X-Message-Number: 9483 Date: Tue, 14 Apr 1998 01:41:15 -0400 From: Mike Darwin <> Subject: Postives I'm sure many people perceive the recent exchanges on Cryonet about CI's human cryopreservation methods as "flame wars" with no useful endpoint and a great deal of "negative impact." I have said what I have to say about the objective scientific issues in a straightforward way. However, this too may be construed as negative, although (in the long run anyway) it is not. There is however a more positive way of dealing with the issues. I believe the content below is as close to totally positive as it is possible to be. (A simple yes or no is all that is required and there are no hidden agendas. I will not be posting further on this issue and have much other work to do.) The arguments on Cryonet are not really about who has the best ultrastructure, but about who has the worst. This is pathetic at best. Pichugin, CI and Bob have acknowledged that improvements are possible in their procedures and have expressed interest and willingness to make changes if such changes are affordable. Rather than engage in challenges, confrontations or polemics I offer these suggestions _and resources:_ 1) Brian Wowk and I are willing to travel to CI's facility in Michigan and instruct their personnel for a 3 day period at no charge other than our airfares and lodging at an economy motel. _This offer is good for 90 days from the trime of this post._ Such instructions would include the option to implement the following: 2) One simple change CI can make is to add an inexpensive colloid to their Ringer's mannitol flush (RMF) solution. Dextran-40 is the cheapest effective colloid. It is not the best, but it is far better than nothing and it goes into solution easily. RMF containing 40 g/l of dextran-40 can be easily prepared and stored in the Ball Food canning jars which is how I observed CI to be storing their RMF when I visited their facility a few years ago. 3) Stepped concentration of glycerol solutions that use an osmotically defensible approach (verified in our lab and many others with tissue slices and organs; including Greg Fahy's lab) to cryoprotectant introduction can be easily implemented by CI using open circuit perfusion. For simplicity here I will deal only with perfusate volumes sufficient for the head. The body can be dealt with by CI as it chooses. Basically the protocol might reasonably go as follows: a) 15 liters of 7.5% (v/v) glycerol in CI's carrier solution (perfusate) plus 40 g/l dextran-40. b) followed by 15 liters of 25%(v/v) glycerol in the perfusate with 40 g/l dextran-40. c) followed by 20 liters of the final concentration solution up to 75% if desired with no dextran or other colloid. The cost of the dextran-40 from Pharmachem will be a few hundred dollars and it will do much to inhibit edema. It is not a prescription item. Generally, you can step-up glycerol concentration in this manner while minimizing osmotic destruction. I would also suggest CI buy an inexpensive hand-held refractometer such as that made by Atago and marketed by Cole-Parmer Scientific for about $300 to monitor effluent glycerol concentrations. 4) Perfusate should be filtered through a 0.4 or preferably a 0.2 micron filter before use. This is to remove the enormous amount of particulate matter which is present in reagent grade chemicals and which will block capillaries. Even the most basic physiology experiments call for filtered solutions. A Pall 0.2 micron pre-bypass filter can be used for this. They cost about $45.00 each and will do about 150 liters of perfusate. These filters will RAPIDLY (1-2 liters a minute) filter perfusate using a standard Sarns roller pump and 3/8" ID tubing such as I observed CI to have when I toured their facility. I will give CI the first 0.2 micron filter free and tell them where they can buy more, or alternatively, where they can get Whatman or Gelman Scientific capsule filters that are nonmedical filters but perform about as well (although they cost more!). 5) The tubing I observed CI using in its roller pump was water spotted and I was told this tubing is re-used from case to case. Medical grade PVC tubing (3/8" x 3/32") is available for a nickel a foot and I can easily provide CI with a 100 foot roll at no charge, and 500 ft. rolls at cost, as needed thereafter. I can also put them in touch with the vendor. This is NOT a medical device so it does not require a prescription. Water spotted tubing can be guaranteed to have pyrogens (which punch holes in cell membranes and enhance edema, as well as chloramines which are cytotoxic). Tubing takes a long time to dry, even when hung-up and grows bugs like crazy. We actually use a tubing dryer to dry our ventilator circuits because we have had animals get respiratory problems from molds and other micro-organisms which grow while the tubing is drying. 6) CI should consider using a good heat exchanger-oxygenator and a 20 or 40 micron filter in-line (before the patient) to allow for consistent control over perfusate temperature and to allow for removing oxygen from the perfusate by passing pure nitrogen gas through the fiber bundle of the oxygenator. I can get good hollow fiber oxygenators for $130 apiece and filters for $35.00 each. If CI just wants to use a heat exchanger, Gish Biomedical markets a simple unit very suited to re-use with a bubble trap. We can easily show CI personnel how to clean, dry and package the Gish heat exchanger (which is all stainless steel and polycarbonate) for re-use (no sterilization involved). For acute studies I get about 50 re-uses out of these things and they cost about $75.00 retail. I will give CI a Gish heat exchanger at no charge. Addition units can be purchased from Gish as nonmedical devices (i.e., nonsterile) without the need for a prescription. 7) I have about 100 nonsterile heparin coated arterial filters still in the manufacturer's over-wrap. These filters have an air vent, which, at moderate rates of flow (say to the head via the carotids) virtually guarantee you can not pump air into the patient's circulatory system. These are _strictly_ disposable devices, but I will give CI 5 of them along with the bleed lines back to the reservoir. The bleed lines cost about $1.30 each. Non heparin coated filters can be purchased non-sterile but ultra-clean for about $35.00 each. Since CI does not use sterile technique this should be acceptable. 8) Aside from osmotic injury from perfusing 75% (v/v) glycerol straight-up one of the most damaging things CI is doing is to wrap their patients in sleeping bags and use slow, passive CO2 convection cooling which means the patient will take a long, long time to freeze and about a week to get to dry ice temperature. We will provide CI free of charge with an IBM XT computer, monitor, software and expertise in the form of Brian Wowk that will allow them to modify their existing air cooling set up to use fans and to control the temperature descent of the patient. The capital cost of this modification in parts is probably in the range of $1,00 to $2,000 with some additional effort on CI's part (Any Zawacki could easily do this in my opinion) to tweak the system to run on their set up. Barring using a computer, the simple expedient of using fan-assisted convection cooling with the patient NOT in a sleeping bag would be far superior to what is being used now. In any event, the expertise is available to implement a more sophisticated system such as something that holds the subject's surface to core temperature a constant at constant delta T of 10 C all the way to about about -60 C to -70 C. We have experience with fan assisted cooling and the engineering problems involved in doing this safely. We also have data on efficacy for head-sized specimens. 9) For patients who are not clotted we would be happy to show CI how to do whole body femoral perfusion using Biomedicus flatwire venous cannulae which will allow for very good venous drainage and excellent distribution of cryoprotectant to the entire brain (validated in dogs and humans). These cannulae are costly: about $400 each, but can be re-used many times. I get about 6 re-uses out of each one, and the dog sized ones are more delicate than the human sized ones. At least 10 re-uses of these cannula can be expected if they are handled with care. More re-uses still if they are not subjected to repeated re-sterilization. This would obviate the need for median sternotomy in some patients (fresh ones) and greatly reduce the likelihood of incomplete perfusion of the brain due to a non-complete Circle of Willis (an anatomical variation present in a minimum of 10% of the population and which results in non-perfusion of the occipital part of the brain if only the carotids are used.) 10) Cole-Parmer Instruments now markets a cheap, highly accurate hand-held data logging thermocouple thermometer. It will log up to 1,000 points from 2 copper constantan probes with the interval between readings logged being highly adjustable. These units cost about $250 each and would allow CI to simply and effectively collect cooling data from patients without a human attendant. BPI bought two and this allowed us to replace a human (quite costly by comparison) who was used being used to continuously record data. 11) We can easily and inexpensively show CI personnel how to do burr holes using hand-held equipment costing about $150 which will allow for determining if blood washout is occurring, if cerebral edema is becoming a problem and to allow for placement of TC probes on the brain surface to determine the CPA concentration in the brain. Further we can give CI safe and effective dyes to add to perfusate when brain edema occurs which will allow them to stop perfusion and start freezing when perfusion of the brain is not occurring. Summing Up While I am critical of CI's procedures, I am also willing to DO something about improving them in a simple, cost-effective manner. I believe in the utility of charity work and volunteer work in making changes in the world, as well as the utility of the profit motive. I have referred a number of clients to CI and would feel far better about referring people if I knew that at least no _active_ harm was being done over and above that which is unavoidable (within the money and technical constraints CI works within) and within the limits of current cryobiological capabilities as they apply to all cryonics organizations.. The costs of the changes I've suggested above is not great. The labor an expertise are free; that's Brian's and my volunteer work for the year :-). Saul is thrilled at the notion of such an offer and has given us leave from 21CM to to this. Nothing suggested above is terribly novel or at all proprietary. There are data to back up the benefits of such changes. As to the future of BPI: there is no "probably" to the 11 month number cited in yesterday's post. BPI has already discontinued services to ACS and has given CryoCare 12 months notice, about 1 month or so of which has already elapsed. Bob is definitely right that I've "lost the faith" in cryonics as it is currently practiced. However, this does not mean that I will stop helping or that I am not working to "get the faith back" by doing research. Certainly I don't intend to act in the nonproductive way so many of the cryobiologists have in the past by refusing to communicate nonproprietary information or to help where I am not hurt for doing so. The above offer is about as positive as I know how to be, right now. I have no ill will towards CI and would like to see the quality of preservation they offer improved. I am not willing to break my back or my bank to achieve this, but I am willing to do what I can. Mike Darwin Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=9483