H&E Troubleshooting Flashcards
Well-stained sections
Well-stained tissue
Blueing was done but not cytoplasmic staining
pale or weak nuclear staining
Muddy stain
Hematoxylin was not differentiated
Blueing agent was not rinsed off adequately; eosin was contaminated with blueing agent
No nuclear staining – Hematoxylin was omitted
Very pale cytoplasmic staining – too short in eosin
No cytoplasmic staining – eosin was omitted
Water droplets under the coverslip is due to contaminated 100% alcohol or contaminated xylene.
White spots seen in the tissue section. Spotty or irregular staining
Patchy staining due to presence of wax in the tissue prior to application of stains. Incomplete dewaxation.
Nuclear staining is not crisp due to
–Incomplete fixation
–Incomplete dehydration, incomplete clearing;
–Heat was introduced in other reagents during tissue processing.
–Prolonged wax infiltration
Pale nuclear staining due to:
–Too short in hematoxylin
–Hematoxylin was over-oxidized or depleted
–Over-differentiation
–Use of acidic fixative
–Tissue was over-decalcified or over-fixed
Dark nuclear staining due to:
–Too long in hematoxylin
–Short differentiation step
–Thick section
Red or brown nuclear staining
–Hematoxylin is breaking down
–Blueing step not properly done
Pale cytoplasmic staining due to:
–pH of eosin >5
–Eosin contaminated with blueing agent
–Too long in low grade alcohol
–Thin sections
Dark cytoplasmic staining due to:
–Eosin - highly concentrated
–Too long in eosin
–Too short a time in low grade alcohols during DCM
–Section is thick
Blue-black precipitation on top of the tissue is due to stain precipitation.
Metallic sheen will be picked up on the slide.
Eosin was not properly differentiated due to:
–Incorrect fixation
–Incorrect dehydration and clearing during tissue processing
–Inadequate time in low grade alcohols during DCM
–Incorrect pH of the eosin solution
Uneven H&E staining due to:
–Water of fixative contaminated the infiltrating paraffin
–Alcohols are contaminated with water
–Reagent levels are insufficient
Poor contrast between nucleus and cytoplasm due to:
—Poor nuclear stain (pale or too dark)
—Poor cytoplasmic stain (pale or too dark)
MICROTOMY PROBLEM
Moth-eaten effect
MICROTOMY PROBLEM
Washboarding
MICROTOMY PROBLEM
Microchatter
MICROTOMY PROBLEM
Patched Earth
MICROTOMY PROBLEM
Folding
MICROTOMY PROBLEM
Contamination- technologist’s skin debris
MICROTOMY PROBLEM
Water bath not on proper temperature
tissue not flat on slide during pickup
MICROTOMY PROBLEM
Bubble on the section
or
Nick on the tissue due to a calcified part
MICROTOMY PROBLEM
debris that has been picked up
MOUNTED SECTIONS TROUBLESHOOTING
Water bubbles
Causes:
Incomplete dehydration
Contaminated 100% alcohol.
Contaminated xylene.
MOUNTED SECTIONS TROUBLESHOOTING
Mountant on slide
Causes:
Incorrect handling of coverglass.
Too much mounting medium applied.
Mounting medium on gloves.
MOUNTED SECTIONS TROUBLESHOOTING
Corn-flaking or Trapped air
Causes:
Too slow in applying the coverslip.
Xylene has evaporated prior to coverslipping.
MOUNTED SECTIONS TROUBLESHOOTING
Retreaction
causes:
Mounting medium was too thin (too much xylene was added to thin it out). Tissue can dry out
