[Histonet] FW: faded H&E stain

Truscott, Tom ttruscot <@t> vetmed.wsu.edu
Thu Aug 23 10:26:29 CDT 2007

Hi Jane, Since other tissues in the same processing run and same
staining run turn out OK, I would look at the differences in handling
the small biopsies. Are the biopsies put in special cassettes or on
sponges that are preventing fluid exchange anywhere along the way? Since
the problem is sporadic, maybe their position in the processor makes a
difference- are they on the bottom or top of the chamber? Specimens on
top might trap air in their cassettes and prevent fluid exchange.  Tom
Truscott USDA-ADRU Pullman, WA

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jane C.
Sent: Thursday, August 23, 2007 5:01 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] FW: faded H&E stain

Recently we have experienced sporadic problems with our routine  H&E
stains of some small GI biopsies and Ultrasound guided needle biopsies
of breast tissue.   The stains appear faded and almost unreadable.
Other tissue processed at the same time, cut, oven dried,  and stained
in the same rack of slides (we hand stain) are stained appropriately.
Usually these are from larger sections of tissue such as appendix,
gallbladder, skin, uterus, colon segment, etc.  This faded stain  may
happen one day and then not again for several and then happen again just
for certain tissues that day.  We are working with the "collecting
techs" to ensure that the tissue is appropriately handled before we
receive it.


For breast tissue, the technique that has been used was to drop the
biopsy in sterile water just to remove it from the syringe and transfer
immediately to formalin.  This has worked well for quite some time.
Today however, we have asked them to develop a method such that the
tissue will be immediately put in the formalin.  The reason it was going
into the sterile water was because the needle is very expensive and
would need to be used again for the second core and obviously could not
be rinsed in formalin. We have also considered asking them to use
sterile saline to rinse the needle, but are not sure of the effect on
the tissue. 


Does anyone have any ideas?  We have had no problems until several weeks
ago and nothing that we know of has changed.


I have obviously watched timing, temperatures, used only fresh stains,
alcohols, xylene, and all products in the processor.


We use Richard Allen stains.


We routinely use the following times but I am adding a xylene on each
end and two more 100% ETOH for hydration and dehydration. 


Xylene-5 min

Xylene--- 5 min

100% ETOH rinse

95 % ETOH rinse

80% ETOH rinse

Distilled water for 1 min

Hematoxylin  3 min

Tap water rinse

Clarifier-45 sec

Tap water rinse

Bluing---1 min

Tap water rinse

Eosin-20-30 sec

80% ETOH

95% ETOH

95% ETOH

100% ETOH

100% ETOH




Thanks in advance for any help.   Jane


Jane Moose

Newberry County Memorial Hospital

Newberry, SC  29108


F- 803-405-7474


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