more H&E troubleshooting Flashcards
What is the process for manual regressive staining
Harris Hematoxylin
RTW
Acid Alcohol for differentiation
RTW
Scotts tap water substitute
RTE
Check differentiation microscopically - (Regressive)
Eosin -counterstain
RTW
DCM
Problem :
No cytoplasmic staining in RBC, muscles and connective tissue fibers
In H&E can be corrected for staining faults
What are causes and CA
Causes:
Eosin was omitted. It was over differentiated in dehydrating alcohols
CA
-TAKE SLIDE BACK UP TO WATER
-rehydrate the slides xylene, xylene>100, 100, alcohol>water
-reapply Eosin and DCM but adjust the dips in low grade alcohol to prevent over differentiation (quicker dips)
-water is the one removing the eosin stain
Problem: ***
Pale cytoplasmic staining in RBC, muscle and connective tissue
Causes:
-wasnt in the eosin long enough
-pH of eosin >
-if above IEP there wont be enough pos to bind to
-Eosin contaminated with blueing agent, not enough rinising after blueing agent, carryover of alkaline will change pH
-section too thin - not enough protein to bind to - cut section again
CA
-Rehydrate slides XY>Al>Water
-check pH of eosin, adjust with acetic acid as needed (4.6 - 5 is best)
-restain with eosin follow staining protocol to DCM
-recut section with proper thickness and restain
-replace old eosin with new
-change 100% alcohols contaminated with water
Problem:
Cytoplasmic tissue components not properly differentiated – 3 shades not
seen.
if problem shows on all the slides
used on same procedure
performed manually or by hand
how did yesterdays stain
Cause:
-inadequate fixation
-incorrect dehydration and clearing
-inadequate time in low grade alcohol during DCM
-incorrect pH of eosin solution
CA
if the error is from tissue processing
-cant be saved but you can prevent by making sure you fix properly with good dehydration and clearing
If the cause is from staining
-take section back to low grade alcohol and differentiate until the staining you want is achieved
-make sure the pH of eosin is correct
-rinse slides properly in water after bluing to avoid alkali carryover into eosin
Problem
Cytoplasmic staining is too dark.
Causes
-too long staining in eosin
-eosin solution is too concentrated
-section passed through dehydrating alcohol too quickly-not enough dips for differentiation
-section cut too thick if higher then many tissue binding site hence harder
-isopropyl was used as the dehydrating agent
-water contaminated with alcoholic eosin
CA
-rehydrate the slides xy>alc>h2o
-give more time in low grade alcohols for proper differentiation - best at 70%
-eosin solution should be diluted if concentrated
-recut with proper thickness and restain
-use ethanol
-change eosin formula
Problem
No nuclear staining
Cause
-hematoxylin was omitted
-hematoxylin was over differentiated; too long in acid alcohol
CA
-remove coverslip
-rehydrate the slides xy>alc>h2o because alcohol will remove most of eosin stain
-do the H&E technique again to DCM
Problem
nuclear staining too pale- cant see defined chromatin patterns
Cause
-too short in hematoxylin
-too long in acid alcohol
-fixative with acid was used
-over decalcified or over fixed in tissue
-efficacy of hematoxylin is lost or reduced
CA
-remove coverslip and rehydrate the slides xy>alc>h2o because the alcohol will remove most of the eosin
-reapply both hematoxylin and eosin, but after HX have short time in acid alcohol
-if acid fixative, over- decal or over fixed you should increase time in Hx or get another way to increase basophila
-replace with fresh hematoxylin
PROBLEM:
* Nuclear stain is too dark.
* Nuclei are overstained.
* Diffuse Hx staining in cytoplasm.
Causes
* Too long in hematoxylin.
* Too short in acid alcohol.
* Section cut too thick.
* Incorrect concentration of Hx
solution.
-HX too concentrated must be 2.5
CORRECTIVE ACTION:
*rehydrate the slides xy>alc>h2o because the alcohol will remove most of the eosin
* Differentiate in acid alcohol then follow H&E protocol to DCM. Blue nuclei and re-apply eosin.
* Re-cut section with correct thickness and re-stain.
-diltute HX with water or glycerin
PROBLEM:
* Muddy stain.
* Eosin is not pink – overall
blue hue on the tissue.
Causes:
* Under-differentiation or omission of acid alcohol differentiation step.
* Hx was not removed from background tissue.
CA:
*rehydrate the slides xy>alc>h2o because the alcohol will remove most of the eosin
*Dip 1-2x in acid alcohol and then do all the h&e steps to dcm . Have to blue nuclei and apply eosin
PROBLEM:
* Weak and uneven cytoplasmic staining
Causes
-Eosin is contaminated if the blueing reagent was not rinsed off properly before eosin was applied
-Insufficient washing after bluing agent leaves residual alkali- this will change pH of eosin
-pH of eosin is too high
CA
*rehydrate the slides xy>alc>h2o because the alcohol will remove most of the eosin
-rinse properly to make sure bluing agent is rinsed off, repply eosin
-check pH of eosin solution and add acetic acid if necessary
PROBLEM:
* Red or brown nuclei.
Causes
-bluing omitted or not enough
-old or over oxidized hematoxylin
CA
*rehydrate the slides xy>alc>h2o because the alcohol will remove most of the eosin
-blue tissue is alkaline bluing reagent so its not possible to over blue and then follow staining to DCM
-check efficacy of HX; replace and freshen if needed
PROBLEM:
* Nuclear staining is not crisp.
* “Smudgy nuclear staining”
Causes
incomplete fixation
-issues with tissue processor ; incomplete dehydration and clearing, introduction of heat to reagents other than paraffin, too long in paraffin infiltration
CA
-no saving - but can prevent
-fix tissue properly
-do not introduce heat in other reagents only paraffin
PROBLEM:
* Blue/black precipitate on top
of tissue
causes:
-stain precipitation; some Hx are formulated to form a metallic sheen when exposed to air for a long time
CA
-filter the staining solution
-dont use past the expiry date
PROBLEM:
* Uneven H&E staining.
Causes
-Water or fixative contaminated the paraffin in the processor
-alcohols were contaminated with water in the processer
-staining solutions not high enough to cover slides
-thick or thin section with banding, chatter or venetian blind
CA
-check tissue processor, do not prolong wax infiltration
-make sure there are enough reagents to cover slides
-have proper techniques in microtomy to prevent artifacts
PROBLEM:
* Poor contrast between nuclei
and cytoplasm.
Causes
* Poor nuclear stain -pale or too dark.
* Poor cytoplasmic stain -pale or too dark.
CA
-determine which stain is causing the issue can adjust timing during staining
-check pH of solution and adjust
