[Histonet] CD10, CD103, FDA, fatty tissues, complement, frozen blocks, fixation

[Bryan Hewlett]

John wrote:

>>My 2 cents on fixation- tissue is fixed when it's fixed.(Formalin
penetrates and fixes 2mm per hour , right?) It's not fixed because the
pathologist wants the tissue out early. I think the sixteen hours is
overkill myself and IHC retrieval methods are in place to counteract long
fixation. If it were my wife I would cut the tissue in at 4 mm, fix for 8
hours and run the tests like I do with all my patients. I've optimized my
lab to give every single person the best possible result. >>

The penetration rate of formaldehyde is largely irrelevant to tissue no more
than 1.5 cm thick.
However, it's NOT 2mm per hour and fixation takes much longer than this!
It's variable. d =K x the square root of time.
K = the coefficient of diffusibility.
Baker determined K=3.6, probably correct and a figure most people accept.
Helander's 1994 data suggests it is at least 2.0 and most likely close to
3.6.
Using K=3.6 in the above formula, the first layer of cells is penetrated at
a rate of over 90 mm per hour.
This slows to 3.6 mm at 1 hour.
It then takes 4 hours to double the depth penetrated to 7.2 mm, a rate of
1.8mm per hour.
At 16 hours, the depth is again doubled to 14.4 mm, a rate of 0.9 mm/hr.
i.e. to double the depth of penetration you must allow 4X the time and so
doing halves the rate!

Your 4mm tissue block would be fully penetrated in less than 1 hour (3.6 mm
from each surface)
What the issue really is, relates to the binding time necessary for
formaldehyde.
A 90% threshold binding, i.e. MINIMAL binding time is 24 hours at 22C and 18
hours at 37C.
It doesn't matter if your tissue is only a few cells thick or 4mm thick!
16 hours is not long fixation, 3 weeks may be, and is certainly NOT
overkill.
The weak initial binding of formaldehyde is freely reversed, 50% in about 12
hours, by the lower alcohols on the processor  and the tissue becomes fixed
by alcohol.
Any thing less than 8 hours formaldehyde fixation reduces the IHC for ER
demonstrably and HER2 markedly.


Bryan



----- Original Message -----
From: "JOHN COLEMAN" <JPCOLEMA <@t> sentara.com>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Wednesday, May 12, 2004 2:02 PM
Subject: [Histonet] CD10, CD103, FDA, fatty tissues, complement,frozen
blocks, fixation


For CD10 we use Novo Castra's antibody at a 1:30 dilution, Trilogy from
Cellmarque (high pH, edta, tween) for 15 minutes in a 98C water bath, and
biogenex's super sensitive concentrate kit for detection. 45 minute primary
antibody incubation.

Question- I need a reliable CD103 antibody for formalin fixed paraffin
embedded material. The clone we have currently is good on coagulative fixed
and air dried material, but is worthless on routinely proccessed bone marrow
and lymphoid tissue.

Pardon my cynical response, but FDA doesn't care if it works. If you follow
the protocol you're in. If you tweak the protcol you're not in FDA land and
can not claim such, even if your controls look great.

For fatty tissues we have a fixative that is 4 gal formalin,1 gal absolute
ETOH and 500 ml Glacial acetic acid . We place the blocks in this after the
tissue is grossed in at the board.

Complement- we stain for C3c, C4c, and C1q on FFPE kidney and skin biopsies
using .1% protease from sigma for 30 minutes @ 40C before DIF labelling.

Does your cryostat have a defrost cycle? if so the blocks are being trashed.
I recommend sealing in oct, wrapping in parafilm and storing in a -70
freezer or processing as usual and doing immunos on formalin fixed tissue,
where the morphology is better.

My 2 cents on fixation- tissue is fixed when it's fixed.(Formalin penetrates
and fixes 2mm per hour , right?) It's not fixed because the pathologist
wants the tissue out early. I think the sixteen hours is overkill myself and
IHC retrieval methods are in place to counteract long fixation. If it were
my wife I would cut the tissue in at 4 mm, fix for 8 hours and run the tests
like I do with all my patients. I've optimized my lab to give every single
person the best possible result. My wife or daughter or mother is no more or
less valuable than anyone else that walks through the door. If anyone would
do differently for people important to them in their lab then you guys need
to bring your standards up! This is medicine- someone's life litereally
depends on our results. If your routine methods are not good enough for your
family you should be ashamed to leave them in place.
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