Histotechnology: staining, special stains and IHC

96 Kerala MSc MLT Entrance practice questions on Histotechnology: staining, special stains and IHC, part of Pathology. The first 25 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

   A 58-year-old woman with rheumatoid arthritis for 20 years has nephrotic-range proteinuria. Her renal biopsy shows glomerular deposits that stain with Congo red and give apple-green birefringence. On a section treated with potassium permanganate before Congo red, the deposits no longer stain. The amyloid is most likely:

   1. AAL (light chain) type
   2. BTransthyretin (ATTR) type
   3. CAA (serum amyloid A) type
   4. DBeta-2 microglobulin type

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   Answer: C · AA (serum amyloid A) type

   Congo red staining with apple-green birefringence confirms amyloid. Pre-treatment with potassium permanganate abolishes the congophilia of AA amyloid, whereas AL and transthyretin amyloid usually keep it. AA amyloid is derived from serum amyloid A, an acute-phase protein, and follows long-standing inflammation such as rheumatoid arthritis, so the history fits. The permanganate test is only a screening clue; the type is confirmed by immunohistochemistry or mass spectrometry.

   • A: AL amyloid usually resists permanganate and is linked to plasma cell disorders.
   • B: Transthyretin amyloid usually resists permanganate and mainly affects heart and nerves.
   • D: Beta-2 microglobulin amyloid occurs in long-term dialysis, mainly in joints and bone.

2. Question 2 · Medium

   A tissue mucin stains with alcian blue at pH 2.5 but not with alcian blue at pH 1.0. What kind of mucin is it most likely to be?

   1. ASulphated mucin
   2. BNeutral mucin
   3. CCarboxylated mucin
   4. DGlycogen

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   Answer: C · Carboxylated mucin

   Alcian blue is a cationic dye that binds ionised acid groups. At pH 2.5, both carboxyl and sulphate groups are ionised, so both kinds of acid mucin stain. At pH 1.0, only the strongly acidic sulphate esters remain ionised, so only sulphated mucins stain. A mucin positive at pH 2.5 alone is therefore carboxylated, such as a sialomucin.

   • A: Sulphated mucins stain at both pH 1.0 and pH 2.5.
   • B: Neutral mucins have no acid groups and do not stain with alcian blue.
   • D: Glycogen is alcian blue-negative; it is shown by PAS with a diastase control.

3. Question 3 · Hard

   An antibody arrives pre-diluted 1:50 by the supplier. The laboratory's validated working dilution is 1:400 of the original concentrate. How much of the pre-diluted antibody is needed for 800 µL of working solution?

   1. A2 µL
   2. B16 µL
   3. C8 µL
   4. D100 µL

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   Answer: D · 100 µL

   The pre-diluted antibody must be diluted a further 400 / 50 = 8 times. The volume needed is 800 µL / 8 = 100 µL, made up to 800 µL with 700 µL of diluent. Dividing 800 by 400 gives 2 µL, which treats the pre-diluted product as concentrate and gives an eight-fold weaker stain. Every new dilution should be checked on a known positive control before patient use.

   • A: 2 µL treats the pre-diluted antibody as concentrate, so staining would be far too weak.
   • B: 16 µL divides by the supplier's 1:50 instead of by the extra factor of 8.
   • C: 8 µL mistakes the extra dilution factor for the volume needed.

4. Question 4 · Medium

   What does Turnbull's blue reaction demonstrate, in contrast to the Perls Prussian blue reaction?

   1. ACalcium salts, using silver nitrate
   2. BFerric iron, using potassium ferrocyanide
   3. CIron bound firmly in haemoglobin
   4. DFerrous iron, using potassium ferricyanide

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   Answer: D · Ferrous iron, using potassium ferricyanide

   Perls' reaction uses acid potassium ferrocyanide, which reacts with ferric (Fe3+) iron to form insoluble blue ferric ferrocyanide. Turnbull's reaction uses potassium ferricyanide, which reacts with ferrous (Fe2+) iron. Ferrous iron is scarce in tissue, so Turnbull's method is seldom positive unless ferric iron is first reduced with ammonium sulphide. Iron in haemoglobin is too firmly bound to react with either method.

   • A: Silver nitrate for calcium is the von Kossa method.
   • B: Ferric iron with ferrocyanide describes Perls' reaction.
   • C: Haemoglobin iron is too firmly bound to react without unmasking.

5. Question 5 · Medium

   Mayer's haematoxylin contains sodium iodate, potassium alum, citric acid and chloral hydrate. What is the role of the sodium iodate?

   1. AIt oxidises haematoxylin to haematein at once
   2. BIt acidifies the solution for nuclear selectivity
   3. CIt preserves the solution against mould
   4. DIt mordants the dye to the nuclear chromatin

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   Answer: A · It oxidises haematoxylin to haematein at once

   Sodium iodate is a chemical oxidant that ripens the solution immediately, so Mayer's haematoxylin can be used on the day it is made. Natural ripening by air and light takes weeks. The alum supplies the aluminium mordant, citric acid lowers the pH to sharpen nuclear staining, and chloral hydrate acts as a preservative. Too much oxidant over-oxidises the dye and shortens its useful life.

   • B: Citric acid lowers the pH and makes the stain more nuclear-selective.
   • C: Chloral hydrate is the preservative in Mayer's formula.
   • D: The aluminium alum, not the iodate, is the mordant.

6. Question 6 · Easy

   In Masson's trichrome stain using aniline blue, what colour are collagen fibres?

   1. ABlue
   2. BRed
   3. CYellow
   4. DBlack

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7. Question 7 · Hard

   After coverslipping in DPX, many H&E slides look milky, and tiny droplets blur the detail at high power. The problem is worst on humid monsoon days, and the last absolute alcohol before xylene has not been changed for two weeks. The most likely cause is:

   1. AMountant too thin and spread unevenly
   2. BAir bubbles trapped under the coverslip
   3. CEosin overstained and poorly differentiated
   4. DWater carried into the xylene and mountant

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8. Question 8 · Easy

   Which cells does the Ki-67 antigen, detected in paraffin sections with the MIB-1 antibody, mark?

   1. AUndergoing apoptosis
   2. BOf epithelial origin
   3. CResting in the G0 phase
   4. DIn the active cell cycle

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9. Question 9 · Hard

   A mastectomy for invasive breast carcinoma reached the laboratory late on Friday and was kept unfixed in a refrigerator until Monday, when it was sliced into formalin. ER immunostaining shows: tumour cells negative; normal breast ducts in the section negative; on-slide external positive control stained as expected. The best interpretation is:

   1. ATrue ER-negative tumour; report as negative
   2. BER result unreliable because of delayed fixation
   3. CValid result; normal ducts lack ER expression
   4. DRun failure; repeat with a new antibody lot

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10. Question 10 · Easy

    Haematoxylin itself is almost colourless. Which coloured compound, formed when haematoxylin is oxidised, actually stains nuclei?

    1. ABrazilin
    2. BCarminic acid
    3. CHaematein
    4. DOrcein

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11. Question 11 · Medium

    Under polarised light, amyloid stained with Congo red shows apple-green birefringence. This is because:

    1. ACongo red fluoresces green in polarised light
    2. BCollagen around the deposit is itself birefringent
    3. CCongo red stains amyloid metachromatically
    4. DDye molecules align in order along the fibrils

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12. Question 12 · Medium

    In silver impregnation methods for reticulin, what does the gold chloride toning step do?

    1. AReduces silver salts to visible metallic silver
    2. BReplaces silver with gold for a stable black
    3. CRemoves unreduced silver from the section
    4. DOxidises the fibres so they take up more silver

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13. Question 13 · Hard

    A substance in a gastric biopsy from a 45-year-old man gives these results: PAS strongly positive; PAS after diastase digestion still positive; alcian blue pH 2.5 negative; alcian blue pH 1.0 negative. The substance is most likely:

    1. AGlycogen
    2. BSialomucin
    3. CNeutral mucin
    4. DSulphated mucin

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14. Question 14 · Medium

    Why does eosin Y stain cytoplasm, collagen and red cells?

    1. AA mordant dye that binds through an aluminium lake
    2. BA cationic dye attracted to nucleic acid phosphates
    3. CA metachromatic dye that changes colour on binding
    4. DAn anionic dye attracted to protonated amino groups

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15. Question 15 · Medium

    A neck lymph node from a 60-year-old man contains a poorly differentiated tumour that is CD45 (leucocyte common antigen) positive, cytokeratin negative and S-100 negative. What is the tumour most likely to be?

    1. ACarcinoma
    2. BLymphoma
    3. CGerm cell tumour
    4. DMelanoma

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16. Question 16 · Hard

    H&E sections from today's run show dark blue-black granules over the tissue and the bare glass. At high power the granules lie in a different focal plane from the tissue. The haematoxylin has not been filtered this week and has a metallic sheen on its surface. The best corrective action is to:

    1. AExtend the dewaxing time in xylene
    2. BExtend the time in acid alcohol
    3. CFilter the haematoxylin before use
    4. DTreat sections with alcoholic picric acid

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17. Question 17 · Easy

    On which type of section are lipid stains such as Oil Red O and Sudan black B normally used?

    1. ACelloidin-embedded sections
    2. BFrozen sections of formalin-fixed tissue
    3. CPlastic (resin) sections
    4. DRoutine paraffin wax sections, dewaxed in xylene

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18. Question 18 · Easy

    A permanent resinous mountant should have a refractive index close to that of glass and fixed tissue, which is about:

    1. A1.47
    2. B1.43
    3. C1.33
    4. D1.52

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19. Question 19 · Medium

    In the Ziehl-Neelsen stain, carbol fuchsin is heated on the section or left on for a long time. Why?

    1. ATo drive the dye through the waxy cell wall
    2. BTo increase the uptake of the counterstain
    3. CTo kill the bacilli before they are stained
    4. DTo make the bacilli resistant to the acid

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20. Question 20 · Medium

    When toluidine blue stains mast cell granules red-purple while nuclei stay blue, the colour change is caused by:

    1. AStacking of dye molecules on closely spaced anions
    2. BOxidation of the dye by enzymes in the granules
    3. CAn aluminium mordant bound to the heparin
    4. DA second red dye that is present in the staining solution

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21. Question 21 · Easy

    Scott's tap water substitute, used to blue haematoxylin, contains:

    1. ALithium carbonate together with sodium nitrite
    2. BSodium bicarbonate and magnesium sulphate
    3. CAmmonium oxalate and potassium alum
    4. DSodium chloride and calcium chloride

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22. Question 22 · Easy

    In breast carcinoma, nuclear staining of tumour cells for oestrogen receptor predicts benefit from:

    1. ATamoxifen (endocrine therapy)
    2. BTrastuzumab (anti-HER2 therapy)
    3. CImatinib (KIT inhibitor therapy)
    4. DRituximab (anti-CD20 therapy)

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23. Question 23 · Medium

    Why has Canada balsam been replaced by synthetic resins such as DPX in most laboratories?

    1. AIt dissolves eosin and must be used wet
    2. BIt turns acidic and yellow, fading stains
    3. CIts refractive index is far below that of glass
    4. DIt is water-based and cannot hold coverslips

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24. Question 24 · Hard

    A liver biopsy from a 40-year-old man with emphysema and liver cirrhosis shows round eosinophilic globules in periportal hepatocytes. The globules are PAS positive and remain PAS positive after diastase. Which condition does this most likely indicate?

    1. AGlycogen storage disease
    2. BHaemochromatosis
    3. CWilson disease
    4. DAlpha-1 antitrypsin deficiency

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25. Question 25 · Easy

    In the PAS-diastase method, the diastase applied to the control section is often obtained from:

    1. AHyaluronidase from testis
    2. BPancreatic trypsin
    3. CBacterial collagenase
    4. DHuman saliva

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