Hematoxylin and eosin staining

8 DOH Abu Dhabi (HAAD) lab licensing practice questions on Hematoxylin and eosin staining, part of Histopathology and Cytology. The first 8 are below; the first 5 with answers and explanations (tap Show answer after you have chosen), the rest in Learn with a free account.

1. Question 1 · Hard

   An automated stainer runs H&E on 400 to 500 slides a day. This week, many sections show irregular pale patches that have taken up neither hematoxylin nor eosin, while the rest of the tissue on the same slide stains normally, with blue nuclei and pink cytoplasm. The patches fall in different places on serial sections from the same block. The stainer log shows: hematoxylin replaced this morning; acid alcohol and bluing reagent changed daily; the two dewaxing xylene stations at the start of the program last changed 6 days ago, with a limit of 1,000 slides per change. Slide drying before staining is unchanged. The most likely cause is:

   1. ABluing step too short after acid differentiation
   2. BWater carried over into the final clearing xylene
   3. CHematoxylin exhausted by the high slide throughput
   4. DWax left in the sections by overused dewaxing xylene

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   Answer: D · Wax left in the sections by overused dewaxing xylene

   Patches that take up neither dye point to a problem before staining starts, because hematoxylin and eosin are applied in aqueous or alcoholic solutions that cannot enter tissue still holding wax. At about 450 slides a day, the dewaxing xylene has handled roughly 2,700 slides in 6 days, nearly three times its limit, so it is loaded with paraffin and leaves wax behind in random areas. Changing the dewaxing stations and keeping to the slide limit fixes the problem, and affected cases are recut or restained. Worn-out hematoxylin gives pale nuclei with normal eosin, short bluing leaves nuclei reddish over the whole section, and water in the final xylene gives a milky haze after coverslipping rather than unstained patches.

   • A: Short bluing leaves nuclei reddish across the whole section; it does not leave unstained patches.
   • B: Water in the final xylene gives a milky haze after coverslipping, not patches without stain.
   • C: The hematoxylin was replaced this morning, and weak hematoxylin would not affect the eosin.

2. Question 2 · Medium

   What role does the aluminum salt (potassium or ammonium alum) play in Harris and Mayer's hematoxylins?

   1. AThe oxidant that ripens hematoxylin to hematein
   2. BThe acid that sharpens nuclear staining
   3. CThe mordant that links hematein to chromatin
   4. DThe preservative that stops over-oxidation

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   Answer: C · The mordant that links hematein to chromatin

   Hematein on its own binds tissue poorly. Aluminum ions form a positively charged dye-metal complex (a lake) that binds the negatively charged phosphate groups of DNA in chromatin. Sodium iodate is the usual chemical oxidant, and acetic or citric acid is added to sharpen nuclear staining. Chloral hydrate in Mayer's and ethylene glycol in Gill's help keep the solutions stable.

   • A: Oxidation is done by sodium iodate or by air, not by the alum.
   • B: Acetic or citric acid is added to sharpen nuclear staining.
   • D: Chloral hydrate or ethylene glycol help stabilize these solutions.

3. Question 3 · Easy

   In a regressive hematoxylin method, the deliberate overstaining of the section is corrected by:

   1. ACounterstaining with eosin
   2. BBluing in an alkaline solution
   3. CLonger dehydration in ethanol
   4. DA brief rinse in acid alcohol

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   Answer: D · A brief rinse in acid alcohol

   Regressive methods stain the section past the end point on purpose and then remove the excess dye with dilute hydrochloric acid in 70% alcohol, commonly 0.5% to 1%. This step, called differentiation, leaves the dye in chromatin and clears it from cytoplasm and background. It is timed in seconds, because too long a rinse makes nuclei pale. Bluing comes after differentiation, not before it.

   • A: Eosin stains cytoplasm and does not remove hematoxylin.
   • B: Bluing turns the bound dye blue and insoluble; it removes no excess dye.
   • C: Ethanol dehydration does not take hematoxylin out of overstained nuclei.

4. Question 4 · Hard

   Eosin staining has become weak and patchy over several days. The working eosin now has a pH of 6.8 (it works best at about 4.5 to 5.0), and the water rinse after the bluing station was recently removed from the program. The most likely cause is:

   1. ABluing reagent carried over into the eosin
   2. BSections dehydrated too slowly after the eosin
   3. CHematoxylin over-oxidized by exposure to air
   4. DToo long a rinse in acid alcohol before bluing

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   Answer: A · Bluing reagent carried over into the eosin

   Eosin is an acidic dye that binds positively charged groups in tissue proteins, and these charges fall as the pH rises. Carryover of an alkaline bluing reagent, with no rinse in between, steadily raises the eosin's pH and weakens staining. Restoring the water rinse and replacing the eosin, often with a little acetic acid added, fixes the problem. Long dwell in low-grade alcohols can also extract eosin, but it would not change the eosin's pH.

   • B: Slow dehydration can pale the eosin but does not raise the eosin's pH.
   • C: Over-oxidized hematoxylin gives pale nuclei, not weak eosin.
   • D: Acid alcohol acts on hematoxylin before bluing, not on the eosin bath.

5. Question 5 · Medium

   After differentiation, H&E sections pass through Scott's tap water substitute or dilute ammonia water. This step:

   1. AMordants the eosin so that it binds collagen
   2. BOxidizes hematoxylin to its active form, hematein
   3. CTurns the aluminum-hematein complex blue and insoluble
   4. DRemoves excess hematoxylin from the cytoplasm

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   Answer: C · Turns the aluminum-hematein complex blue and insoluble

   The aluminum-hematein lake is red and fairly soluble under the acid conditions of the dye bath and the acid alcohol. A weakly alkaline solution converts it to the blue, insoluble form that gives nuclei their final color. If bluing is too short or the reagent is exhausted, nuclei stay reddish-purple. Running tap water blues sections only if it is alkaline enough.

   • A: Eosin needs no mordant; it binds tissue proteins by charge.
   • B: Oxidation to hematein happens when the solution is made, with sodium iodate or by air.
   • D: Removing excess dye is differentiation, which comes before bluing.

6. Question 6 · Hard

   Nuclei on every slide in today's H&E run show sharp chromatin detail but are reddish-purple rather than blue. The eosin is normal, and the same hematoxylin gave blue nuclei yesterday. The most likely cause is:

   1. APoor fixation of the tissue
   2. BIncomplete bluing after the acid rinse
   3. CExhausted hematoxylin solution
   4. DOver-long differentiation in acid alcohol

   Answer and explanation in LearnFree account

7. Question 7 · Medium

   How does a progressive hematoxylin method differ from a regressive one?

   1. ANuclei are stained after the eosin counterstain
   2. BAn iron salt replaces the usual aluminum mordant
   3. CThe section is overstained, then cleared with acid alcohol
   4. DStaining stops at the end point, with no acid differentiation

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8. Question 8 · Hard

   Over two weeks, H&E nuclei have become steadily paler although the stainer program is unchanged. The hematoxylin has not been replaced in that time and has turned brownish, while the acid alcohol and bluing reagents are changed daily. What should be done?

   1. AIncrease the bluing time to five minutes
   2. BChange to a more concentrated eosin solution
   3. CReplace the hematoxylin and change it on a slide count
   4. DLengthen the acid alcohol differentiation step

   Answer and explanation in LearnFree account