As mentioned throughout this course, proper tissue processing hinges on a variety of factors and variables that must be addressed in order for the outcome to be consistent and free of artifacts. Accurately troubleshooting tissue processing is a mark of a well-seasoned technician, since most troubleshooting skills are gained through first-hand experience. The most evident processing problem in histology laboratories is under-processed tissue samples. Although some tissues suffer from incomplete fixation, which in turn may lead to improper dehydration, clearing, and infiltration, troubleshooting fixation is best saved for a detailed course on tissue fixation.
Processing problems may be noticed at various points from embedding to slide quality control (QC). Histotechnologists and pathologists have the responsibility of noting any problems seen with the tissue and reporting it to the laboratory supervisor for correction. With each problem there may be several sources to troubleshoot. Starting with the simplest solution first not only takes the least effort, but may save a lot of time. Troubleshoot by process of elimination, from least labor intensive to the most involved solution to the problem. Verifying that the correct processing program was used is a simple solution, compared to changing out all of the processing reagents.
The table below provides some troubleshooting tips for poor processing:
Tissue feels soft or mushy during embedding | - Tissue may have been grossed in too thick
- Tissue may have been processed on a program that was too short for that tissue type
- Processing reagents may be saturated with water
- Paraffin may be saturated with xylene or isopropanol
| - Reprocess tissue on proper program
- Reprocess tissue on correct processing protocol
- Change reagents and reprocess tissue
- Change paraffin and reprocess tissue
|
| Tissue bounces out of paraffin block during microtomy or tissue does not adhere to block or slides (Commonly experienced with uterus and prostate tissue, as well as dense organ core samples) | - Poor dehydration and paraffin infiltration due to water left in the tissue
| - Change reagents and reprocess tissue on proper processing protocol
|
Tissue looks greasy and "explodes" or separates rapidly when ribbon is placed on water bath
| - If the temperature of the water bath is between 45-50° C, then the tissue is under-processed
- Tissue may have been grossed in too thick
- Tissue may have been processed on a program that was too short for that tissue type
- Processing reagents may be saturated with water
- Paraffin may be saturated with xylene or isopropanol
| - Reprocess tissue on correct processing protocol
- Reprocess on proper program
- Reprocess tissue on correct processing protocol
- Change reagents and reprocess
- Change paraffin and reprocess tissue
|
Tissue does not adhere to slide or falls off easily
| - If tissue slides are placed in oven prior to deparaffinization in xylene, tissue is under-processed
- Reagents saturated with water or contaminated with the preceding reagent
| - Reprocess tissue on correct processing protocol
- Change reagents and paraffin and reprocess tissue on proper processing protocol
|
Hematoxylin and eosin (H&E) stained tissue section shows uneven nuclear staining and "blue blobs" lacking distinct chromatin patterns
| - If tissue was fixed properly, then sample was improperly dehydrated and infiltrated with paraffin
| - Change reagents and reprocess tissue on proper processing protocol
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