Hello,Michelle:

Do I understand that your manual stain set-up is absolutely identical to your
automatic stainer set-up?...is that in time values as well as reagent set-up?

You commented that when you stain from your manual set-up the staining results
are fine.   I would recommend that you "manually" stain using your automatic
stainer set-up.    If you are able to acheive the desired results, then we can
identify the mechanical differences between human and machine staining.     It
would be helpful to compare your stain programs (Manual procedure and automatic
times).   

One of the biggest differences between hand and machine staining is how the
surface tension of the reagent currently on the slide is broken and then
replaced by the next reagent.  When we stain by hand we exert much more and
varied force than a machine does when plunging the slides into the reagent.  We
also knock off more reagent when time is done so less reagent clings to the
slide.      A stainer (machine, not human) simply lowers the slides slowly, in
a
single plane, into the reagent.  Even the agitation of the machine staining is
in that careful, single plane (up and down) movement.   When we stain by hand
we
cause the reagent in the dish to bombard the slide from several angles and with
greater force that breaks the surface tension in less time than a machine can
accomplish.  Therefore, a longer exposure time (of tissues to stain) may be
required on a machine to yield the same results as hand staining.       

When programming the machines I find it necessary to watch the hand staining
carefully in order to make an accurate translation of a "dip" to a time value
that the machine could reproduce.  A "dip" in acid alcohol in manual staining
may not be able to be reproduced by a machine.  I may be able to use 1% acid
alcohol in hand-staining but have to use 0.5% acid alcohol on the staining
machine with a 2-second timing value to get the same results.       Ten "dips"
in a manual stain may require 30 seconds on a machine.   Ten "dips" in a manual
alcohol step may require 1 minute on a machine for the same results.       

One of the things we need to remember is that the machine will move the slides
exactly the same way for the programmed time.    We humans (consciously or
unconsciously) adjust our handling of the slides based on how the tissues or
even the reagent looks.   

Additional questions:
Which model of Tissue-Tek stainer do you have?    Is it a Sakura DRS-60 or
DRS-601 or DRS-2000?   Analyzing the stain, is the nuclear stain OK but the
counterstain is too light?   Is the nuclear stain too light but the
counterstain
is OK?  Is the nuclear stain too light AND the counterstain is too light?
Are
the stains consistant in their lightness throughout the specimen and throughout
all sections on the slide?   Do you notice an improvement in the stain after
the
new reagents have become somewhat diluted?

Please feel free to call our technical help group (histologists) who will be
happy to review staining variables.  They can be reached at 800/725-8723, menu
#2.

Best regards,
Nancy

Nancy Klemme, HT(ASCP)
Customer/Product Support Mgr.
Sakura Finetek USA, Inc.
1750  West 214th Street
Torrance, CA  90501

Web Page = www.sakuraus.com 
e-mail = nancy.klemme@sakuraus.com
Phone = 800/725-8723

____________________Reply Separator____________________
Subject:    H&E help
Author: ,"Skelton, Michelle" <mskelton@anthc.org>
Date:       9/27/99 4:44 PM

From: Skelton, Michelle
Date: Mon, Sep 27, 1999 4:44 PM
Subject: H&E help
To: 'histonet@pathology.swmed.edu'
   We are having a problem with our H&E being inconsistent (sometimes from
day to day, sometimes from batch to batch).  We have an automated stainer
(Tissue-Tek) and purchase the hematoxylin and eosin from Surgipath.  We do
not change program times or reagents, yet sometimes our stain is light and
sometimes it is dark (preferred).
   We had experimented with the times and thought that we had found one that
was "perfect"....then something happened and we have had problems ever
since.  The stain seems to be especially light after changing the alcohols
and xylenes.  We have not changed any processes, vendors, or manufacturers.
But our stain is continually changing. Some days it is great and other days
it is too light.  The same hematoxylin, eosin, alcohol, and xylene are on
our manual stain line.  We stain those following the same times as on the
auto stainer and they come out perfect every time. Has anybody else had this
problem and does anybody know what to do about it?
  We have tried everything that we can possibly think of and are at our wits
end. Any help would be greatly appreciated.

Thanks in advance,
Michelle Skelton
Alaska Native Medical Center


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