Re: [Histonet] Iron leaching out during decal

From:"Lee & Peggy Wenk"

1. Make certain that it is hematopoetic bone. That is, bone with marrow (RBC, WBC, etc.). Vertebrae, rib, or sternum are fine. But NOT adult femur heads, or bones from arms or legs from amputations from adults. These are all fat and connective tissue. No iron stores in these bones in adults.
2. Use a mild decal for minimal time. We like to use FASC (formic acid, sodium citrate) for 2-3 days. Stronger acids tend to leach out the iron stores. If using hydrochloric or nitric acids, use 5% for a few hours to overnight, depending upon the size (see #3).
3. Use minimum size. The larger the size you gross, the longer it has to be in decal, and the more iron that gets leached out. The HTL exam asks for bone 1 cm long. There is no requirement for width. So at needle core biopsy would be acceptable, as long as there is cortex and hematopoetic marrow.
4. Realize that not all adults have large amounts of iron stores. If you are getting it from an autopsy from someone who has been sick a long time, or who has been undergoing treatment for cancer, they may have very little to no iron stores. Try several different sources.
5. Make certain the formalin is OK. Acidic formalin will also leech out the iron, so use newly made, buffered formalin. And fix for minimal time. If the bone is being stored in formalin for a length of time (weeks to months), the formalin can go acidic also.
Just some hints we've learned along the way.
Peggy A. Wenk, HTL(ASCP)SLS
School of Histologic Technicians and Histotechnologists
William Beaumont Hospital
Royal Oak, MI
----- Original Message -----
From: Jeannie Heck
To: Histonet
Sent: Monday, August 18, 2003 3:27 PM
Subject: [Histonet] Iron leaching out during decal

Please help! I am working on my HTL Practical test and I am required to do a Prussian Blue iron stain on a 1.0 cm piece of bone. My problem is that everything that I use to decal my bone (so that it cuts nicely) leaches out all of the iron. HCL decal solutions were obviously too powerful (tried them anyways) so I used a milder selection that included acetic acid (after fixing w/ formaldehyde), and a combination of formic acid and formaldehyde (to fix and decal in the same step). I am leary to get Zenker's solution (which was recommended by Frieda Carson's Histology: A Self Instructional Text) because if it doesn't work, then I am left with the expensive and arduous task of disposing of the mercury. Does Zenker's really fix and decal effectively to preserve the iron AND allow me to cut a nice section? What about Zinc Chloride to replace the mercury in Zenker's? Any suggestions would be greatly appreciated, I really need some help. Thank you!
Bethany J. Krafels

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