[Histonet] B5 substitute

Lee & Peggy Wenk lpwenk <@t> sbcglobal.net
Thu Nov 8 19:04:12 CST 2007

Couple of hints with any B5 substitute (which are usually zinc formalin),
from a research project 2 of our students did on various brands of zinc
formalin at different time intervals -

1. If you are going to place zinc formalin in the tissue processor, use zinc
sulfate, not zinc chloride. When zinc chloride cross-links with protein, it
releases hydrochloric acid, which can chew up the retort chamber of the
processor (hint from a Journal of Histotechnology article a lot of years
ago, one of which authors was Liz Chlipala, who contributes to Histonet).

2. However long you are used to fixing the tissue in B5, multiply that
number by 1.5, and that will be about right for the minimum time in zinc
formalin. Zinc binds slower than mercury - plain simple chemistry. So if you
were doing bone marrow biopsies for 2 hour in B5, 2 x 1.5 = 3 hours in zinc
formalin. If the lymph nodes were taking 4 hours in B5, try 4 x 1.5 = 6
hours in zinc formalin. 

The first time we tried this experiment with different brands of zinc
formalin, we kept the times the same as B5. All the tissues had awful
looking nuclei. So we dropped the idea of switching. A couple of years
later, we tried the experiment again, with time intervals of something like
1, 2, 3, 4, 6, 8 and 24 hours. And we did it on bone marrow biopsies and on
lymph nodes. That's when we came up with the "multiply it by 1.5" factor. It
seemed pretty universal, regardless of which company.

After coming up with the best time, we conveyed the information to the med
techs who go up on the bone marrow biopsies. Most of the biopsies looked
fine, but every once in a while, one would have nuclei that looked like a
bad alcian blue on goblet cells. Turns out, one of the med techs didn't like
the change in routine - "but I've always done it for 2 hours" and was
refusing to change to 3 hours, until we showed her other tech's well fixed
zinc formalin bone marrows and her zinc formalin-fixed bone marrows, and
asked her which one she would like the hematopathologist to diagnosis from,
if this was her daughter's biopsy for possible leukemia. No problems after

3. You may need 2 different zinc formalins. We found one worked better for
tissues that were going to go into acid for decalcification, and a different
one worked better for all other tissues (tumors, lymph nodes). 

Sorry, I just don't remember which companies we liked. I'd have to dig out
the research papers.

Peggy Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48073

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Joanne
Sent: Thursday, November 08, 2007 11:51 AM
To: gvdobbin <@t> ihis.org; Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] B5 substitute

Hi ,

What are you using to replace B5 fixative with mercury? I want the best for
hematopoetic tissues, and for immunostains.


Jo Mauger

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