X-Message-Number: 9462
Date: Sun, 12 Apr 1998 00:58:30 -0400 (EDT)
From: Charles Platt <>
Subject: From Mike Darwin, re Ettinger/CI

Mike Darwin avoids CryoNet and sci.cryonics these days, but since he has 
access to reference materials that are unavailable to me right now, he 
agreed to comment on Bob Ettinger's statements re Cryonics Institute 
procedures for freezing human patients.

Since Mike's post is long, I will extract from it just a couple of
pertinent points. In a communication to Mike Darwin from Yuri Pichugin,
Pichugin wrote, regarding single-step perfusion with a high concentration
of glycerol: 

"Strong osmotic stresses were developed while implementing the CI method
of perfusion and reperfusion." 

Thus, Pichugin agrees that single-step perfusion as practiced by CI 
does cause osmotic stress. This was one of my primary points.

The Immortalist, April 1995, contains find the following text, in which
Pichugin explains why he ABANDONED single-step perfusion with high
concentration of glycerol: "A new perfusion procedure was used ...
Perfusion with Hank's solution plus mannitol and GRADUALLY INCREASING
CONCENTRATION OF GLYCEROL." (emphasis added.) Thus, contrary to Bob's
repeated statements, Pichugin DID ramp up the concentration of glycerol in
an attempt to reduce damage. Since CI does not do this in human cases,
Pichugin's results, using this protocol, cannot be applied to human
cases--which is what I said originally. 

--Charles Platt


From: Michael G. Darwin

Opening Remarks:

I do not typically post to Cryonet or Sci.Cryonics any longer because in
my opinion the quality of the debate and the absence of a reasonable
signal to noise ratio where science* is concerned is negligible. 

(*i.e., rigorous, well documented, reproducible experiments which provide
feedback in a meaningful time frame.)

Recently Charles Platt asked me some question about a report I had written
regarding CI light and electron microscopy performed on glycerolized and
glycerolized-frozen-thawed-fixed brains (sheep).  I believe Charles
contacted a number of people in CI as well.  I told Charles what I knew
first hand from examining the micrographs and what I understood of the
procedures used to prepare them based on my correspondence with the
Principal Investigator who had done the work for CI, Dr. Yuri Pichugin of
Ukraine.  Some of the information Charles got about the methods used to
prepare the sheep brains used in the study he obtained from my report and
from conversations with me.  He has asked me to re-state the information I
gave him, document my sources where possible, and re-post my original
commentary on the CI/Pichugin work. He has also asked me to comment on
some of Ettinger's posted remarks and claims as they relate to research,
BioPreservation and 21st Century Medicine.  Because Charles is a friend,
and I owe him a number of favors I will do this.  I do not intend to get
into any extended debate over this issue or any others on the Internet or

The Results:

When I first obtained the Pichugin micrographs as high quality prints I
wrote the following commentary as part of a lengthy report to CI which I
believe was also published on Cryonet:

>What I find most surprising is Pichugin's evaluation of the 
>EM results.  This tissue is grossly disrupted on every 
>level.  A first year neurophysiology graduate student would 
>be able to tell this as would someone who was briefly 
>oriented to what normal brain ultrastructure looks like.  
>Particularly disturbing is the absence of control photos 
>showing normal brain architecture in the absence of 
>ischemia, cryoprotective treatment, or freezing and thawing.

>The notion that this kind of injury is compatible with any 
>resumption of functional indices of organized brain 
>activity/metabolism is completely unsupported by even the 
>best of the micrographs.  This is further confirmed by the 
>inability of the investigators to reperfuse the brains after 

Recently, I re-examined the Pichugin EMs and I note that many of the claims
Pichugin makes are simply wrong, including the absence of  chromatin
clumping in the cell nuclei (a typical post-ischemic change).  Further, in
re-examining these EMs I am even more struck by the overwhelming degree of
disruption of brain ultrastructure.  I would rank the ultrastructure as
comparable to or worse than obtained with slow (4-5 degrees C per hour
cooling rate) straight freezing in our laboratory or others.

About a week ago I reviewed the Pichugin/CI EMs with Dr. Greg Fahy, a
leading organ cryobiologist with extensive experience in evaluating the
appearance of normal (control)  and cryopreserved brain tissue and he
concurred with this estimation deeming the CI results amongst the worst he
has ever seen.

Thus, debate over whether 75% (v/v, w/v or w/ww) glycerol was directly
perfused into these brains or not is somewhat immaterial if these results
are what CI clients can expect to get (i.e., "the CI Method').

However, my word, or Dr. Fahy's word need not be the last one.  You can
judge for yourselves if you care to take the time.  Request a copy of the
CI micrographs (as prints) and go to your local medical/biomedical library
and obtain a copy of _The Fine Structure of Nervous System: Neurons and
Their Supporting Cells, Third Edition by Alan Peters, Sanford L. Palay, and
Henry deF. Webster, Oxford University Press, New York, 1991, ISBN#
0-19-506571-9, and compare the CI micrographs to _any_ which are in this
standard reference work.  Judge for yourself.  A child can tell this
difference between massive destruction and conservation of fine brain

Now, as to exactly _how_ the sheep brains were prepared.  I asked Yuri
Pichugin this on several occasions and have obtained several different
answers which I'll quote briefly from below:

In a communication dated 10 February, 1995 entitled Cryoconservation of
Sheep Heads by the Cryonics Institute's Method, Pichugin and Zhegunov

"The first stage and the second one were jointly analyzed. Washout of
blood, perfusion with 82% (w/w) glycerol and reperfusion did not result in
the significant alterations both in the grey and white substance of the
sheep brain tissue and in the pituitary body. Strong osmotic stresses were
developed while implementing the CI method of perfusion and reperfusion."  

These statements contradict each other, (no changes versus strong osmotic
stresses) however, the EMs speak for themselves: large scale destruction
with occasional small islands of structure sufficiently well preserved as
to be recognizable.

When I pressed Pichugin on which protocol the EMs I had obtained from him
had been subjected to he noted that the results of this work very
unsatisfactory no doubt due to the tremendous osmotic stress of single-step
exposure of brains to  82% (w/w) glycerol and referred me to THE IMMORALIST
April, 1995 vol. 26 # 4 noting that he had altered the protocol as
referenced there to minimize the massive, glycerol related osmotic injury. 
To quote from the published text in THE IMMORTALIST:

" Sheep Heads. A new perfusion procedure was used. Washout was with 1 liter
of Hank's solution, pH 7..2 with heparin as anticoagulent and with 20 g/l
of mannitol at 10 degrees C.  Perfusion with Hank's solution plus mannitol
and gradually increasing concentration of glycerol to 25% (w/w). Then 25%
glycerol solution was injected into the sheep heads until fluids from veins
reached 25% glycerol..." 

In any event, as I stated at the beginning of this communication the
results were massive disruption of brain ultrastructure at the EM level.
Most of the EMs arrived unlabeled in any meaningful fashion and the osmotic
injury was so bad I was unable to distinguish the glycerol perfused
"control" tissue from that which had been frozen and thawed even though it
had just been glycerolized using the CI technique.  There were no
non-glycerolized controls (i.e., animals evaluated for the efficacy of the
fixation and electron microscopy techniques in the absence of critical
experimental interventions such as freezing thawing, glycerolization,

Light micrographs showed many "apparently" intact neurons with multiple
(every 30-50 microns) tears in the neuropil from ice formation.  I say
"apparently", because, as with our own work, this light-level preservation
is misleading and is not borne out by EM level examination. 

Now to comment on Ettinger's specific remarks:

> Just very briefly to repeat: Platt's main and repeated complaint, that
> CI human procedure differs substantially from the sheep procedures
> resulting in the published micrograms, is 100% wrong. The procedures are
> essentially the same; see previous post. 

So much the worse.  As I've repeatedly stated these EM results are very bad
and I would not consider them acceptable let alone desirable.

> Concerning openness of procedure, Charles says CI has not provided
> detailed documentation of results with individual patients. When I said
> CI has been more open than others--both in our cryobiological work and in
> our cryostat work--I meant in providing information about our procedures,
> as compared with others. We have supplied this information to many people
> at many times, and have never hidden it. 

> As to CryoCare's openness, there seems to be a bit of a contradiction.
> Yes, they have published large amounts of data--but they (or BP/21 CM)
> still apparently have many secrets requiring signing of a non-disclosure
> agreement before they are released, if they are released at all. 

These statements are either false, misleading, or both.  BPI has provided
complete disclosure of every area of the protocol used to prepare and
evaluate cryopreservation techniques used on human clients.  This included
composition of perfusate,  A-V glycerol introduction curves, all
medications used, cooling and warming rates, terminal glycerol
concentration reached, and procedures used for fixation and evaluation.  By
contrast, as Pichugin himself stated in his report :

"Our devices (sic: thermocouples/recorders) are not adapted for automatic
registration (sic at) such very low rates, therefore we registered
temperature by hand and only by day.  Thus we could only partially
determine cooling curves using periods of measurement."

I never received, despite repeated requests, ANY of this data.  And, as
Pichugin points out it is fragmentary at best.  No data is, after all, no
data and cannot be evaluated.

Nondisclosure agreements regarding the data on BPI methods for perfusion
and freezing have not been required.  This data (i.e., as to the effects of
existing techniques) has been openly reported on the Internet and copies of
micrographs have been sent to CI, to Dr. Pichugin, to Dr. Fahy and others. 
These data, including EMs are substantially better than the Pichugin/CI
results.  In fact, there is no comparison in the quality of the
preservation of the neuropil, neurons capillary endothelium and overall
brain ultrastructure: BPI "wins" by a mile.  However, this isn't saying
much.  The BPI data still show massive ice damage as well as
ultrastructural alterations from glycerol.

Bob also alleges BPI was selective in picking micrographs.  Not so.  Keep
in mind that we have over a thousand micrographs and reproduction costs
forthe high quality images required for a meaningful evaluation are high. 
If Bob wants to look at all or most of them, I'll personally load the boxes
in the car along with the supporting experimental data and drive to Phoenix
and go over it with him picture by picture.

Bob goes on to say:

> Charles asks why I don't offer to sign a non-disclosure agreement in
> order to try to get the information. The answer is that, sure as a bear
> poops in the woods, if I am provided information and there is a leak, I
> will be on the list of suspects. No thanks; I'll wait for publication. 

I think apples and oranges are being compared here.  Yes, we have much
improved ultrastructure and viability data using proprietary compounds and
these do require nondisclosure agreements.  However, this bears little
relevance to the "head-to-head" comparison of CI methods versus those used
by BPI on its human cryopreservation patients (and by other conventional
cryobiologists): this is all public domain.

> Charles asks in what way we got poor results ramping up glycerine, and
> what new approaches we will try. To the former, the edema problems were
> much worse; to the latter, this is still being investigated. 

I have perfused hundreds of animals and dozens of humans with asanguineous
(blood free) solutions with and without cryoprotectant.  Edema will occur
rapidly if colloid is not present and CI does not use colloid (examples of
colloids are the dextrans, the hetastarches, Hemaacell, Ficoll, albumin,
etc.).  Edema rapidly develops during colloid free perfusion. We have
recovered many dogs from 5 hours of asanguineous perfusion but NEVER
without colloid.  In fact, the choice of colloid is critical to achieving
survival of the animal.  Attempts to omit colloid in blood washed out 
perfused _human_  brains exposed to any significant ischemic insult result
in the development of rapid cerebral (brain) edema and failure of
subsequent cryoprotective perfusion.

> Concerning openness of procedure, Charles says CI has not provided
> detailed documentation of results with individual patients. When I said
> CI has been more open than others--both in our cryobiological work and in
> our cryostat work--I meant in providing information about our procedures,
> as compared with others. We have supplied this information to many people
> at many times, and have never hidden it. We have not reported detailed
> results with individual patients because there was nothing especially new
> or interesting to report, and we try not to waste time and paper. When
> detailed reports will serve a useful purpose, we will provide them. 

If CI has not noted differences between patients in response to
resusciutation, perfusion, etc. something must be very wrong or they must
not be looking very closely.  We have found very significant differences
between patients in terms of cerebral edema and many, many other markers
for injury and cryoprotection based on ischemic time and the patient's
ante-mortem (agonal) course.  Of course, if the brain is not being
evaluated for blood washout, freezing point (an indication of brain
glycerol concentration) via thermocouples placed in or on the brain, and
tissue-specific enzymes levels in the effluent then there would be little
to report other than that some patients have "good" venous drainage and
some swell and drain poorly.  In the end, every patient gets frozen and the
"results" are the same for everyone: their temperature is -196 degrees
Celsius: and the rest is for someone in the future to worry about.  This
NOT science. It is not medicine.  It is not, arguably, even sane cryonics. 
However, it _is_ cheap, and if that's what some of you folks want, have at
it and God bless you.  I refer people looking for that kind of service to
Bob Ettinger at CI at a rate of about 2-3 per month.  A number have become
CI "patients." (and VERY patient indeed will they have to be).

> As to CryoCare's openness, there seems to be a bit of a contradiction.
> Yes, they have published large amounts of data--but they (or BP/21 CM) 
> still apparently have many secrets requiring signing of a non-disclosure
> agreement before they are released, if they are released at all. 

If data is published there is full disclosure.  I can think of only one
exception to this: in the Gallagher cas- report novel and proprietary
anti-ischemia drugs were used and were referred to by 21CM in-house code #.
These drugs do not materially affect cooling curves, or other published
results in this case.  Several drugs, based on our dog-lab experience, may
improved brain perfusion time during CPR and mean arterial pressure during
CPR, however, these results might also have been due to the use of high
impulse active compression-decompression CPR.  There is a large literature
on these modalities of CPR and BPI has published (see previous issues of
CryoCare Report) detailed descriptions of the hardware used to carry out
this mixed-mode kind of CPR as well as photos of the equipment and
descriptions of how results were obtained and evaluated.

> Charles had asked why we don't drill burr holes in patients' skulls to
> monitor edema. I said it wasn't necessary, because the brains don't
> swell, but rather shrink a bit. He now says that Andy Zawacki said we do
> sometimes get edema, and have to back off. This was a misunderstanding
> based on a telephone conversation. (That is one of the reasons I prefer
> communication in writing.) We occasionally get edema in the body, not the
> head, and it is in the washout phase, not the perfusion phase. Therefore
> we occasionally have to terminate body washout, and begin perfusion,
> earlier than usual. 

How would CI know if the brain always shrinks rather than swells in their
human patients without EVER having donme a burr hole.   Alcor and BPI
patients generally experience cerebral dehydration during glycerolization,
but this is not always so.  Many develop cerebral edema despite very
aggressive increases in the rate of glycerolization, including using sharp
step-ups.  We have in particular found that blood washout with colloid free
mannitol Ringers or M(annitol)HP-2 perfusate in the severely ischemic
patient causes severe cerebral edema during subsequent glycerol perfusion
and we now begin the glycerol ramp at once in these individuals and keep
our arterial to venous gradient at the highest "safe" (i.e., below cell
lysing limit) of about 1,100 to 1200 mOsm. We let the glycerol containing
solution begin the blood washout.  We have documented better results with
this by light and electron microscopy as well as by carbon perfusion

> Charles says our measurements (e.g. of glycerine concentration in
> effluent) were on sheep heads and hence prove nothing about results with
> human patients; and suggests that we cannot learn much just by visual
> external observation of the patient. 

> As to the former, certainly the sheep model is not ideal; but I believe
> CP or/and 21 CM or/and Alcor have found results with humans reflect
> pretty well results with dogs, and there are reasons to think the sheep
> model is at least as good as the dog model. 

There is no substitute for gathering human clinical data.  Our dogs do not
die before cryoprotection perfusion and freezing in the plethora of ways
that humans do.  In fact, we try to control for the very variables CI and
other cryonics organizations MUST confront with their human patients:

a) Duration of ante-mortem shock and the underlying disease.  A patient
with diffuse intravascular clotting (DIC), renal failure, dehydration, and
many, many other conditions will respond different to resuscitation and
cryoprotective perfusion.  Our dogs and other research animals are
controlled for these variables (and are generally absent them!).

b) Ischemic time before perfusion (both cold and warm).

c) Age ( a critical factor).  Our dogs are all between 1 and four years old
with most being 1-3 years old.

d) Presence of premedications such as aspirin, coumadin, dilantin and other
therapeutic drugs that can powerfully affect ischemic injury, clotting,

e) Duration and quality of post arrest cardiopulmonary support and response
of the patient to it.

f) Weight: our dogs are between 20 kg and 25 kg in weight and have similar
body morphology.  Humans can be emaciated or obese and this greatly effects
response to cooling.

These are just a few uncontrolled variables in human cases.  Ettinger and
CI are seriously misleading themselves and others if they think these
things don't matter.

>As to the latter, we have found with the sheep heads that visual external
>observation does correlate well with observation of the brain through a
>window cut in the skull. Fact, not conjecture. 

Sheep aren't people: see the discussion above.

> Incidentally, some of us have problems with infliction of suffering on
> dogs or other animals, even if the eventual goal is to save human life.
> Certainly this is a gray area, but I want it on the record, again, that
> Cryonics Institute does no experiments on live animals. When they are
> killed for our research, it is either routinely at a slaughterhouse where
> it would have happened in any event, or by euthanasia under the
> supervision of a veterinarian, and not on CI premises in any case. 

Dogs undergoing cryoprotective experiments do not suffer.  They are
completely (Plane III) anesthetized before the start of any painful
procedure.  This is thus a bogus objection.  However, I'll deal with it. 
But first WHO are the "some of us" that Bob Ettinger refers to, _exactly_? 
Please ANSWER this question Bob. 

Further, if you eat food of any kind (including fruit) you inflict untold
and horrible suffering on many, many animals.  Egg and milk production are
nightmares of cruelty (I've seen both up close).  Agriculture involves
plowing of land and the killing, maiming and dislocation of millions of
animals who suffer powerfully.  As the poet Robert Burns noted in the poem
"To a Mouse" (1786) which was composed on "turning up her nest with a
plow, November 1785": 

But Mousie, thou art no thy lane,       [not alone]
In proving foresight may be vain:
The best laid schemes o' mice an' men
               Gang aft a-gley          [go oft awry] 
An' lae'e us nought but grief an' pain,
               For promised joy.

Still, thou art blest compared wi' me!
The present only toucheth thee:
But och!  I backward cast my e'e
                   On prospects drear!
An' forward though I canna see,
                   I guess, an' fear!

Here Burns seems to exercise more insight than Ettinger: at least mice
don't (presumably) know they are going to die whereas people do.  Doing
animal research is painful to the investigators and emotionally draining. 
Far more so than dining on the battery raised chicken or the feedlot beef
that Bob ate at the recent Alcor banquet.  At least the investigators at
21CM have to SEE the consequences of their work and are motivated
(powerfully so) to mitigate pain and suffering.  I would also note that I
keep my own free range chickens for eggs, do not eat meat and generally
avoid infliction of unnecessary and wasteful cruelty to animals which is
more than I can say for the "some of (sic) those who have problems with
infliction of suffering on dogs or other animals" that Ettinger refers to. 
Such men and women are cowards and hippocrites being unwilling to take
responsibility for the intrinsic suffering their continued existence
inflicts on other emotionally sensitive living things and being unwilling
to have the strength to change their lives to minimize such suffering.
They are also in massive denial: animals and plants are nice to each
other, either. It is, after all, a dog-eat dog world out there. 

> Platt says cracking "should" not occur above dry ice temperature. If he
> means it does not or cannot occur, he is mistaken. Cracking can occur at
> any sub-freezing temperature, depending on several variables, not all
> controllable. Our slow cooling avoids cracking, as verified by
> independent professionals. 

Ettinger says "cracking can occur at any subfreezing temperature."  This is
an unqualified throw-away statement.  Cracking most unequivocally does NOT
occur with cooling rates of up to 8 degrees C per hour in whole dogs down
to -90 C.  We have done this repeatedly.  We have also repeatedly rewarmed
dogs from -90 C by immersion in 0 C alcohol baths and found not only no
cracking but much improved carbon particle (india-ink in fixative)
reperfusion using such rapid (10 C/hour) rewarming.  By contrast, using the
CI protocol we have, much as has Pichugin, failed to achieve reperfusion. 
We have NEVER observed cracking in any of these experiments.

The fastest rate you can cool a human is about 4-5 C/HOUR.  This will not
cause cracks if the surface to core tempeperature differential is kept to
about 10 C from the freezing point of the water glycerol solution on down
to -79 C.  Slow cooling from -79 C to -196 C does seem to minimize, but not
stop cracking even with cooling periods 300 hours long!

Shell  freezing and subsequent cracking can occur at very high external
cooling rates.  We have never observed this in glycerolized animals cooled
at rates well over that achievable in humans.  Direct immersion of animals
and humans loaded with 7.5 M glycerol (about 55% w/v) in a -40 C bath does
not result in cracking;  nor should it since this is only a few degrees
above the freezing point of 7.5 M glycerol.  

It no doubt takes CI's typical 60 kg patient a week to reach -79 C which
means _days_ at high subzero temperatures with post-mortem autolysis often
continuing during this interval.  In experiments we've done here using
similar slow cooling rates on post-mortem animals that have been
cryoprotected we have seen near total ultrastructural disruption. 

If this is what CI clients want, then that's  fine and they are entitled to
it.  Since people call me here often and want ritual with a veneer of
science and a strong authority figure to tell them everything is "probably"
going to be OK, that's fine me with. People are free to practice their
religion as the choose and I strongly defend that right and I refer them to

Because of the lack of meaningful feedback ALL of cryonics is mostly
religion.  As long as it is understood as such, and not confused with
real-time science that is fine.  Freedom makes life worth living and
freedom means responsibility, judgment and discretion.  To each his own.

Finally Bob comments on the proposed formation of BTI (BioTransport), a new
service provider company by saying:

> Try as I may (and I have asked those involved to correct me or inform me
> if they have relevant numbers or estimates available), I cannot see BT as
> raising either the investment capital or the revenues needed for the kind
> of readiness and expertise envisioned, any time in the next several
> years. On the other hand, 21 CM (including BPI) will already have the
> capital and the equipment and many or most of the personnel, and could
> provide cryonics emergency services as a sideline, not relying on that
> for its main revenues

BioPreservation (BPI) probably will be leaving the human cryopreservation
business in about 11 months.  My research schedule (torturing animals, you
know) does not allow my continued full-time responsibility for cryonics. 
Further, my own grave doubts about cryonics make it untenable for me to
continue operating a business with the primary goal of freezing corpses. 

Similarly, the staff at 21CM unanimously feel that they wish to devote
their primary attention to research--into cardiopulmonary-cerebral
resuscitation, organ preservation, and if possible, suspended animation.
Assuming they are successful, the traditional practice of cryonics (which
I would characterize as stacking up frozen corpses) will thus be left
pretty much to CI. 

I mention this to emphasize that my remarks about CI's protocol are made
without any prejudicing competitive zeal. 

Mike Darwin

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