TABLE

Table 5.2.
Automated Tissue Processing Schedule

Station Reagent Purpose Time
1 10% neutral buffered formalin Fixation 1 hour
2 70% alcohol Dehydration, using ascending 1 hour
3 80% alcohol grades of alcohol 1 hour
4 95% alcohol 1 hour
5 95% alcohol 1 hour
6 100% (absolute) alcohol or methanol 1 hour
7 100% (absolute) alcohol or methanol 1 hour
8 Xylene or substitute Clearing, 1st change 1 hour
9 Xylene or substitute Clearing, 2nd change 1 hour
10 Xylene or substitute Clearing, 3rd change 1 hour
11 Paraffin wax Infiltration at 55°C-58°C 1 hour
12 Paraffin wax (~2°C-3°C above melting point) 1 hour
TIPS AND TROUBLESHOOTING GUIDES IN TISSUE PROCESSING

(Bancroft, Carson, Winsor, Zimmerman)
Problem Cause Prevention and Correction
Microchatter around edges Excessive dehydration Process small biopsy tissues separately from larger tissues, carefully controlling
of tissue on small biopsy the time of dehydration.
specimens Decrease time allowed in the dehydrating solutions.
Poor or absent nuclear Underdehydration Implement complete fixation and increase dehydration time.
staining in H&E Processor: There may be Ensure no condensation in the processor, with either fixative or alcohol droplets
a clearing agent in the dripping onto the tissue; no mechanical problems with the processor; good
paraffin, too much heat schedule or reagent rotation is developed and maintained; and use heat only
during processing, or for the paraffin.
mechanical problems with
the processor.
Tissue inadvertently Open processors dry up If tissue is fixed, morphology can be recovered and diagnosis rendered.
desiccated the tissue accidentally If tissue is in paraffin, blot off wax with paper towels. Then, place it in
rehydrating solution composed of 50 mL water, 30 mL absolute alcohol, and 20
mL of 5% aqueous solution of sodium carbonate. Soak overnight, and then
reprocess as usual.
Tissues that have dried prior Problems with automated 70 mL of 70% ethanol30 mL of glycerol and 1g of dithionite.
to wax infiltration processors
Tissues are immersed in the solution for several hours or overnight. Processing
begins with the dehydrating solutions and continues to completion. Tissues may
be hard to section, and coated slides should be used.
Inadequate processing of Routine schedule and Fatty tissues should be sampled and cut thinly (~2-3 mm) for easy reagent.
fatty tissues reagents may not process action. Ethanol is a poor fat solvent. To ensure complete dehydration, far better
fat adequately lipid solvent (acetone or isopropanol) should be inserted before using the final
absolute ethanol, and chloroform or 1,1,1,-trichloroethane as the transition
solvent.
Hard, dense tissues Tough keratin component Dense tissues may be softened by treating fixed tissues using 4% aqueous
of certain tissues phenol for 24-72 hours or by dehydrating tissues using phenol in the first bath
of absolute ethanol, or in all dehydrant baths.
Xylene turns cloudy in the Xylene with excess water Xylene reagent needs to be changed immediately when it appears cloudy.
presence of water and during
processing.
On embedding, tissue is soft Inadequate processing At gross, cut thin sections, especially fatty tissues.
and mushy in the center due to very thick or fatty
sections Ensure complete fixation.
Cut the tissue into thinner sections, then deparrafinize in xylene. Repeat
processing and re-embed.
Deparaffinize, refixthen reprocess.

On embedding, tissue is Special orientation not Mark the cutting side or cutting face of tissue with ink.
oriented incorrectly clearly indicated
Write special instructions on the worksheet.
Tissue carry-over (floaters or Dirty forceps Clean forceps between specimens.
pick-ups)
Tissue cross-contamination Opening all cassettes at Open one cassette at a time.
during embedding one time and leaving
them open
Tissue not embedded at the Tissue not uniformly Press tissue down uniformly and firmly in mold.
same level pressed down
Melt paraffin evenly so tissue pieces get embedded at the same level.
Tissue pieces embedded
at different levels in the Work at an even, rapid rate so tissue pieces get embedded at the same level
same mold before wax starts to harden.
Tissue pieces missing from May be overlooked during Record number of tissue pieces.
the block embedding
Check lid of cassette or lens paper and inspect all pieces.
Ink minute tissues with eosin.

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Table 5.2.

Automated Tissue Processing Schedule

TIPS AND TROUBLESHOOTING GUIDES IN TISSUE PROCESSING

Deparaffinize, refixthen reprocess.

Ink minute tissues with eosin.

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