[Histonet] Mucin overstained with Hematoxylin in G.I. specimens

Lee & Peggy Wenk lpwenk <@t> sbcglobal.net
Mon Nov 5 04:10:13 CST 2007

Check the pH of the hematoxylin. It should be 2.2-2.9. We like to keep it at
about 2.4-2.6. If the pH goes above 3.0, mucin starts to stain. Add acetic
acid to the hematoxylin, and bring the pH back down. (Note: have to use a pH
meter. The hematoxylin stains the pH strip blue, so they don't work when
having to compare color of the strip to the color on the pH range. Personal
experience with a "duh" factor at the end on my part.)

Check the pH of the hematoxylin right from the bottle. If the hematoxylin pH
is the problem, make certain you contact the manufacturer, tell them the lot
number, and explain the problem. The manufacturers usually keep samples of
each lot, and and see what the problem is (poor QC on their end, rapid
decomposition, improper storage, etc.).

Since the 2.2-2.9 is an acceptable range, sometimes a batch/lot will arrive
that is closer to the 2.9 end. As water is carried over into the
hematoxylin, the water (having a higher pH), will increase the pH of the
hematoxylin, causing the hematoxylin to go over the 3.0 pH side, and thus
stain the mucin. It also then starts to stain the slide, too. Increasing
time in the acid wash afterwards will remove some of the mucin staining. But
it's better to reduce the pH of the hematoxylin.

If you are using tap water to rinse, you might also want to check the pH of
the tap water. Sometimes the water companies have to add other chemicals to
the water supply, to take care of problems on their side (increase bacteria,
inversion of the water table levels stirring up debris, etc.). So possibly
your water supply is more alkaline these past couple of weeks. Thus the
carry over of water into the hematoxylin is increasing the pH of the
hematoxylin a lot faster than it usually does. If this is the problem, you
might need to add a step of using distilled or deionized water, after the
tap water and before the hematoxylin.

Let us know what the problem is. It will help other techs in your area if
the problem is the water pH. It will help other techs in the country, if the
problem is a lot/batch of a particular brand of hematoxylin.

Good luck.

Peggy A. 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 DNon
Sent: Saturday, November 03, 2007 10:42 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Mucin overstained with Hematoxylin in G.I. specimens

Hello Histonet, 

     Over the past several weeks our lab has had problems with hematoxylin
overstaining in the mucin of our gastric specimens.  The degree of
hematoxylin overstaining is alarming in the mucin of the gastric specimens
but absent or negligible in other specimens.  One pathologist reviewing the
slides indicated some of the overstained specimens appeared to be
inadequately fixed.  We have addressed that issue which showed some
improvement, but the problem persists.  We are making changes to increase
fixation time (although the specimens now appear adequately fixed yet retain
a large measure of the overstaining), but any further suggestions for
corrective action would be much appreciated.

    We've been hesitant to increase acid alcohol clearing time because our
other specimens are staining well with the current protocol.

We are using the following reagents and stain times:

-  Surgipath specimen containers pre-filled with 10% neutral buffered
-  Surgipath Gills III Hematoxlyin - 6.5 minutes followed by running water
-  .75% HCL in 70% ETOH for 1 second (1 dip) followed by running water wash
-  Surgipath Scott's Tap Water for 1 min followed by running water wash

Sakura automated stainer set to agitate ( up and down dipping of racks )
once every 2 seconds, which is the fastest speed available on it.  

Dick Non
Pathology Department
Ocean County Medical Lab
Brick, New Jersey
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