[Histonet] RE: (reply) silly questions.---PFA

Tue Dec 16 16:07:41 CST 2008

tf,
=20
Answers as follows:
Seems obvious that the 4% formaldehyde (made from PFA) was incorrectly made=
. What they did, I have no idea nor did I have the time to show them how to=
 make it up. My advice: "make it from concentrated 38% formaldehyde (formal=
in)".
=20
=20
You wrote: "I do think most biomedical labs currently are using PFA to prep=
are the fixatives!" - I do not think so. A sweeping statement if ever I hav=
e heard one. This s the type of misinformation that we have to deal with. M=
y experience (over 30 years) indicates that all pathology labs I have worke=
d in as well as those I have visited, as well as several Pathology Colleges=
 (eg CAP and RCPA) surveys indicate that MOST (if not all) histopathology l=
abs prepare their 10% formalin fixative from concentrated formalin not poly=
formaldehyde as you have incorrectly stated.=20
=20
Be careful of misinformation.
=20

Regards=20

Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)=20
Laboratory Manager & Senior Scientist=20
Tel: 612 9845 3306=20
Fax: 612 9845 3318=20
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA=20

	-----Original Message-----
	From: tf [mailto:tifei <@t> foxmail.com]=20
	Sent: Friday, 12 December 2008 7:35 PM
	To: Tony Henwood; Pat Flannery; histonet <@t> lists.utsouthwestern.ed
	Subject: (reply) silly questions.---PFA
=09
=09
	"I looked at the sections and the cell shrinkage (and prominent spaces
	between cells and connective tissue) indicated that most of the
	"fixation" seemed to have occured in the processing ethanols. I asked
	him for some of the fixative he used, tested the formaldehyde
	concentration and found it to be less than 0.5%!!"
	=20
	Tony: Do you think this is because of inproper preparation of PFA in his l=
ab, or the common problem in all researchers using PFA?
	         I do think most biomedical labs currently are using PFA to prepar=
e the fixatives!
	        =20
	So, anyone has the idea on a correction preparation procedure of 4% PFA?
	I noticed some of you dissolve PFA powder in NaOH-conditioned alkaline wat=
er, then add concentrated PB solution.
	We here dissolve PFA in concentrated PB solution directly (heat & stir for=
 2-3 hours), then adjust pH to 7.4.
	=20
	We dont have big problem in tissue quaility....except when one want to cut=
 the brain in a cryostat rather sliding microtome.
	Many times the brain sections from the cryostat have "cheese" like holes/c=
avities, which almost never appear on sliding microtome-prepared sections.
	=20
	2008-12-12=20
=09
________________________________

	tf=20
=09
________________________________

	=B7=A2=BC=FE=C8=CB=A3=BA Tony Henwood=20
	=B7=A2=CB=CD=CA=B1=BC=E4=A3=BA 2008-12-12  06:18:47=20
	=CA=D5=BC=FE=C8=CB=A3=BA Pat Flannery; histonet <@t> lists.utsouthwestern.edu=20
	=B3=AD=CB=CD=A3=BA=20
	=D6=F7=CC=E2=A3=BA RE: [Histonet] Silly Question?=20
=09
=09
	Pat,
	I agree with you.
	In a routine diagnostic histopathology laboratory, it makes little
	difference what you use. Around the world for over 100 years most labs
	use 10% neutral buffered formalin made from concentrated 38%(or there
	abouts) formalin (or formaldehyde).
	Researchers, though, are a different kettle of fish. They will tend to
	hang on to misinformed, "mystical" methods believing they are being
	scientific. Funny, you would think that they, as a group, would be the
	ones pushing the boundaries and critically assessing each step of their
	research, ensuring that they understand what and why they are doing it.
	(Disclaimer - not all researchers are like this, thank heavens!!)
	Using a formaldehyde solution made from polyformaldehyde can cause
	problems. One researcher used it and wondered why their morphology was
	sub-optimal and their p53 immunohistochemistry was negative. He assured
	me that he had fixed small samples of tissue for 6 hours in 4%
	formaldehyde and then processed them using ethanol, xylene and wax.
	I looked at the sections and the cell shrinkage (and prominent spaces
	between cells and connective tissue) indicated that most of the
	"fixation" seemed to have occured in the processing ethanols. I asked
	him for some of the fixative he used, tested the formaldehyde
	concentration and found it to be less than 0.5%!!
	This also explains the loss of p53 staining. I gave him some of our
	routine 10% phosphate buffered fomalin, asked him to fix overnight, and
	try agin. Low and behold problem solved.
	How's that for a Friday Flamming!!!
	Regards
	Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC)
	Laboratory Manager & Senior Scientist
	Tel: 612 9845 3306
	Fax: 612 9845 3318
	the children's hospital at westmead=20
	Cnr Hawkesbury Road and Hainsworth Street, Westmead=20
	Locked Bag 4001, Westmead NSW 2145, AUSTRALIA=20
	-----Original Message-----
	From: histonet-bounces <@t> lists.utsouthwestern.edu
	[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Pat
	Flannery
	Sent: Friday, 12 December 2008 3:59 AM
	To: histonet <@t> lists.utsouthwestern.edu
	Subject: [Histonet] Silly Question?
	Please humor me on this if it's obvious (to everyone but me):  why do =20
	we use paraformaldehyde (which is so inconvenient to make up) rather =20
	than buffered formalin or just diluted formaldehyde itself?
	It seems that around here, some folks prefer paraformaldehyde (either =20
	2% or 4%) and others use formalin, while some others stick to diluted =20
	formaldehyde (I see all 4 on labels for specimens submitted for =20
	histology).  Is it mostly a matter of personal preference or where you =20
	were trained (i.e. force of habit) or is there a valid reason to use =20
	each solution (basically the same chemical once in solution, merely =20
	buffered or not)?  The only answer I've gotten when I've asked is, =20
	"That's what we always use."
	Thanks.
	-Pat Flannery (not a "real" histologist - I just play one in the lab)
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Views expressed in this message and any attachments are those of the indivi=
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