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-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Gudrun Lang Sent: Saturday, September 26, 2009 2:32 AM To: histonet@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet