a United States Army Medical Department
continuing education program.
Hist o compatibility testing
a panel discussion with dr braun
doctor cell dr gary
boy and dr species
moderated by Roberti.
Lord in lieutenant Colonel U.
S.
Air Force Medical corps assistant chief reno
service Wilford Hall U.
S.
Air Force Medical Center Lackland Air Force Base.
We have several good questions that have been
sent up here and having quickly glanced at them.
I don't have the answers to all of them will begin
by.
Although this wasn't specifically directed all
direct this to dr braun
dr brown.
Would you use a one haplotype living related donor with a
mildly abnormal M.
L.
C.
A mildly abnormal
mlc.
Well we've used a lot of one haplotype
identical uh transplants
living related.
I still feel that the
there are significant advantages in using living related
donors and looking at serial renal blood flows in
these combinations.
It usually turns out that if you use a living
related Sibling even
mismatched that you've got enough of a renal blood
flow buffer.
That even if they go through an annual rejection and we've
seen two of these you can get them through with a functioning
graph.
Pulling them out with serum creatinine of less than
two and still have about 400 mils of
blood flow left to work on.
So they can tolerate a serious
rejection much better than a cadaver graft will
where you might start out with 400 mils of blood flow.
And after they get belted with a serious rejection
they've got 200 their creatinine is 10
and you can't keep them off dialysis.
So I think there's enough of a
physiologic buffer in there that you can tolerate
a serious rejection in a single haplotype
sibling transplant that you can't
tolerate in a cadaver graft.
Now that's not a an immunological answer or based
on typing,
which is what the question was aimed at.
But I think it's a practical approach
to these.
And we've looked at some of our living related
graphs now who have been functioning for 10 years and
they are mismatched,
living related in some cases and
unmatched cadavers so that they
certainly have the potential for long term
survival.
And the fact of the matter is that we
still don't have ways of
evaluating the ability of
a recipient to tolerate a graft.
We still are unsure really of what the
significance of blocking factors are beforehand and we still
don't know how to evaluate the presence or absence
of suppressor cells in these situations.
But I certainly would
preferentially go ahead with a living related
single appetite mismatched sibling transplant
from a young age group in preference
to a cadaver waiting list.
There are two questions that uh are
quite interesting.
The first is,
what is the current status of our liberal transfusion
regimen to identify non
and or hyper responders or I think what they
mean is people who do not form antibodies when sensitized versus
people who form a lot of antibodies are sensitized
and a very similar question is do you think
pre sensitization might be useful because it would
identify people who are quote hyper
responders and should be and should transfusions
uh,
and I presume they mean transfusions of whole blood be
restarted?
Well,
I'll ask several people on the panel to comment on that.
And I'll begin with myself.
We have had several instances in our
own transplant program where people did not have
identifiable or known or at least
willing living related
individuals uh for purposes of giving
them a kidney.
And these people have been put on the cadaver list and
over a period of time have received
transfusions in the course of a chronic dialysis
regimen.
And then lo and behold up comes a missing
brother or absent father who indeed
suddenly has realized that a
transplant for the particular patient in hand would
be useful and is willing to do it.
And we find that we've sensitized this individual to the living
related person.
So you've eliminated that person and from potentially being
transplanted with a living related graft,
which is far superior in my mind under any
circumstances than any cadaver graft.
So,
for that reason alone,
I don't feel we should liberally transfuse anybody in the
fact,
even if they are responders,
we may at least prevent them from being sensitized
against a given individual who they're related to
whether or not it's useful to know if someone has
the capacity to form antibodies or not to form antibody
is as I tried to indicate when I in my talk
a matter of opinion and clearly not settled
I think that if someone has the
capacity to form antibodies
and they've never been sensitized
the capacity of form and a body is obviously still there when
they are first sensitized say by a kidney
graft.
And it may well indeed although not
clearly shown it may well indeed make this
individual less likely to
have a graft survival compared to someone who
doesn't have this capacity.
It may indeed be
possible that if this individual response
is a responder or forms antibody that
he would be the best candidate for a
for energy and match craft or a good energy match
graft versus someone who is not a responder
because there's certain individuals who
feel that if someone is not one who
forms antibody and if these antibodies are H.
L.
A.
Specific which by definition of the H.
L.
A.
Antigens they are.
Because the H.
L.
A.
Antigens have been defined by people's reactive
reactivity to these antibodies.
Therefore that knowing that someone's responder would then
make him a candidate to wait for a better graft versus someone
who isn't a responder should
be transfused that transplanted at
the first available organ that comes along and to
disregard typing or H.
L.
A.
Matching in these people.
I don't think that this has been well
established and agreed upon.
I think there's arguments for both sides which I may be in a very
confused way trying to present to you know I don't
feel we should start transfusing people until there's convincing evidence
that knowing whether someone forms antibody is
worthwhile.
Dr.
Braun,
What might be your opinion on this 1?
I agree with you completely.
I don't think it's approved at all that you can establish
acceptable recipients by attempts to
sensitize them in some unknown fashion and it's
very uncertain as to
whom you should sensitize with what sort of
antigens in order to evoke this benevolent
situation of acceptability of all kinds of
graphs.
And until we know that I think the greater likelihood is
one of harm and risk to the patient of
advantage.
I think there's some evidence now that there's a way to
compromise to find out what sort of delayed hypersensitivity
response an individual has without using
uh Lucas cider agent lee antigens.
And I'm thinking of the work of mel Williams
who did D.
N.
C.
B.
A.
Coral Benzene delayed hypersensitivity testing
on pre transplant patients who are on the waiting list.
And if I remember his figures right he found that
those individuals who gave a delayed hypersensitivity
response to dynamic or Benzene,
of them rejected their transplant for as the group
that did not show a skin response.
Only 27% rejected their transplant
interestingly enough.
He used only the cutaneous hypersensitivity
method.
Others have shown that if you look at the in vitro and in
stimulation you can detect some individuals who really
do have a response to D.
N.
C.
B.
That does not show up in the skin test.
In other words,
perhaps if he had looked at the in vitro response to the
26% of the D.
N.
C.
B.
Negatives who rejected the skin grafts,
you might have found that some of those actually were DNC responders who were
not being identified.
So no one knows what the
actual molecular analogy is between D.
N.
C.
B.
And the H.
L.
A.
Sensitization.
But it seems to be an interesting phenomenon and several people
have reported.
So I guess the answer that I'm giving is that
I think we should look into other antigen stimulation
responses rather than a sensitization so that we can keep our
options open for transplant and not develop
hyper acute rejection.
Do you have any comments?
Yes.
The same sort of situation holds in the
studies of Coulson and Shumway
who have shown that some patients
are poor responders in
Mlc.
Probably based on some sort of circulating inhibitory
substance in their serum and in fact this
whole discussion really should be aimed at why
an individual is not responding.
Is it a genetically inherited ability to
respond?
Is it a acquired inhibitor of some
sort?
Is it a product of the disease?
It could be related to the stage or of disease or the
quality of the clinical
treatment regimen for that patient.
I think those things needed to find and I would agree with dr
braun that willy nilly giving blood
transfusions as a way to see whether or not someone will respond is an
extremely risky way to measure this as yet
very poorly understood phenomenon.
I must say that in a study that we're doing
with first with Dr Belser non with
dr salvatore at the university of California in san Francisco
were carefully analyzed.
100 patients were actually studying and lymphocyte
serum treatment.
About 37 of those patients had pre for and
antibodies obviously not to the cadaver donor
because that would provide a positive cross match and would be
excluded but 37% had antibodies against some other
H.
L.
A antigens.
And in that series we see no difference between those who
had made antibodies and those that did not
since you've brought that up.
We have a question here for dr brian.
And I'll have you comment out to Dr Dr brian,
how do you rationalize the disparity and transplant results between
institutions regarding preformed antibodies?
Mhm.
Well there's a whole list of
things there,
many of them technical um
one that depends on a complete analysis of all
available positive syrah from the recipient and this is
getting to be a horrendous cataloging and
library storage type of problem where
sometimes in our patients who look presumptively
negative on a cross match.
We will have to haul out as many as 10 or 12 old
syrah in order to actually confirm this many times,
we found that patients who are being held on satellite programs
elsewhere,
who are being screened by another laboratory if they
come to us without those serum specimens will have been
shown to have spikes of antibody which would
give positive reactions with prospective
donors with which we were going to transplant
them for a kidney.
So it one is you must have available to
you a tremendous library of syrah
on any recipient on any potential recipient,
cadaver waiting list.
Secondly,
you should do all your tests with delusions because
undiluted there are some anti complementary factors that interfere.
Uh thirdly,
you should use some tests of increased sensitivity
such as dr Lorton described.
And dr deer avoid described with
the detection of sensitization either by
serological methods or by cellular defined
methods.
Uh fourth,
there's also the possibility that in some individuals
and we saw this happen on the table and reported it in
transfusion a couple of years ago,
patients who may have preformed antibody not directed
against the kidney donor may have antibody
reacted reacted against lymphocytes in the blood
transfusion given at the time of transplant.
And we saw a daughter hyper acutely reject her mother's
kidney,
even though the cross match against the mother was repeatedly
negative before and after transplantation.
But her white cell antibodies reacted
with a unit of two of the
three units of blood that she was given at the time
of surgery.
And because there was a hemorrhagic problem,
one unit happened to be given as whole blood with
abundant lymphocytes and she hyper acutely rejected
on the table.
Uh The only antibody that we could find
reactive in this situation was the white cell
antibody reactive against blood donors,
not the kidney donor.
This is another uncommon,
rare but still uh remote
possibility.
The fifth reason I would say there's differences
between reporting centers and this doesn't hold for the boston group because
that's relatively a concise group.
But in accumulation of data from
uh by dr tara sake.
He's relying on the heterogeneity of testing
methodologies existing in probably 80 some
laboratories,
many of whom do or do not adhere to many of the
criteria that we discussed this morning.
And that makes for a very
unstable type of reporting in my
estimation,
doctor.
So you got any comments
here's a question which is directed
to no one in particular and that is what do
biopsies look like And Group
three or people who have excessive amounts of
antibodies or strong responders as a
cause of rejection is its cellular versus funeral.
Uh I'm not aware of any published data that
delineates this.
It's been clearly shown that in individuals who
have a chronic vascular
rejection that this is humanly mediated,
which was shown by the group of Emma at
MGH we have seen
individuals with high levels of antibody
with both acute cellular as well as
evidence for human rejection
manifested as
uh proliferation of their
cells and uh
also by
immunosuppressants.
We've seen immunoglobulins on the vessel walls which may
or may not be uh significant.
Clearly the ones who have hyper acute rejections.
Its characteristic of a
a policy cellular infiltrate of the interstitial man.
Why would anyone else care to comment regarding that
question?
I'm not aware that these people classically
have au Meral pathologic change
or a cellular pathologic change.
Uh There's a question here that's of interest which
I'll throw up into any volunteer on the panel.
What is the clinical significance of the two a.m.
L.
C.
And the use of the recipient serum in the test system?
Mhm.
Any volunteers?
The same study that we're undertaking with Belcher's group in SAn
Francisco is the location of the first report of the
significance of the two a.m. L.
See that as we do at two a.m. L.
C.
In the macro system and you get a stimulation index of greater
than eight.
That is the response between the two is eight times
the control responses that this
correlates with rejection.
This work was done I think about two years ago
was considered highly significant and is highly
significant when you look at it statistically data was
reviewed carefully by Dr Van Rood who agreed with the
significance in looking at the last 100 cases
where we've been where our statisticians have been
handling all of the data on the case including the M.
L.
C.
S.
You know the significance becomes less
obvious.
I'd like to comment that they had most of their
data.
That was uh that could be
at least the stuff that was published two years ago.
Most of the data was that was statistically significant
and large enough numbers to calculate
in a statistical way we're on the cadaver donors.
And indeed it did show that uh they
however uh in the living related had
only three individuals who had a two way stimulation
ratio greater than 1-8.
And on the basis of this they felt that this was
also significant.
I I think that that's very weak.
I personally would not refuse
to do a living related transplants
in individual that had a greater than one day
stimulation on two a.m. L.
C.
Anyone else I'd just like to expand a little further
to.
And that is that the two a.m. L.
C.
S.
That are being done are being done by a macro mlc system.
It's interesting that we moved to a micro mlc system.
This correlation apparently doesn't hold at all.
Even in Kent Cochran's hands.
The fellow who first reported this and also even at the
time he first reported this when he tried to do one way ml
CS both ways that didn't
correlate making an extremely confusing picture.
And so I would think that to M.
L.
C.
Sees for decisions about transplantation should be
one of those items to be held
awaiting further evidence.
Question for dr gary boy.
We're in the experiments.
You sided with the enhanced animals challenged with skin grafts at
intervals following the successful kidney graft.
And if not what would you predict would happen
in the experiments that I showed this morning?
These were passively enhanced animals.
These animals were not subsequently challenge the skin
graft.
However,
in animals who enhanced by the
active process these animals became one
and two year survivals survivors.
Those animals did get skin grafts and those skin
grafts were accepted very well.
A question directed to me,
the studies of the pelts and Tara sake have found that
only about 50% of the transfused patients developed
preformed antibodies.
In contrast the data from Perkins.
Uh I think that
This is a variable percentage in
various centers and it probably relates to the number of transfusions
that people have had and uh
the time in catching these individuals
following their sensitization.
Uh the ones who were greater than
50% were numbers the
85% figure where people would receive greater than
20 transfusions on dialysis.
Which is not a common practice
in in my experience in talking with
others so that I think the amount of
exposure is going to determine the percentage of your population
where you'll see this.
There's another question which is directed to me and that is really Lucas pierce
and Williams have incriminated subside.
A toxic levels of antibody determined by
standard tests as an important factor in graft
rejection.
Do you believe their results and should their techniques be incorporated in the
pre transplant cross match?
I myself am not familiar with the sub
toxic levels of antibody determination that is being referred to
as someone else here on the panel with.
I don't know if they're referring to the immunity tests on kidney
cells.
Uh when Williams was working in hume's
laboratory and the group down there was
evaluating the sub toxic levels of
antibody.
They did show that by the immune adherence technique which was number five
on dr bronze rating scale
that you tested kidney
cells from the donor with serum from
the recipient.
By this technique,
they have apparently could pick up more sensitively
the presence of antibodies than they could by lymphocytes
toxicity.
Which is not too surprising because lymphocytes
toxicity is not a very sensitive
assay.
There are very few human serially diluted more than a couple of times.
Will still kill human lymphocytes which is
distinctly unlike any other side of toxicity
system that I know of in biology.
So that almost any other technique
should be more sensitive in picking up these antibodies than the one that we're
using
enough.
If the question is referring to the the
late cross match and the
L.
G cross match and the
anti g mediated cross
match in my mind.
There's no sufficient data to say that these
uh techniques are
are clinically significant as far as
showing a greater or lesser survival
in patients who have been transplanted with these being negative or
positive.
With the individual who asked this question.
Care to comment on Zoltan Lucas has
published on subliminal sensitization.
Not detectable by standard sido toxicity but the
detectable by more sophisticated techniques
which employ variants of anti
endothelial testing and that's what's really has
done.
And they've both shown uh people in the
retrospective work primarily the
reject ear's of their graphs.
Uh The syrah from patients who have rejected
transplants.
Let's start over the pre transplant
syrah examined
retrospectively uh and
patients who have rejected grass were found to be positive
by these augmented side a toxic techniques or
primarily by using a different target cell.
Either kidney cell in the theater or something else.
And that's what the question is really intended to
get at.
The problem with these techniques is preparation of the
material or the length of the test
required,
which we're talking about 68 or 10 hours of
cross match technique instead of three or four
hours.
As with the standard techniques for five hours.
With standard techniques of course,
Belser suggests,
then explains that the fact that his
patients do just as well who have antibody as
those who don't.
Is that his technique for cross matching is better And that's
the framework from which the question was intended.
The test you're referring to against what ophelia cells and
kidney cells.
Are these a sido toxic test
uh,
side of toxicity against kidney cells and then Williams published
on immune adherence and Cirelli was
against was anti endothelial antibody.
I've forgotten what Lucas target cells.
Yes,
we've been looking at that using lymphocyte blasts
with uh Peroni and
scripts group and it is true you can pick up a toxic
antibodies more sensitively.
As I said before,
I think you do it by almost any technique because the human lymphocyte is a
very resistant cell.
The question is,
do they correlate with success?
And that is a little harder to be sure of.
I think all of these techniques should be used
in a prospective study.
We did a dr philo when he was in our
laboratory before he went to University of Indiana,
did a very nice study where he pre sensitized dogs
and he found that even when he could detect sido toxic
antibody at low levels there was no permanent
rejection and no permanent loss of kidneys.
It took a certain way level of antibody response
in order to have permanent damage to kidneys.
So that you would question just a little bit if
the psycho toxic levels are so low that you can't detect them
by our present techniques.
Just how much significance is there,
really?
And when you look at the great variability and clinical
results.
It's very hard to correlate those kinds of data.
I think you're getting at the question that I really wanted you to
get at.
And that's uh,
is there are we talking about too sensitive
techniques or false positive cross match is in a sense,
in a sense of whether they're clinically significant.
Because if you take it to continuing extremes,
you will get to the point where no one can be transplanted
because they have some kind of positive
reaction to one of these ultra sensitive tests.
I think that's a very valid point.
I mean,
what you're saying is ultimately we'll be doing nothing but identical
twins.
Yeah.
Dr gary boy,
your test of of self
sensitivity
Have received a great amount of
enthusiasm.
How do you feel as far as their significance in
renal transplantation is concerned at the present time?
Well,
at the present time,
I think it's still a two.
It needs a lot of work.
We really haven't done a large enough series of
prospective patients to say what the clinical
significance is.
When we first did this series of tests up in
Boston two years.
So now we ask about 25 patients
prior to transplantation at that time,
five patients had a positive LPC
reaction prior to transplantation.
Three of them lost their kidney in the next two
months was a very small number of patients to make
any any conclusion of.
And since I've been down here in San Antonio,
we've also looked at another 20 five
Patients and four or five of them again
have also been positive and two or three of them
have also lost a kidney in a very short period of time.
But these survival results are
indeed identical to the survival results.
Most patients who did not have the positive prior
to transplant.
So I can't say that this group has a
greater risk.
I think it's of note that
several instances now individuals who are
H.
L.
A identical siblings who are M.
L.
C.
Negative have been found to
have sensitivity or pre
sensitization to each other.
And this has primarily been in patients with a plastic anemia who have
received transfusions from H.
L.
A,
identical sibs.
But in these instances they've been able to find
that they can develop a positive
LMC or cell mediated side uh license
test as well as an antibody
induced cell cell mediated psycho
toxicity.
And this would imply therefore that
the sensitizing
uh an agent or agent
or inheritance of the sensitizing agent
is one which is not uh
inherited on the chromosome or a
wheel of the H.
L.
A.
System.
And since the renal survival and H.
L.
A identical sibs is
pretty uniform throughout the world as being certainly greater
than 90%.
And excluding technical failures,
certainly greater than 95% would
imply that at least in certain instances pre
sensitization is detected by the L.
M.
C.
Or the A.
I.
L.
M.
C.
R.
R.
Sensations that are perhaps not significant with
regards to kidney transplant.
They may well indeed however have a great significance in
bell marrow transplants.
And I'll ask dr sell his comments on that.
Well.
We have only done about 11 or 12 bone marrow
transplants and we've looked for these reactions in those
patients and in our identical
matched but non identical,
otherwise not identical.
Sibs we've not seen these kinds of reactions
but I've been very intrigued by a recent as yet
unpublished report from Bruce comida which
showed that there are killer
lymphocytes present in the marrow of
sibs directed against other sibs in
the family without pre sensitization and being
genetically inherited apparently
through chromosomes other than those that handle the
mlc locus.
And uh this kind of
genetically occurring spontaneously occurring
CML reaction may well have
tremendous significance in the bone marrow
transplant situation.
We've only recently learned of this and now we
and others thomas.
I think everyone is now looking for those reactions
because they may be extremely important in
graft survival.
As you know,
fully 25% of a
plastic anemic patients will reject their graphs
despite the fact that they're mlc negative and
perfectly identical at the H.
L.
A.
Locust.
25% will reject their graphs.
Hist o compatibility testing
a panel discussion with dr braun,
doctor Cell Dr Gary boy
and Dr species
was moderated by Roberti Lord in
Lieutenant Colonel U.
S.
Air Force M.
C.
Assistant Chief reno service.
Wilford Hall U.
S.
Air Force Medical Center,
Lackland Air Force Base and produced
through the mobile facilities of the television division,
Academy of Health Sciences,
United States Army,
Fort SAm Houston texas.