Re: alcohol/xylene processing

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From:BB racing <>
To:Adrienne Vair <>
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Thank you for your note to me and your interest in my paper.
1.  Is it possible to use BB's fixative for everything?  Yes it is, but it is some what expensive
and time consuming to make the large volumes of this reagent that the OR's would need.
In our experience the combination of the alcohol/xylene processing and minimum of 24
hours 10% fixation gives more than adequate results for large specimens.
2.  The processing time is not 21 hours, but rather 12 hours with the delay in station #6.
On our processors we set the start and end times and the processor automatically
calculates the delay time in station #6.  This can range from as short as 4 hours to as long
as 52 hours, as over a four day holiday.  We normally start our work day at 7 am, with the
processors finishing at that time, and most often are finished the days work at 3 PM with
the processors started at that time.
3.  As to shorting the program.  Again the answer is yes, but it depends on what you are
processing.  Small biopsies fixed in BB's fixative can be adequately processed in a few
hours, but if you have large sections taken from breast or other types of tissues fixed in
10% formalin you will be inviting problems such as inadequately processed tissues which
will be difficult to cut, and will have poor morphology.
4. After hours biopsies.  Biopsies that are received after hours, and that have been fixed for
only one or two hours in BB's fixative can simply be added to the processor at any time as
long as it has not gone past station #6, with the proviso that they will get at least a couple
of hours in station #6 to dehydrate before the processor goes on to the next station which
would be pure xylene.
5.  Using BB's fixative and standard graded alcohol processing.  For biopsies you will find
that your results will be significantly superior to what you would get with formalin fixation.
This is because the specimens are small and they will be adequately fixed and dehydrated using this procedure.  Larger specimens will not give as good a results due to the less than
adequate dehydration that graded alcohol processing gives.
6. The purpose of formaldehyde in BB's fixative.  The addition of formaldehyde to BB's
fixative is to alter the morphological appearance of these tissues, and give them more of a
formalin fixed appearance.  Most pathologists are use to seeing only formalin fixed tissues,
and find that tissues fixed in other fixatives can be more difficult to interpret,  This makes
them more at ease with this fixative.  Once they get use to looking at tissues fixed in this
fixative they quickly get to the point where they prefer it over formalin, as the morphology
and nuclear detail is far superior, and approaches that of B5 fixed tissues.
7.  Foamy Muddy Poor Nuclear detail.  In my experience this is a function of 10% formalin
fixation and graded alcohol processing.  It was this never ending problem that prompted
me to try and find a solution to better tissue processing, and resulted in the paper I just
published.  The problem centers around the slow fixation rate of formalin.  This is easy to
confirm, by simply taking a piece of fresh tissue, placing it in 10% formalin, and
sequentially taking sections from it at 12 hour intervals,  It will be noted that as the time in
formalin increases the quality of the morphology improves.  By the 96 hour mark much of
the nuclear foaminess and muddy appearance will have disappeared, and the sections will
look pretty good, but in to days world, who can have a surgical specimen sitting around for
96 hours in formalin waiting to be processed in order to have good morphology?
8.  Slide Drying.  Overheating your sections during the slide drying process can lead to a
whole host of problems including ruining the sections morphology.  I use a small amount of
granulated gelatin in my water bath as an adhesive, and dry the slides in a 65C hot air
oven only long enough to melt the wax, usually 15 - 20 min, then into xylene.  We never have a proble with the sections comming off the slides.
Kerry Beebe ART

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