Trevor and Jennifer,
This method originally came from Mawhinney et al. Control of rapid nitric acid decalcification J Clin Pathol 1984 37:1409-1415 and was cited by Cathy Sanderson (Mayton)in a publication using EDTA, found in Biotechnic and Histochemistry. Mawhinney acutally did a chemical test on final acid change to see that calcium was not present, but we never had to do that. If you end up with a bit of residual calcium in block, I would surface decalcify at microtomy. I used it for years when we downsized and gave away our FAXITRON. Radiography is still the most accurate, and if you had either a micro CT or digital FAXITRON available, it would be a better test. I used Cathy's method and will be happy to send the original publication for anyone's files/future referencing. A chemical test for EDTA is a pain to do if a FAXITRON is not available. I have put the full method below, with a bit more detail. WEIGHT LOSS/WEIGHT GAIN ENDPOINT TEST This is a method of choice for EDTA decalcification although it was originally used by Mawhinney et al for testing nitric acid decalcification. Many samples can be decalcified together in one container, i.e. 25 mouse femurs in 1 liter of 10% formic acid. If all the samples are the same size, i.e. mouse femurs, tibia, paws, choose several as representative samples and test only those to save time. Always suspend bones in the decalcifier. Requires a balance that reads in milligrams to 3 places for greatest accuracy. Specimen must be blotted free of fluid for accurate weighing each time you weigh the sample. We suspend bones in nylon specimen bags for easy removal to weigh. Bags can be marked with pencil too. Technique: 1. Rinse NBF off bone, blot with paper towel, WEIGH BONE, RECORD BEGINNING WEIGHT. Suspend bone in acid or EDTA decalcifier. During acid decalcification CO2 bubbles are given off, so stir during decalcification to release bubbles or small samples will float. EDTA does not create CO2 bubbles, only acids. Large bones can be started at end of day in acid decalcifier and sit overnight with testing the next morning. 2. After 4 to 5 hours in acid or overnight in EDTA, remove bone, rinse with water, BLOT, weigh. RECORD WEIGHT. If bone shows loss of weight, change acid decalcifier to refresh acid. Return samples to resume decalcification, and repeat as many times as necessary. EDTA should be changed but not as often as acid. Always use a large volume of decalcifier i.e. 20:1 or more. Remove bone from specimen bag, and place in weighing boat to protect balance from acids/EDTA. 3. When bone begins to GAIN WEIGHT, the bone is decalcified. Once calcium is removed, water is taken on and the weight increases. This water does not affect the bone. 4. Rinse bone with running tap water for an hour or longer to remove these decalcifiers. Either store in 70% alcohol or process. Store endpoint tested decalcified bones in 70% alcohol while waiting for other samples to finish decalcifying and mass processing run. Reminders: For EDTA, one can suspend bones and check every day for accuracy but bones can be left in the EDTA over a weekend or several days without damage as long as the bones were well fixed. Acid decalcified bones cannot be left over a weekend, remove from acid, put in NBF to stop decalcification. Bones should be endpoint tested before stopping decalcification so you can resume decalcification on the next working day. Rinse off NBF briefly before resuming decalcification. Do not overexpose bones to acids or you will damage antigens and nuclear staining. Enjoy the method, as it truly is fast and easy. Gayle M. Callis HTL/HT/MT(ASCP) ____________________________________________________________________________ _____ Ha Wow...that's almost too easy. Thank you for this! Trevor Jordan Wait University of Texas Health Science Center, San Antonio Class of 2017 MD Candidate Abilene Christian University Class of 2013 Graduate B.S. Biochemistry ________________________________ From: Jennifer MacDonald < <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> JMacDonald <@t> mtsac.edu> Sent: Monday, August 11, 2014 2:33 PM To: Wait, Trevor Jordan Cc: <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet <@t> lists.utsouthwestern.edu; <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet-bounces <@t> lists.utsouthwestern.edu Subject: Re: [Histonet] Weight Loss/Weight Gain Decal I believe this was originally from Patsy Ruegg Decalcification End Point: Weight Loss, Weight Gain 1. Blot sample to remove excess fixative 2. Weigh bone in mg, record as beginning weight 3. Next day, rinse bone, blot and weigh bone daily, record weight. Change decalcifying solution to refresh acid OR EDTA. Return bone to fresh decalcifying solution. 4. When bone begins to GAIN weight, remove from decalcifying solution, rinse and process. YOU MUST WEIGH IN MILLIGRAMS FOR ACCURACY Once calcium is totally removed (bone loses weight as this happens), water replaces the calcium and weight begins to go up. This is the point at which calcium should be totally gone. The original method used a chemical test at the end to insure no calcium was in the decalcifying solution. If you do this, you cannot stir the solution during decalcification. Be sure to suspend bone in the solution to insure all sides of bone are in contact with decalcification solution. From: "Wait, Trevor Jordan" < <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> WaitT <@t> livemail.uthscsa.edu> To: " <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet <@t> lists.utsouthwestern.edu" < <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet <@t> lists.utsouthwestern.edu> Date: 08/11/2014 12:29 PM Subject: [Histonet] Weight Loss/Weight Gain Decal Sent by: <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> histonet-bounces <@t> lists.utsouthwestern.edu ________________________________ Hello all! I'm currently doing some decalcification and was curious if anyone had some particular advice about the weight loss/weight gain method. I understand that when the decalcification process is complete, the tissue block will begin to increase in weight. However, I'm confused when I should record the weight for the block once they have been taken out of the EDTA solution. You see, for the times I weighed the blocks before... the weights were a little skewed because there were differing amounts of solution on the blocks while they were sitting on the balance. I just want to standardize my protocol a little more so that I can be sure the block is actually gaining weight due to the calcium loss rather than just extra solution sitting on the outside of the block. Would letting the blocks sit out of solution for about 30 minutes before being weighed help with the matter? I know that the blocks take on water once they are completely decalcified so I'm not sure how much this will affect that. Trevor Jordan Wait University of Texas Health Science Center, San Antonio Class of 2017 MD Candidate Abilene Christian University Class of 2013 Graduate B.S. Biochemistry _______________________________________________ Histonet mailing list <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> Histonet <@t> lists.utsouthwestern.edu <http://lists.utsouthwestern.edu/mailman/listinfo/histonet> http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet