Leica Biosystems PELORIS_PELORIS II - Manual (Page 75)

Reagent Setup
Leica PELORIS™ User Manual Rev K © Leica Biosystems Melbourne Pty Ltd 2011 75
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Absolute ethanol (100% dehydrant) contaminated by carryover from absolute IMS (100%
dehydrant) has a concentration of 100%, as both the original reagent and the contaminant are
dehydrants.
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A new xylene (100% clearer) contaminated by carryover from absolute ethanol (100%
dehydrant) has a reduced concentration — typically around 94% after one cycle — as it will
consist of 94% xylene (clearer) and 6% ethanol (not a clearer).
A reagent that is used early in a sequence of the same group will have a rapid concentration decline
as most of the contamination it receives will be from the previous group. A reagent that is used late
in a sequence will have a slow concentration decline as most of the contamination it receives will be
from the same group.
Managing Concentrations
The Peloris software uses reagent concentrations to select stations when protocols are run (unless
the protocol uses station reagent selection). It selects the station with the lowest (in-threshold)
concentration of a reagent group or type for the first step using that group or type, then stations of
increasing concentration for following steps. It always uses the highest concentration reagent for
the last step before changing to another reagent group or type. The software also uses
concentration information (amongst other factors) to prompt you to change reagents that have
exceeded purity thresholds.
It is very important, then, for high quality processing and efficient reagent use, that the
concentration information the software uses is accurate. The software automatically tracks the
concentration of the reagent in each station, updating values after each run. For it to do this
effectively you must enter accurate information for it to work with, e.g. set realistic protocol
carryover values and enter the correct number of cassettes in each run. It also means you should
update the software properly whenever you change reagents.
Under default settings the software assigns concentrations “by calculation”. This uses information
such as the retort fill level, the number of cassettes processed, the carryover setting and the
reagent groups involved to calculate concentration in each station.
Concentrations can also be assigned “by cycles” or “by position” (i.e. by station). These methods
rank station concentration by the number of processing runs each station has been used for or the
physical order of the stations, respectively.
Leica Microsystems does not recommend the “by cycles” or “by position” options, which must be
set by a technical support representative.
For consistently high quality processing always replace reagents as soon as you are prompted, with
fresh reagent at the default concentration. If you remove a bottle from the instrument, always
check that you enter the correct reagent information for the bottle when you return it. Supervisors
can manually change station concentration values in the Reagent stations screen if they believe
the value there is incorrect. Be sure that you independently verify the concentration if making such
a change.