 0 degrees Celsius after cryoprotective perfusion but not to a temperature where the vitrification agent solidifies into a glass. For this reason the procedure discussed in this article should be named field cryoprotection (or field cryoprotective perfusion) and not field vitrification or field cryopreservation.
The idea of field cryoprotection is not new and various proposals to introduce the technology have been introduced in the past (including proposals for real field vitrification and shipping below the glass transition temperature). In June 1990 Alcor patient A-1239 received a field cryoprotection with glycerol in Australia prior to shipment on dry ice to Alcor in the USA. In addition, on October 23, 2004, the cryonics company Suspended Animation performed a field cryoprotection with glycerol for the American Cryonics Society prior to shipping the patient on dry ice to the Cryonics Institute for long-term care. The Cryonics Institute also has authorized field cryoprotection for select (international) cases.
There are number of distinct protocol differences between this current implementation of field cryoprotection and cryoprotection at the Alcor main facility. These protocol differences are not intrinsic to either field cryoprotection or facility cryoprotection however. By historical standards, today’s field cryoprotection protocols by Alcor are often more sophisticated than older facility cryoprotection protocols and even contemporary protocols at other cryonics organizations.
One concern that has often been expressed about field cryoprotection is that shipping the patient at dry ice temperature after introducing the vitrification agent could result in ice formation en-route to the cryonics facility. While this concern cannot be completely eliminated yet, independent results from at least three research labs indicate that this issue does not seem to be a problem for CPA solutions currently used for vitrification in cryonics. The cryobiologist Yuri Pichugin stored large volumes of VM-1 (the vitrification agent used by the Cryonics Institute) and cryoprotected cortical rat brain slices at dry ice temperature without observing ice formation after days of storage. Similar results have been observed in other animal models at 21st Century Medicine. In 2012 Advanced Neural Biosciences collaborated with Alcor to specifically validate Alcor’s proposed field cryoprotection protocol in the rat model and again no ice formation was found after up to 48 hours of storing the brains at dry ice temperature prior to further cooling.
These encouraging research results and experience with this protocol in companion animal cases led Alcor to authorize field cryoprotection for overseas cases that otherwise would end up being “straight freeze” cases (i.e., cryopreservation without cryoprotection).
WHOLE BODY AND NEURO FIELD CRYOPROTECTION
In principle, field cryoprotection can be conducted in both whole body and neuro cases. Whole body field cryoprotection presents a number of distinct challenges. For starters, a lot more cryoprotectant is needed for whole body cases which for most locations would require the shipping of large volumes of perfusate to the location where the patient will be cryoprotected. Usually, though, there should be ample time for this because most cases in which field cryoprotection is feasible and productive involve patients with a prolonged agonal “dying” phase which allows the timely shipping of perfusate.
An additional complication involves shipping the patient. Because the patient needs to be shipped on dry ice it is crucial that the cryonics organization comply with airline regulations concerning dry ice and potential weight restrictions. Of course, since cold ischemia is basically eliminated during shipment it would also be possible to transport the patient by ground to the cryonics facility (in whole body cases).
While it is sometimes claimed that one major difference between whole body and neuro cryoprotection involves a difference in surgical procedures this is not necessarily the case. In case a median sternotomy is chosen to cannulate the heart or aorta both neuro and whole body cryoprotective perfusion can be conducted by just making minor adjustments. A more detailed discussion of potential surgical protocols follows.
There are basically three options for obtaining vascular access in field cryoprotection.
(1) Femoral cannulation
In femoral cannulation a “femoral cut down” is performed to cannulate the femoral artery and vein in a single leg to perfuse the patient. One advantage of this approach is that femoral cannulation used to be the preferred approach for remote blood washout and the cannulae can just remain in place for subsequent cryoprotectant perfusion (even in field cryoprotection, stabilization usually benefits from a washout to accelerate cooling and removing the patient’s blood). This approach, however, would not constitute an attractive option for neuro cryoprotection because a lot of perfusate is wasted in perfusing the rest of the body. Another potential disadvantage is that in conditions of ischemia-induced edema perfusion of the brain could be suboptimal. In addition, not all patients have a healthy, patent, femoral artery that will ensure good flow.
(2) Heart (aorta) cannulation
In a median sternotomy the chest is opened to cannulate the heart or the ascending aorta. This procedure can be used to either perfuse the whole body or, when the descending aorta (and arms) are clamped, to limit perfusion to the upper body. A major advantage of this approach is that a large organ (the heart) or the widest vessel in the body (the aorta) is selected for perfusion which reduces challenges associated with cannulating patients with no flow (such as collapsed vessels) and ensures good flow. In a very basic version of the procedure, venous cannulation is not necessary and an opening in the right atrium will suffice for venous drainage. A concern about this approach is that too much perfusate is wasted in neuro cases. Median sternotomy used to be the standard surgical approach for both whole body and neuro cases at Alcor prior to going to isolated head perfusion for neuro patients, and as of this writing is the default approach for all cases at the Cryonics Institute.
(3) Carotid cannulation
Carotid cannulation involves cannulating the carotid arteries, and sometimes the vertebral arteries, in the neck of the patient. This procedure is primarily designed to allow cryoprotective perfusion of the head. As such, this surgical approach is used primarily in neuro cases. It is the simplest cannulation to perform. It focuses on the head (brain) of the patient and minimizes required perfusate volumes. Another advantage is that if the cephalon is perfused separately the whole stump of the head can be used for venous drainage. Disadvantages include the lack of an easy “downstream” fall-back option in case errors are made or the vessels are too fragile or damaged for perfusion. There is also the issue that a determination would need to be made about whether a patient has an intact Circle of Willis. Without this, the vertebral arteries would need to be cannulated, too, for complete perfusion of the brain.
One argument against the carotid approach is that unless cephalic isolation is used as an approach for cryoprotection, washout will also need to be restricted to the head unless the team performs two separate cannulations. This may introduce temperature differences between the head and the rest of the body. There is also a risk of introducing blood to the brain during cryoprotective perfusion if there is some blood remaining after washout. In the opinion of the author, the most decisive argument against the carotid approach is that there are limited fall-back options in case of failure. If the femoral or heart/aortic approach is used, the field team could decide to terminate efforts to conduct cryoprotectant perfusion and transport the patient to Alcor where professional surgeons can attempt carotid cannulation. Field cryoprotective perfusion should allow for a back-up plan in case of failure, which the carotid approach does not permit. The heart / aortic approach also has the advantage that it permits both neuro and whole body cryoprotection.
Designing a protocol for field cryoprotection presents 4 challenges:
1. Ensuring a gradual introduction of the vitrification agent (CPA) to reduce osmotic injury to the cells. When a patient is cryoprotected at the main Alcor facility this goal is achieved by gradually mixing the “carrier solution” with the cryoprotectant in a recirculating reservoir and terminating perfusion when the desired terminal concentration of the agent has been consistently observed in venous fluid. In field cryoprotection such a recirculating setup would be complicated and current field cryoprotection protocols involve introducing a series of bags with increasing concentrations of the vitrification agent. Terminologically, the current field cryoprotection protocol is “open circuit” perfusion in which venous flow is discarded, while Alcor’s facility protocol is “closed circuit” perfusion in which venous flow is recirculated. In Alcor’s field cryoprotection protocol bags can be (and are) overlapped using a “teeter-totter” which blurs the jump between steps, further smoothing the introduction of different concentrations.
2. Temperature control. At the Alcor main facility cryoprotective perfusion is started at 0 degrees Celsius and lowered to about -3 degrees Celsius for the final half of the procedure to mitigate the cryoprotectant toxicity associated with higher concentrations. In field cryoprotection subzero perfusion presents a bigger challenge and would require an enclosure with circulating nitrogen gas and running the perfusate through a heat exchanger (HEX) capable of reducing the temperature below the freezing point of water. Alcor’s current field cryoprotection protocol involves keeping the temperature of the patient and the perfusate as close to 0 degrees Celsius as possible.
3. Monitoring the refractive index (or Brix reading) of the vitrification agent as the concentration increases. At the Alcor main facility the concentration of the vitrification agent is continuously monitored in the perfusion lines to observe trends. Decisions as to whether to continue or stop perfusion are made using a benchtop refractometer. In field cryoprotection continuous inline monitoring of concentration of the vitrification agent would be challenging and the current protocol requires the use of a handheld digital refractometer to make frequent refractive index (or Brix) readings to observe trends and to decide whether to continue or end perfusion.
4. Controlling flow rate and pressure. There are two options for controlling flow of the perfusate in the patient: a pump or a hanging bag system. The major advantage of using a pump is that it provides precise control over flow rates and pressure. The advantage of a hanging bag system is that no priming of the pump and other associated challenges need to be performed. Another advantage is that pressure spikes are limited by the height of the bags. In reality, the choice of either a pump or a bag will greatly depend on the degree of expertise and experience in the field.
For example, the current Alcor protocol for field cryoprotection under discussion employs an 8-step bag system (including washout with B1 carrier solution):
|Bag||nM22 Concentration||Refractive Index (BRIX)|
nM22 denotes a solution make by diluting 125% M22 solutes prepared in LM5 carrier solution with B1 carrier solution to achieve the stated concentrations. The percent concentration scale is not concentration of solutes, but percent full concentration of M22, which has defined solute concentrations. 100% M22 or 100% nM22 is also sometimes called 100% CNV (concentration needed to vitrify) to express the idea that tissue is ideally to reach full M22 solute concentration before stopping perfusion and attempting vitrification by cooling. The endpoint for perfusion in this protocol has been measurement of jugular effluent of nM22 over 49.9 Brix refractive index (100% CNV) for over 30 minutes. This protocol ensures a concentration necessary to vitrify (CNV) in the cells without prolonged exposure to even higher concentrations.
TWO VISIONS OF FIELD CRYOPROTECTION
While Alcor has authorized field cryoprotection for overseas cases (see the announcement in Part 2 below) there is still an ongoing debate about the desirability of introducing field cryoprotection for most Alcor members who are pronounced legally dead in the United States and Canada. Issues that have been discussed include scientific, technological, and financial concerns. Alcor’s facility cryoprotection procedures are designed to closely replicate laboratory research protocols that have shown published efficacy for brain cryopreservation. They are based on established principles of organ cryopreservation for minimizing osmotic and cryoprotectant injury while eliminating ice formation. To what extent do simplified and shorter open-circuit field cryoprotection protocols compromise cryopreservation quality? Alcor’s facility infrastructure includes computerized control and recording of multiple perfusion parameters, and personnel for observation and note-taking. To what extent will field cryoprotection quality suffer because of decreased perfusion parameter control, decreased data recording, and resulting decreased quality control feedback? Can a patient be shipped on dry ice without risking ice formation during transport to the cryonics facility? What is the easiest and safest surgical approach? How many concentrations of the vitrification agent need to be used? Can we lower the cost of our procedures by embracing field cryoprotection? Perhaps the most difficult question of all is: At what distance and transport time from Alcor do the disadvantages of current field cryoprotection procedures (especially no cryoprotection for the body of whole body patients) become outweighed by the advantages of avoiding long transport times at 0°C? There are some who worry that simplified field cryoprotection procedures with limited monitoring are driven by a desire to reduce costs, complexity and oversight rather than strict improvement of care and cryopreservation outcome. Yet clearly there are distances for which even the simplest field cryoprotection protocols are beneficial, such as locations with multiday transport times.
A sensible approach to evaluate these issues is to ask whether the primary aim of field cryoprotection is improvement of patient care or simply reduction of cost. While it is indisputable that the elimination of two separate deployments can lower the costs associated with Alcor’s procedures (assuming field cryoprotection protocols that are deliverable by current standby teams), these different perspectives can lead to different views on how to conduct field cryoprotection. If field cryoprotection is primarily advocated as a means to improve patient care the most likely implementation for Alcor right now is to request its standby provider (currently Suspended Animation for non-local cases) to add field cryoprotection to its washout procedure. While it would be simplistic to argue that this would just involve simply adding a few bags of perfusate to the washout procedure, it should be recognized that an organization that employs professional surgeons to establish surgical access and professional perfusionists for running the pumps should be able to perform this procedure without formidable challenges. If the aim, on the other hand, is to just reduce cost and involve Alcor staff and volunteers in field cryoprotection, the most conservative surgical protocols and cryoprotection protocols would need to be followed to reduce errors.
In the opinion of the author, it is not possible to have a sensible discussion about the nature and scope of field cryoprotection without asking the question who is going to perform it. If Alcor entrusts the conduct of remote blood washout to qualified independent contractors then concerns about the absence of relevant surgical and perfusion skills may not be all that relevant. If field cryoprotection is seen as a replacement of these contracts, however, Alcor would be making a challenging leap into the unknown.
ALTERNATIVES FOR FIELD CRYOPROTECTION?
The only credible alternative for field cryoprotection would be to validate and introduce organ preservation solutions aimed at securing viability of the brain, or at least perfusability of the brain, for much longer than is possible with Alcor’s current organ preservation solution (MHP-2). In essence, this would require the design and successful validation of “brain preservation solutions” that can preserve cerebral viability for up to 24 or 48 hours of cold ischemia. While the cryobiology company 21st Century Medicine has made a number of breakthroughs in organ preservation solution design that permit securing viability of the brain for much longer periods than is possible with MHP-2, these protocols require either continuous or intermittent perfusion of the patient (or the patient’s brain) en route to the main cryonics facility. This fact by itself necessitates ground transport of the patient under supervision of qualified staff, which in some cases could involve many days.
Another concern with continuous perfusion protocols is that there is little information available on their effects in cases of preceding warm ischemia. Prior research in the art would indicate, however, that continuous perfusion in an ischemic patient, especially in a whole body patient, will produce severe edema over a long period of time. This edema could prevent any meaningful cryoprotective perfusion at the main facility, defeating the main objective of blood substitution.
In conclusion, the most basic question is rather straightforward. If cryoprotectant perfusion can be done competently in the field without much sacrifice in quality, and with much better outcomes in terms of elimination of cold ischemia and ice formation, why continue the tradition of transport on ice after remote washout? In the long term, there is no theoretical reason that everything currently done in Alcor’s facility operating room couldn’t be done at remote locations. However the cost of establishing such infrastructure would be very high, raising the question of how sophisticated field cryoprotection really needs to be to be beneficial at various distances.
By Max More
For decades Alcor has welcomed members residing overseas, and pledged to attempt to cryopreserve members who suffer legal death while traveling outside the United States. However, options for responding to overseas cases have been very limited. Historically there has been a choice between shipping on water ice near 0 degrees Celsius (with or without blood replacement) and attempting subsequent cryoprotective perfusion at Alcor to eliminate or minimize ice formation, or so-called “straight freezing” to dry ice temperature of -79 degrees Celsius without cryoprotective perfusion and shipping to Alcor.
Cryoprotective perfusion after a prolonged period of cold ischemia is usually compromised, typically leading to the difficult decision to “straight freeze” overseas cases to dry ice temperature prior to shipping. Freezing without cryoprotectant is extremely damaging to tissue. About all that can be said for it is that it is better than the alternative of not being cryopreserved at all.
There is now a better alternative. As described in Part 1 above, Alcor has developed a simple system for perfusing cryoprotectant solution in a remote field setting instead of requiring patients to first arrive at Alcor’s facility. After completion of this field cryoprotection, patients can be cooled to dry ice temperature (-79 degC) for shipment to Alcor with less time urgency and a slower rate of biological damage than at 0 degrees. Once at Alcor, cooling is resumed to the temperature of liquid nitrogen (-196 degC) at which temperature tissue is stable for practically unlimited lengths of time.
Alcor’s initial implementation of field cryoprotection is still crude compared to cryoprotective perfusion in Alcor’s operating room. Temperature and pressure control are limited, the cryoprotectant concentration rises more rapidly than is ideal, and the perfusion time is comparatively brief. Very importantly, the present field cryoprotection procedure only perfuses the head and brain with cryoprotectant, so the body of whole body members receiving field cryoprotection will still be frozen without cryoprotectant. However, this is obviously a better outcome than the entire body, including the brain, being frozen without cryprotectant.
UPGRADED CANADIAN AND EUROPEAN RESPONSE
The logistical challenges of implementing field cryoprotection internationally are substantial. Only future experience will reveal whether we are able to apply FCP in a majority or a minority of international cases. Here is the current situation: The Alcor board has authorized the use of FCP for cases taking place outside the United States. We have stationed a kit in Canada in the Toronto area, and another one in London, England. The contents of the two kits is similar, with minor differences that depend on supplies already present locally.
The Toronto kit includes stabilization equipment and the equipment, supplies, and solutions for FCP. Although the perfusate can be stored indefinitely when refrigerated, we will check its condition every six months by ensuring that a visual inspection is performed locally.
In England, we will conduct a training session with members of Cryonics-UK on November 15, and hope to conduct an additional training session the day before in London with the international morticians where the FCP kit is currently stored.
Having a kit located in Canada means that we can respond to Canadian members needs without worrying about essential supplies being held up in customs. This is equally important in England. The England kit may also be used to respond to members throughout Europe. Even so, we aim to eventually store another kit in Continental Europe, perhaps in Germany.
Who responds to a critical member in England or elsewhere in Europe? The answer depends on how much advance warning is available. In some cases, we would expect our Medical Response Director to fly to London and pick up the FCP kit and take it to the location where a standby or immediate stabilization is required. If time is too short, we may make use of the staff of the international mortician where our kit is stationed, including their highly skilled embalmer. A third option is to call upon the trained members of Cryonics-UK. Of course, we may use two or all three of these options, as circumstances indicate.
We are still in the very early stages of using field cryoprotection. However, assuming that we respond quickly, our Canadian and European members should benefit from a very substantial improvement in the quality of their cryopreservation, should they need it. Our first priority is to ensure that all necessary items of the FCP technology are in place and that local persons are adequately trained in using them (in case we cannot reach them quickly enough). Beyond that, we would like to position new kits in more locations. We would also like to improve the FCP technology to address some of the shortcomings of this approach in comparison to conducting closed-circuit cryoprotection in Alcor’s operating room. For instance, we may be able to improve chilling of the perfusate, and improve control over flow rate and pressure.