17 replies to this topic

[caliban]

patients will likely have their blood replaced with an organ preservation solution for shipment to the U.S.A. at 0 degC.

[huh]
Why not use a proper solution and ship on dry ice?

[bgwowk]

Good question. The answer is that both CI and Alcor are now switching to vitrification solutions that aim to prevent ice formation completely during cooling to liquid nitrogen temperature. Dry ice temperature (-79 degC) is a particularly bad temperature to hold at for vitrification. It is cold enough to nucleate and drive ice growth, yet not so cold that ice growth is inhibited by solution viscosity. There is an excellent chance that tissue held at dry ice temperature for dozens of hours will freeze, especially imperfectly perfused tissue.

So why not just revert to old-style glycerol perfusion and freezing? That's a judgement call that involves trading cold ischemic injury for freezing injury, bearing in mind the logistical difficulties of doing a proper cryoprotective perfusion and cooldown remotely. Proper cryoprotection and cooling are much more complicated than blood washout, needing specialized equipment and expertise. Both CI and Alcor have decided, for now, to try ice-temperature transoceanic shipment followed by cryoprotective perfusion and cooling in the U.S. In the long term, both organizations acknowledge the superiority of local cryoprotection and cooling followed by overseas shipment below the glass transition temperature (-120 degC). If it is discovered that long cold ischemic times are compromising cryoprotective perfusion, the urgency to develop remote perfusion and cryogenic shipment capability will be increased.

---BrianW

[caliban]

Just had a meeting over here discussing that advice.

I have conflicting instincts: on the one hand it would make matters sooo much easier. On the other hand it sounds like the classic US-centric fudge where international customers are at best an apologetic footnote.

Could you present some more data supporting the argument? It is a hughe decision to make.

[bgwowk]

Frankly, Caliban, it's a bit academic. I am not aware of any international groups with the resources available currently to setup a remote vitrification perfusion and cooling capability. An LEF principal has been trying to create such a capability in Florida for the past several years, and $2 million later is still not there yet. Granted not all of that has been concentrated on just the perfusion problem, but it really is a non-trivial undertaking. I'm pinning my hopes on the Florida (SA) effort, because once finished it will be a template of what can be done remotely.

Of course it is unfortunate that Alcor lost the Eastbourne facility that Allan and others poured so much into, and that at its Zenith had a glycerol perfusion and dry ice shipping capabiltiy if I'm not mistaken. I don't know much of the history of how the facility was lost, and what role Alcor had in its demise, as I myself left Alcor during that time period for other reasons.

The final fallback is what CI used to do, which is let a local mortician briefly perfuse a cryoprotectant solution open circuit with no monitoring followed by shipment in dry ice. But that's such an uncontrolled situation, it's hard to say what you would get.

---BrianW

[caliban]

Academic?

Only the building was lost. Vitrification can't be done over here, but dry ice shiping (or better) was the agreed protocoll until... yesterday morning... for both CI and Alcor patients. Alcor has recently provided perfusion equipment and meds. It was (is) also what the German standby team is training towards.

There is also the option of a straight freeze, plastination or to just not bother. (Which is what learned people were suggesting when water ice shipping was discussed in the past)

So, this 'academic' evidence would be appreciated to facilitate decision making on investments of time, money and emotion.

[bgwowk]

Now I'm really confused. Is not Alan Sinclair part of the UK cryonics group? I've had a lot of corespondence with Alan over the past year or more concerning the difficulties of building a dewar capable of overseas shipment at temperatures below -120 degC (the approximate glass transition temperature) for vitrified patients. Alan generously spent a lot of money having a dewar designed to solve this problem, but apparently the engineering firm that was to build it had unanticipated problems scaling up dry shipper designs to human-sized dewars.

Anyway, with all that going on, I've assumed that the difficulties of shipping vitrified tissue at dry ice temperature were common knowledge. Are you saying that everyone in Europe is proceding on the assumption that patients will be perfused with vitrification solution locally and shipped on dry ice? I didn't even know there was any vitrification solution situated in Europe.

Are you sure that the perfusion equipment and meds supplied by Alcor weren't just for blood replacement with organ preservation solution? If not, what kind of cryoprotective perfusate and equipment are over there?

The choice between straight freezing and 48 hours shipment at ice temperature is a no-brainer. Straight freezing causes enormous structural damage. Brains promptly washed out and cooled to 0 degC and maintained for 24 hours are indistinguishable from controls in transmission electron micrographs.

---BrianW

[caliban]

Alan is and remains the heart and soul of Cryonics UK.

I said

Vitrification can't be done over here

The protocol trained was "glycerol+extras" perfusion, shipping at very cold because of toxicity.

The evidence you elude to is the sheep brain trial? (I think it was sheep)
Have you tried vitirtification on a body that was washed out and kept reasonably cold for three days?

PS: Appreciate your help with this. I might not be able to follow this up the next few days. Maybe others could chip in as well?

[bgwowk]

The hypothermic storage research I spoke of was recent unpublished research in rabbits (not done at CI). The real wild card is cerebral edema, which could compromise cryoprotective perfusion if the blood brain barrier is compromised by long cold ischemic times. Data aren't available yet. If the recent Alcor M22 case is any guide, edema in the rest of the body could be a limiting factor that sets in sooner. More data are needed.

I'm curious to know what equipment and protocol you use for glycerol perfusion over there. I didn't know you still had that capability. Let me know when you get a chance.

---BrianW

[torsten]

I am a member of the German team. We are still in early stages, but the current goal is to be able to perform glycerol-based cryopreservation and then shipment to the US on dry ice. No vitrification is currently planned.

The question I have (which was also raised by caliban) is to the comparison of glycerol cryopreservation and vitrification after 0C shipment. I agree that vitrification is superior to glycerol-based preservation when both can be performed directly on the spot. But I am unclear if the damage thru glycerol is larger than that occured by >24h storage at 0C? Brian, could you comment some more on the reasoning behind preferring remote vitrification to direct glycerol preservation in regard to this question?

[caliban]

I'm curious to know what equipment and protocol you use for glycerol perfusion over there. I didn't know you still had that capability.

We recently did a full inventory list which should have been send to SA.
CUK has a mobile perfusion unit that can be used anywhere in the country, thumper (the first one ever I think) , portable ice bath, etc.

Eluding to Torstens question, would you be moved to reconsider your important advice when taking into account that these capabilities exist?

More data are needed.

Are any trials planned in this regard?

[bgwowk]

Caliban and torsen, I need to know what your glycerol perfusion and cooling capabilities are. I need to know

* surgical approach

* cannula used

* pumps and tubing

* circuit design

* how pressure is monitored

* how cryoprotectant concentration is monitored

* what carrier solution and cryoprotectant solution concentrate are used (who makes them, how long are they stored?)

* what nominal time/temperature/concentration profile is followed

* what facility will be used (mortuary?)

* what personnel are used and what experience they have

I ask these questions not to give you a hard time, but because field cryoprotection and cooling are difficult operations. We cannot look to laboratory experiments comparing glycerol cryoprotection and freezing with cold storage and vitrification if what happens in the field is completely different from laboratory conditions. So let's talk about what reasonably can be expected to happen out in the field.

Once again I'll just say that if it turns out that patients are unperfusable with vitrification solution after 48 hours of 0 degC blood substitution and storage, then the question is moot and whatever kind glycerol cryoprotection can be done will be better than nothing.

---BrianW

[jonano]

When we are cooling the brain to liquid nitrogen temperature, are we doing it gradually or rapidly ? With which instrument can we do it gradually?

The best is to ship a brain at 0 degree, which material should we use for that? Only glass ?

[torsten]

Hi Brian,

Speaking for the German team, we do not have any capability yet, as we are still in early stages of organization. However, I do not a agree that a theoretical (laboratory) analysis is irrelevant, because the data from this will be important in deciding
a) what we aim for
b) how to proceed in specific circumstances

To explain b): If for some reason we can only reach a deanimated patient after say 12 hours, we would need to make an on the spot decision whether to do glycerol cryonisation or cool to 0C. This sort of decision can only be made if we are informed by experimental data, as stated in your post:

Once again I'll just say that if it turns out that patients are unperfusable with vitrification solution after 48 hours of 0 degC blood substitution and storage, then the question is moot and whatever kind glycerol cryoprotection can be done will be better than nothing.

On the other hand, if it turns out that cooling to 0C, shipping and the vitrifying are superior in any case, we would not need
to develop expertise and obtain equipment and supplies for glycerol perfusion.

Caliban and torsen, I need to know what your glycerol perfusion and cooling capabilities are. I need to know

* surgical approach
* ...

I can outline the current early stage of planning:

Surgical approach: Perfusion via access to femoral vein and artery.
Cannula used: Not yet decided.
Pumps and tubing: A hose pump
Circuit design: Undecided
Monitoring: Temperature feelers at input and output to/from body, pressure monitoring planned
Solutions: Undecided, probably similar to Alcor UK
Temperature Profile: My suggestion: Immediate cooling to 0C by perfusion as quickly as possibly (if circumstances allow); this approach and subzero approach are still in debate
Facility Used: If possible, direct surgical access at hospital, cooling to 0C by perfusion, then transfer to mortuary; otherwise external cooling during transfer to mortuary.
Personal: One or two medical doctors, several layman helpers with rudimentary cryonics training

As you can see, quite a lot is still in planning. I hope you can still give us some suggestions based on the data available, and I greatly appreciate your input!

[bgwowk]

Lab experiments on the effects of long periods of hypothermic storage are being done as time permits. However please note that nobody has expressed any interest in funding such experiments, so most of the data is probably going to come from actual cryonics cases over the next couple of years.

I'll just make a couple of brief comments about your perfusion plans. You want to cool to 0 degC as quickly as possible if your objective is just blood washout and shipment with organ preservation solution. However 0 degC is too cold for glycerol perfusion. To effectively perfuse glycerol, you want to be between +5C and +10C during the whole perfusion. Circuit design needs careful planning because multiple pumps are reservoirs are necessary to achieve a gradual concentration ramp.

You should study every case report on the Alcor website, and all technical information available in the website Library. Also read all BPI Tech Briefs on the the CryoCare website. While much of this is tedious narrative, there is invaluable technical information scattered throughout. This reading project will also help prevent you from underestimating the difficulty of what you are trying to do.

What is the budget of your group?

Also, if I'm not mistaken, Caliban's posts indicated that the UK group already has a glycerol perfusion capability. Perhaps someone from that group can post details of their capability.

---BrianW

[torsten]

Hi Brian,

thanks for your tipps. To our situation: At the moment we have minimal budget, and are still in the process of sighting and reading the literature and reports to specify our protocol. We will be working together with Alcor UK to learn from the expertise they have already developed.

Do I understand correctly that no (or almost no) data exist on
a) the extent to which prolonged hypothermia at 0C may cause irreversibly damage
b) interfere with success of vitrification?

In this case, the decision to ship at 0C is of course risky. You describe it yourself as a judgement call. I personally do think it is a valid approach, and would be a good first goal for the German team, since -- as you state -- it is much easier to implement than a full protocol with glycerol. I am looking forward to hearing the self-assessment of the UK team, as to whether they feel their training and approach are so that their implementation of the glycerol protocol is better than 0C shipping and vitrificaition in the US.

[bgwowk]

I'm not sure what you mean by "irreversible." The best cryonics methods today cause damage that is irreversible with present technology. Hypothermia at 0 degC under ideal conditions is reversible for up three hours with present technology, but not longer. What's important to know is whether long storage times at 0 degC result in damage that is substantially greater than cryopreservation under ideal conditions, or cryopreservation under not-so-ideal conditions, like field perfusion and cooling with glycerol. Whether that damage will be reversible in the future without amnesia is neuroscientific speculation, not something that can be easily quantified. All that is certain is that the more damage is reduced, the greater the likelihood remaining damage will be reversible in the future without memory deficits.

---BrianW

[torsten]

I'm not sure what you mean by "irreversible."  The best cryonics methods today cause damage that is irreversible with present technology.  Hypothermia at 0 degC under ideal conditions is reversible for up three hours with present technology, but not longer.  What's important to know is whether long storage times at 0 degC result in damage that is substantially greater than cryopreservation under ideal conditions, or cryopreservation under not-so-ideal conditions, like field perfusion and cooling with glycerol.
---BrianW

You are right of course. Sorry for the sloppy terminology. The proper question is indeed the comparison of the different conditions you mention (i.e. vitrification after prolonged hypothermia, good glycerol perfusion, and non-ideal glycerol perfusion).

[jonano]

What concentration should be used with glycerol ?