[Histonet] CISH on formic acid decalcified bonemarrow biopsies

[gayle callis]

To All,

Gudrun, thank you for the references plus I liked your protocol where the BM
are well fixed before decalcification.   If anyone is interested I have a
simple decalcification endpoint check that works as long as you have a
balance that weighs in mg to three places.  We use this faithfully for all
bone calcification methods either acid or EDTA.  It takes very little time,
and certainly prevents over exposure to any acid, our preference being 10 to
15% formic acid on totally fixed murine or other species samples.  Using
buffered formic acid (commercial sources) is very handy for those in
clinical laboratories and the buffered acid is very gentle for BM biospsies.
The concentration of formic acid in sodium formate or sodium citrate
buffered solutions is approximately 4.5% (I calculated this one time out of
curiosity).  

Gayle Callis
HTL,HT,MT(ASCP) 



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gudrun Lang
Sent: Saturday, September 26, 2009 2:32 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] CISH on formic acid decalcified bonemarrow biopsies

I agree with Gayle, that EDTA is the best way for preserving DNA and RNA.
But we have switched to formic acid decal of bonemarrow biopsies for the
speed-reason. And we have found that IHC has become better with some
markers.

This year I tested CISH (Kappa/Lambda) on 25 BM and compared the results
with the IHC. With our protocol of BM processing the CISH was successful in
all cases. And the results were "clearer to read".

The point is, that concentration and duration of decal in formic acid must
not exceeded. There are several publications on this.

 

Our protocol: one day fixation in NBF, next day decal in 5-10% formic acid
with 5-10% formaldehyde (commercial product) for 6-8 hours, then processing
over night in VIP like all other specimen.

 

These are some of the publication, I've looked up:

 

Brown RSD, Edwards J, Bartlett JW, Jones C, Dogan A; Routine Acid
Decalcification of Bone Marrow Samples Can Preserve for FISH and CGH Studies
in Metastatic Prostate Cancer; J. of Histochem. and Cytochem. Vol. 50 (1):
113-115, 2002

Janneke CA, Krijtenburg P-J, Vissers KJ, van Dekken H; Effect of Bone
Decalcification Procedures on DNA in Situ Hybridization and Comperative
Genomic Hybridization: EDTA is Highly Preferable to a Routinely Used Acid
Decalcifier, J. Histochem. and Cytochem Vol. 47(5): 703-709, 1999

Korac P, Jones M, Dominis M, Kusec R, Mason DY, Banham AH, Ventura RA;
Application of the FICTION technique for the simultaneous detection of
immunophenotype and chromosomal abnormalities in routinely fixed, paraffin
wax embedded bone marrow trephines, J Clin Pathol 2005;58:1336-1338

Miranda RN, Mark Hon Fong L, Medeiros LJ; Fluorescent in Situ Hybridization
in Routinely Processed Bone Marrow Apirate clot and Core Biopsy Sections;
Am. J. of. Pathology, Vol 145, Nr. 6, December 1994

Naresh KN, Lapert I, Hasserjian R, Lykidis D, Elderfield K, Horncastle D,
Smith N, Murray-Brown W, Stamp GW; Optimal processing of bones marrow
trephine biopsy: the Hammersmith Protocol, J. Clin. Pathol. 2006:59;903-911

 

Gudrun Lang

Histolab, AKH Linz, Austria

 

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