Leica Biosystems PELORIS_PELORIS II - Manual (Page 151)

Troubleshooting
Leica PELORIS™ User Manual Rev K © Leica Biosystems Melbourne Pty Ltd 2011 151
C. Slow Reverse Process
Process using a modified cleaning protocol for a period equal to the time required to forward
process the tissue (see Slow Reverse Cleaning Protocol below). Then reprocess from formalin using
a schedule suitable for the size and nature of the specimen (see 8.2.1 Specimen Type and Protocol
Duration).
A long reverse process is very gentle on the tissue. If time permits this is the favored option.
D. Direct Reprocessing
In this method wax is not removed before reprocessing. Place cassettes directly back into formalin
and then, with no other pretreatment, reprocess using a schedule suitable for the size and nature
of the specimen (see 8.2.1 Specimen Type and Protocol Duration).
This method is the quickest, however it causes wax contamination of the reagents on the processor.
Change all reagents (except wax) after direct reprocessing.
7. Tissue Affected by Formalin During Clearing or Wax Infiltration
This problem can occur if a leaking valve allows backflow of formalin into the wax bath. Contact
customer support to test your instrument if you see this problem.
Formalin contamination is characterized by “blue hue” in the nuclei, loss of chromatin detail,
nuclear shrinkage, variable eosinophilia and cytoplasmic swelling and/or shrinkage.
First melt down the blocks, blot off excess wax, and then place the specimens in new cassettes.
This minimizes wax contamination of the processing reagents. Then reverse-process the blocks by
any of the methods listed for problem 6. After this, soak in a high pH Tris-HCl retrieval solution (e.g.
Bond Epitope Retrieval Solution 2) for 2–12 hrs at room temperature.
This treatment improves the staining qualities of the H&E, the cutting qualities of the tissue and the
cohesiveness of the section. Nuclear detail, cytoplasmic swelling and definition are unlikely to
improve.
8. Inadequately Fixed, Under-Processed Tissue
A possible remedy is to slowly reverse-process the tissue (see 6C), apply additional formalin
fixation, then reprocess using a schedule suitable for the size and nature of the specimen (see 8.2.1
Specimen Type and Protocol Duration).
Unfixed or poorly-fixed tissue is damaged by processing – the alcohol and high temperatures exert
a fixative effect different to that of formaldehyde. As a result blocks become more susceptible to
hardening and shrinkage. When combined with under-processing, blocks may be unusable.