Fixation, tissue processing and sectioning

45 Lab technician recruitment practice questions on Fixation, tissue processing and sectioning, part of Histopathology and Cytology. 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 · Medium

   Biconcave microtome knives were designed mainly for cutting:

   1. AResin sections for electron microscopy
   2. BCelloidin sections on a sliding microtome
   3. CParaffin sections on a rocking microtome
   4. DFrozen sections in a cryostat

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   Answer: C · Paraffin sections on a rocking microtome

   Both faces of a biconcave knife are hollow-ground, giving a thin edge suited to soft paraffin blocks on the rocking (Cambridge) microtome. Plano-concave knives were used for celloidin on sliding microtomes, and plane-wedge knives suit harder blocks and cryostats. Glass and diamond knives are used for resin ultrathin sections. Disposable blades have largely replaced all steel knives for paraffin.

   • A: Resin sections for electron microscopy need glass or diamond knives.
   • B: Celloidin was cut with plano-concave knives on sliding microtomes.
   • D: Cryostats use plane-wedge knives or disposable blades.

2. Question 2 · Medium

   A muscle biopsy for enzyme histochemistry is mounted on the cryostat chuck so that the fibres run:

   1. ACoiled, so a long length is sampled
   2. BFlat, so sections run along the fibres
   3. CUpright, so sections cut across the fibres
   4. DObliquely, so both views appear in one section

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   Answer: C · Upright, so sections cut across the fibres

   Muscle diagnosis depends on transverse sections, where fibre size, shape, central nuclei and fibre types can be judged. The block is oriented under a lens so that the fibres stand perpendicular to the chuck surface, often on cork with a mounting medium. It is then snap frozen in chilled isopentane. Oblique fibres give false variation in fibre size.

   • A: Coiling is used for bowel strips, not for muscle.
   • B: Longitudinal sections hide differences in fibre size and the checkerboard of fibre types.
   • D: Oblique sections distort fibre diameter and mislead the reader.

3. Question 3 · Medium

   Paraffin infiltration on a processor uses two or three changes of wax mainly to:

   1. ACool the tissue slowly before embedding
   2. BRaise the melting point of the wax step by step
   3. CLet any remaining fixation finish
   4. DWash out the clearing agent carried over

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   Answer: D · Wash out the clearing agent carried over

   Each basket carries clearing agent such as xylene into the first wax bath. Xylene left in the wax softens it, so blocks become soft and crumbly. Successive changes dilute it out so that the last bath holds nearly pure wax. The first wax bath is therefore changed most often.

   • A: The baths are all kept just above the melting point; cooling happens at embedding.
   • B: All the wax baths use the same grade of paraffin.
   • C: Fixation is finished before dehydration; wax does not fix tissue.

4. Question 4 · Medium

   Frozen sections offer each of these over paraffin sections EXCEPT:

   1. AEnzyme activity well preserved for histochemistry
   2. BFat kept in the tissue for staining
   3. CA result within minutes during surgery
   4. DBetter nuclear detail than paraffin sections

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   Answer: D · Better nuclear detail than paraffin sections

   Frozen sections are fast, keep lipids that processing would dissolve, and preserve enzymes and many antigens. Their weakness is morphology: ice crystals, thicker sections and uneven staining give poorer nuclear detail than paraffin sections. This is why every frozen section is followed by paraffin sections of the same tissue. Intraoperative reports are therefore limited to what changes the operation.

   • A: Most enzymes are destroyed by fixation and heat, so frozen sections are needed.
   • B: Fat is lost in alcohol and xylene, so it is shown only in frozen sections.
   • C: Speed is the main reason frozen sections are used during surgery.

5. Question 5 · Medium

   Small biopsies left for many hours in absolute ethanol on a stalled processor tend to cut as:

   1. AMilky tissue with white chalky patches
   2. BSoft, mushy and smeared tissue
   3. CSwollen tissue with pale nuclei
   4. DHard, brittle and crumbly tissue

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   Answer: D · Hard, brittle and crumbly tissue

   Absolute ethanol removes bound water as well as free water when left too long, so tissue shrinks and hardens. Brittle tissue crumbles, chatters or cracks at the microtome. Small biopsies suffer most because they reach equilibrium quickly. Processing schedules are matched to tissue size to avoid this.

   • A: Milky patches show water left in the tissue, the opposite fault.
   • B: Soft, mushy tissue points to too little dehydration or poor wax infiltration.
   • C: Ethanol shrinks tissue; it does not swell it.

6. Question 6 · Medium

   Adding about 0.1% urea to aqueous nitric acid decalcifier helps to:

   1. ASpeed the removal of calcium
   2. BPrevent yellowing caused by nitrous acid
   3. CNeutralise the acid after use
   4. DProtect enzymes in the bone

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7. Question 7 · Medium

   A gallbladder arrives in normal saline instead of formalin, 2 hours after surgery. What should the laboratory do?

   1. AReturn it to theatre as an unacceptable specimen
   2. BFreeze it and store it until reported
   3. CProcess it directly from the saline
   4. DOpen it and fix it in formalin at once

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8. Question 8 · Easy

   Perenyi's fluid, a slow decalcifier that also softens tissue, contains nitric acid, chromic acid and:

   1. AEDTA
   2. BFormic acid
   3. CPicric acid
   4. DEthanol

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9. Question 9 · Hard

   A processor's delayed start failed, so endoscopic biopsies stayed in the formalin station over the weekend (about 60 hours) before processing. Sections look normal. What is the main effect to expect?

   1. ASome antigens may need longer epitope retrieval
   2. BThe tissue will show severe autolysis
   3. CFormalin pigment will appear over all the sections
   4. DThe blocks will be soft and crumbly

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10. Question 10 · Hard

    A bone slice of about 15 mL is decalcified in 20 times its volume of 10% formic acid, changed daily for 4 days. Taking the stock as 100% formic acid, how much stock is used in all?

    1. A300 mL
    2. B30 mL
    3. C120 mL
    4. D1,200 mL

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11. Question 11 · Medium

    Celloidin embedding is chosen for whole eyes and large brain sections mainly because it:

    1. AIs finished overnight like routine paraffin
    2. BKeeps tissue enzymes active for histochemistry
    3. CGives thin ribbons quickly on a rotary microtome
    4. DNeeds no heat, so tissue shrinks very little

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12. Question 12 · Hard

    A tooth decalcified in formic acid gives good sections of dentine and pulp, but no enamel is seen at all. Why?

    1. AThe block was faced too deeply before cutting
    2. BEnamel was lost because fixation was too short
    3. CEnamel is almost all mineral and dissolves away
    4. DThe acid was too weak to soften the enamel

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13. Question 13 · Hard

    In a breast lumpectomy, fibrous areas and lymph nodes cut well, but fatty areas are mushy, smear and fall out of the sections. Other tissues on the same run are fine. What is the best change?

    1. ASlice fatty tissue thinner and extend dehydration and clearing
    2. BFix the fatty tissue in Carnoy's fluid
    3. CCool the blocks longer before cutting
    4. DRaise the paraffin oven temperature by 10 °C

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14. Question 14 · Easy

    Carnoy's fluid (6:3:1) contains absolute ethanol, glacial acetic acid and:

    1. AMercuric chloride
    2. BChloroform
    3. CFormalin
    4. DPicric acid

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15. Question 15 · Medium

    All of the following are needed in a clearing agent for paraffin processing EXCEPT:

    1. AMiscibility with ethanol
    2. BMiscibility with water
    3. CLittle hardening of the tissue
    4. DAbility to dissolve paraffin wax

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16. Question 16 · Hard

    A lung lobe for a small peripheral nodule was put intact into formalin. Next day the lung is collapsed, its centre is soft and poorly fixed, and the nodule is hard to find. What should be done for the next such specimen?

    1. AFreeze it before slicing
    2. BInflate it with formalin through the bronchus
    3. CUse a larger volume of formalin
    4. DFix it in Bouin's fluid

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17. Question 17 · Medium

    Many rotary microtomes retract the block on the return stroke in order to:

    1. AFeed the ribbon onto the water bath
    2. BChange the section thickness on every turn of the wheel
    3. CStop the block face rubbing on the knife
    4. DCool the block between sections

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18. Question 18 · Easy

    A white sediment forms at the bottom of a stock formalin bottle kept in a cold store. The sediment is:

    1. AParaformaldehyde
    2. BFormic acid
    3. CSodium phosphate
    4. DCalcium carbonate

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19. Question 19 · Medium

    Sections floated on the water bath stay wrinkled and do not flatten out. The most likely cause is that the bath is:

    1. ABelow its correct temperature
    2. BAbove the wax melting point
    3. CMissing an albumin adhesive
    4. DFilled with fresh distilled water

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20. Question 20 · Hard

    Six gastric biopsy fragments are embedded in one block. The first level shows only three fragments; deeper levels show the other three, but by then the first three have been cut away. What should be done next time?

    1. ACut thicker sections to reach all fragments
    2. BFace the block much deeper before cutting
    3. CPress all fragments flat to the mould floor together
    4. DEmbed each fragment upright on its edge

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21. Question 21 · Easy

    A vibrating-blade microtome (vibratome) is useful because it cuts:

    1. ALarge undecalcified bone blocks in resin
    2. BCelloidin blocks with an oblique knife
    3. CUltrathin sections of resin-embedded tissue
    4. DFresh or fixed tissue without freezing or embedding

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22. Question 22 · Easy

    Liquid nitrogen, used to cool isopentane for snap freezing, has a temperature of about:

    1. A-20 °C
    2. B-78 °C
    3. C-40 °C
    4. D-196 °C

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23. Question 23 · Medium

    Fresh tissue for frozen section is blotted free of saline before freezing because surface fluid:

    1. AFixes the tissue surface unevenly
    2. BRaises the temperature needed for cutting
    3. CFreezes into ice that cuts poorly and splits from the tissue
    4. DDissolves the embedding medium completely

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24. Question 24 · Medium

    How much paraformaldehyde powder is needed to make 500 mL of 4% (w/v) paraformaldehyde fixative?

    1. A40 g
    2. B20 g
    3. C2 g
    4. D200 g

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25. Question 25 · Easy

    Fresh renal or skin biopsies for immunofluorescence that cannot be frozen at once are best sent in:

    1. AMichel's transport medium
    2. BBouin's fluid
    3. CNormal saline
    4. DNeutral buffered 10% formalin

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