H&E Troubleshooting

Question 1

Answer 1

Well-stained sections

Question 2

Answer 2

Well-stained tissue

Question 3

Answer 3

Blueing was done but not cytoplasmic staining

Question 4

Answer 4

pale or weak nuclear staining

Question 5

Answer 5

Muddy stain

Hematoxylin was not differentiated

Blueing agent was not rinsed off adequately; eosin was contaminated with blueing agent

Question 6

Answer 6

No nuclear staining – Hematoxylin was omitted

Question 7

Answer 7

Very pale cytoplasmic staining – too short in eosin

Question 8

Answer 8

No cytoplasmic staining – eosin was omitted

Question 9

Answer 9

Water droplets under the coverslip is due to contaminated 100% alcohol or contaminated xylene.

Question 10

Answer 10

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.

Question 11

Answer 11

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

Question 12

Answer 12

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

Question 13

Answer 13

Dark nuclear staining due to:
–Too long in hematoxylin
–Short differentiation step
–Thick section

Question 14

Answer 14

Red or brown nuclear staining
–Hematoxylin is breaking down
–Blueing step not properly done

Question 15

Answer 15

Pale cytoplasmic staining due to:
–pH of eosin >5
–Eosin contaminated with blueing agent
–Too long in low grade alcohol
–Thin sections

Question 16

Answer 16

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

Question 17

Answer 17

Blue-black precipitation on top of the tissue is due to stain precipitation.

Metallic sheen will be picked up on the slide.

Question 18

Answer 18

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

Question 19

Answer 19

Uneven H&E staining due to:
–Water of fixative contaminated the infiltrating paraffin
–Alcohols are contaminated with water
–Reagent levels are insufficient

Question 20

Answer 20

Poor contrast between nucleus and cytoplasm due to:
—Poor nuclear stain (pale or too dark)
—Poor cytoplasmic stain (pale or too dark)

Question 21

MICROTOMY PROBLEM

Answer 21

Moth-eaten effect

Question 22

MICROTOMY PROBLEM

Answer 22

Washboarding

Question 23

MICROTOMY PROBLEM

Answer 23

Microchatter

Question 24

MICROTOMY PROBLEM

Answer 24

Patched Earth

Question 25

MICROTOMY PROBLEM

Answer 25

Folding

Question 26

MICROTOMY PROBLEM

Answer 26

Contamination- technologist's skin debris

Question 27

MICROTOMY PROBLEM

Answer 27

Water bath not on proper temperature tissue not flat on slide during pickup

Question 28

MICROTOMY PROBLEM

Answer 28

Bubble on the section or Nick on the tissue due to a calcified part

Question 29

MICROTOMY PROBLEM

Answer 29

debris that has been picked up

Question 30

MOUNTED SECTIONS TROUBLESHOOTING

Answer 30

Water bubbles Causes: Incomplete dehydration Contaminated 100% alcohol. Contaminated xylene.

Question 31

MOUNTED SECTIONS TROUBLESHOOTING

Answer 31

Mountant on slide Causes: Incorrect handling of coverglass. Too much mounting medium applied. Mounting medium on gloves.

Question 32

MOUNTED SECTIONS TROUBLESHOOTING

Answer 32

Corn-flaking or Trapped air Causes: Too slow in applying the coverslip. Xylene has evaporated prior to coverslipping.

Question 33

MOUNTED SECTIONS TROUBLESHOOTING

Answer 33

Retreaction causes: Mounting medium was too thin (too much xylene was added to thin it out). Tissue can dry out