Histotechnology: reception, fixation and decalcification

68 Kerala MSc MLT Entrance practice questions on Histotechnology: reception, fixation and decalcification, 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 · Medium

   A renal biopsy for light microscopy, immunofluorescence and electron microscopy arrives in a single container of formalin. Which study can no longer be done by the standard method?

   1. ASilver (Jones) stain for basement membranes
   2. BDirect immunofluorescence on frozen sections
   3. CCongo red stain for amyloid
   4. DH&E and PAS for light microscopy

   Show answerHide answer

   Answer: B · Direct immunofluorescence on frozen sections

   Routine direct immunofluorescence for immunoglobulins and complement is done on cryostat sections of unfixed tissue, because formalin cross-linking masks many of these antigens. The fresh cores should have been divided at once, with the immunofluorescence portion sent in Michel's transport medium and the electron microscopy portion in glutaraldehyde. Light microscopy stains, including PAS, silver and Congo red, work well on formalin-fixed paraffin sections. Protease-digested paraffin sections can sometimes rescue immunofluorescence, but they are less sensitive.

   • A: Silver methenamine stains basement membranes well after formalin fixation.
   • C: Congo red works on formalin-fixed paraffin sections.
   • D: H&E and PAS are routine stains on formalin-fixed paraffin sections.

2. Question 2 · Medium

   Tissue for electron microscopy in 2.5% glutaraldehyde is cut into cubes no more than about 1 mm across. Why?

   1. AGlutaraldehyde dissolves membrane lipids
   2. BSmall cubes do not need post-fixation
   3. CGlutaraldehyde penetrates tissue very slowly
   4. DLarger blocks will not fit the ultramicrotome

   Show answerHide answer

   Answer: C · Glutaraldehyde penetrates tissue very slowly

   Glutaraldehyde has two aldehyde groups and cross-links proteins rapidly and strongly, giving excellent ultrastructure, but it penetrates only a fraction of a millimetre in a reasonable time. In larger pieces the centre autolyses before the fixative reaches it. Tissue is therefore minced into cubes of about 1 mm, fixed cold in buffered glutaraldehyde and post-fixed in osmium tetroxide to preserve lipids. It should be placed in fixative within minutes of removal.

   • A: Glutaraldehyde does not dissolve lipids, though it fixes them poorly.
   • B: Post-fixation in osmium tetroxide is needed whatever the size.
   • D: Ultramicrotome blocks are trimmed later; the size limit is set by penetration.

3. Question 3 · Easy

   Formaldehyde fixes tissue mainly by:

   1. AForming methylene bridges between proteins
   2. BOxidising unsaturated lipids
   3. CForming metal-protein complexes
   4. DCoagulating and precipitating the tissue proteins

   Show answerHide answer

   Answer: A · Forming methylene bridges between proteins

   Formaldehyde reacts with reactive side groups of proteins, mainly the amino group of lysine, first adding on and then forming methylene (-CH2-) bridges between neighbouring molecules. The cross-linked network preserves structure with little shrinkage. The same cross-links can mask antigens, which is why heat-induced epitope retrieval is used in immunohistochemistry. Alcohols, by contrast, fix by coagulation.

   • B: Oxidising and blackening lipids is the action of osmium tetroxide.
   • C: Metal-protein complexes form with mercuric chloride fixatives.
   • D: Coagulation and precipitation is how alcohols and acetone fix.

4. Question 4 · Medium

   Formalin pigment forms in tissue when:

   1. AFormalin oxidises haemosiderin in old haemorrhages
   2. BFormalin reacts with bilirubin in jaundiced tissue
   3. CAcidic formalin acts on haemoglobin in blood-rich tissue
   4. DBuffered formalin acts on melanin in heavily pigmented tissue

   Show answerHide answer

   Answer: C · Acidic formalin acts on haemoglobin in blood-rich tissue

   Formalin slowly oxidises to formic acid, and below about pH 6 formaldehyde reacts with haem from haemoglobin to form acid formaldehyde haematin. The brown-black, birefringent granules lie over red cell-rich areas such as spleen, haemorrhages and congested organs, and they increase with long fixation. Buffering formalin to about pH 7 (neutral buffered formalin) prevents it. The pigment is Perls-negative, which separates it from haemosiderin.

   • A: Haemosiderin is a separate, iron-positive pigment present before fixation.
   • B: Bilirubin colours tissue yellow-green; it does not form formalin pigment.
   • D: Melanin is not changed into a pigment by buffered formalin.

5. Question 5 · Medium

   Glacial acetic acid is added to compound fixatives such as Bouin's fluid mainly because it:

   1. APreserves cytoplasmic lipids and granules
   2. BPrevents the formation of formalin pigment
   3. CSwells tissue and offsets shrinkage by other agents
   4. DCross-links proteins more strongly than formalin does

   Show answerHide answer

   Answer: C · Swells tissue and offsets shrinkage by other agents

   Acetic acid swells collagen and other tissue and precipitates nucleoproteins, so nuclei are sharply fixed. Alcohol and picric acid shrink tissue, and the acid's swelling balances their effect in Bouin's, Carnoy's and Zenker's fluids. It does not fix cytoplasmic proteins, it dissolves some cytoplasmic granules and it lyses red cells. That is why Helly's fluid leaves it out.

   • A: Acetic acid damages cytoplasmic granules and lyses red cells.
   • B: Formalin pigment is prevented by buffering formalin to neutral pH.
   • D: Acetic acid does not cross-link proteins; it precipitates nucleoproteins.

6. Question 6 · Hard

   A fresh, unfixed cervical lymph node 2.5 cm across, from a 30-year-old man with suspected lymphoma, reaches specimen reception at 11 am. What is the best way to handle it?

   1. AFreeze the whole node for frozen-section diagnosis
   2. BFix the whole node at once in neutral buffered formalin
   3. CSlice it, send fresh tissue for flow cytometry, fix the rest
   4. DFix the whole node in Bouin's fluid for nuclear detail

   Answer and explanation in LearnFree account

7. Question 7 · Easy

   Which fixative both preserves and blackens unsaturated lipids, making it useful in electron microscopy?

   1. AMercuric chloride
   2. BPotassium dichromate
   3. COsmium tetroxide
   4. DGlutaraldehyde

   Answer and explanation in LearnFree account

8. Question 8 · Hard

   A lymph node fixed in B-5 (mercuric chloride-formalin) gives H&E sections with coarse, dark brown-black granules scattered randomly over the tissue, including areas with no red cells. Which treatment of the sections before staining removes them?

   1. APotassium permanganate and oxalic acid
   2. BIodine followed by sodium thiosulphate
   3. C1% acid alcohol for 10 minutes
   4. DSaturated alcoholic picric acid

   Answer and explanation in LearnFree account

9. Question 9 · Easy

   Helly's fluid differs from Zenker's fluid in that it contains formalin in place of:

   1. APotassium dichromate
   2. BSodium sulphate
   3. CMercuric chloride
   4. DGlacial acetic acid

   Answer and explanation in LearnFree account

10. Question 10 · Hard

    To save time, a fresh femoral head slice was put into 10% formic acid within 20 minutes of surgery, without prior fixation. The used acid later tested negative for calcium, and the slice was washed in running water before processing. Sections show smudged, poorly stained nuclei and swollen, frayed marrow and cartilage. What is the most likely cause?

    1. ADecalcification started before the tissue was fixed
    2. BDecalcification was stopped before its true end point
    3. CThe slice was not neutralised before processing
    4. DFormic acid is too weak for femoral head bone

    Answer and explanation in LearnFree account

11. Question 11 · Medium

    The end point of decalcification cannot be checked by radiography if the bone was fixed in:

    1. ANeutral buffered formalin
    2. BZenker's fluid
    3. CBouin's fluid
    4. DFormal-alcohol

    Answer and explanation in LearnFree account

12. Question 12 · Medium

    Tissue fixed in Bouin's fluid is moved straight to 70% ethanol rather than being washed in water, because water:

    1. APrecipitates mercury pigment
    2. BDissolves the soluble protein picrates
    3. CTurns the residual picric acid explosive
    4. DReverses formaldehyde cross-links

    Answer and explanation in LearnFree account

13. Question 13 · Hard

    Penetration of a fixative follows d = K√t (d in mm, t in hours). Taking K = 0.78 for formaldehyde, about how long does formalin need to reach the centre of a 6 mm thick tissue slice that it enters from both faces?

    1. AAbout 59 hours
    2. BAbout 8 hours
    3. CAbout 4 hours
    4. DAbout 15 hours

    Answer and explanation in LearnFree account

14. Question 14 · Medium

    A lymph node block cuts well except for a few small, gritty calcified foci that tear the sections. The usual remedy is to:

    1. ADecalcify the block for 3 days in EDTA solution
    2. BRe-process the whole block from absolute ethanol
    3. CSoak the block face briefly in acid, then recut
    4. DCut the block at a section thickness of 15 µm

    Answer and explanation in LearnFree account

15. Question 15 · Medium

    Glutaraldehyde preserves ultrastructure better than formaldehyde mainly because it:

    1. ADissolves lipids that blur fine structure
    2. BPenetrates much faster into the centre of the tissue
    3. CHas two aldehyde groups and forms more cross-links
    4. DContains osmium, which stains membranes

    Answer and explanation in LearnFree account

16. Question 16 · Medium

    Zinc formalin is often used in place of B-5 for lymph nodes, mainly to:

    1. APrevent formalin pigment in blood-rich tissue
    2. BAvoid mercury while keeping good nuclear detail
    3. CSpeed up penetration into the tissue
    4. DAllow electron microscopy on the same block

    Answer and explanation in LearnFree account

17. Question 17 · Hard

    A liver biopsy from a 4-year-old with hepatomegaly and fasting hypoglycaemia is sent for suspected glycogen storage disease. A PAS stain with diastase control is planned. Which fixation of one core is best for showing the glycogen?

    1. ANormal saline overnight, then formalin
    2. BZenker's fluid with later washing
    3. CAn alcohol-based fixative such as Carnoy's
    4. DGlutaraldehyde for 24 hours

    Answer and explanation in LearnFree account

18. Question 18 · Easy

    A specimen sent for intraoperative frozen section should reach the laboratory:

    1. AFrozen in a domestic freezer
    2. BFresh, without any fixative
    3. CIn 70% ethanol
    4. DIn 10% neutral buffered formalin

    Answer and explanation in LearnFree account

19. Question 19 · Medium

    Some laboratories keep marble chips (calcium carbonate) in stock formalin bottles. Their purpose is to:

    1. AMake the solution isotonic with tissue
    2. BPrevent polymerisation to paraformaldehyde
    3. CNeutralise formic acid that forms on standing
    4. DAbsorb water and keep the formalin strong

    Answer and explanation in LearnFree account

20. Question 20 · Medium

    Acetic acid is left out of fixatives meant to preserve mitochondria, such as Regaud's fluid, because it:

    1. APrevents the tissue from being decalcified
    2. BTurns the tissue yellow and brittle
    3. CForms a black pigment with dichromate
    4. DDestroys mitochondria and dissolves lipids

    Answer and explanation in LearnFree account

21. Question 21 · Medium

    A breast core biopsy was left in formalin for only 1 hour before going onto the overnight processor. What is the main risk for its ER immunostaining?

    1. AStronger staining, giving a false positive
    2. BWeak or patchy staining, giving a false negative
    3. CNo effect, since processing completes fixation
    4. DLoss of tissue from the slide during staining

    Answer and explanation in LearnFree account

22. Question 22 · Hard

    A container labelled 'gastric biopsies x 3' arrives at reception. The technician sees only two fragments floating in the formalin. What is the best action?

    1. APour off the formalin and process the two pieces received
    2. BRecord three pieces as stated on the label
    3. CFilter the fixative, check the lid, and record the count
    4. DAsk the endoscopist for a new biopsy

    Answer and explanation in LearnFree account

23. Question 23 · Medium

    Formalin is added to Helly's fluid only immediately before use because:

    1. AIt evaporates out of the mixture
    2. BIt makes the fluid too acidic
    3. CIt reacts with the dichromate on standing
    4. DIt precipitates the mercuric chloride

    Answer and explanation in LearnFree account

24. Question 24 · Medium

    Adding an ion-exchange resin to formic acid speeds decalcification because the resin:

    1. ARaises the acidity of the solution
    2. BWarms the solution by its reaction
    3. CChelates calcium within the bone crystals
    4. DTakes up calcium ions released into the acid

    Answer and explanation in LearnFree account

25. Question 25 · Medium

    Tissue for chromosome analysis, such as products of conception, should be sent to the laboratory:

    1. AIn 10% neutral buffered formalin
    2. BUnfixed, in sterile saline or medium
    3. CFrozen dry at -20 °C
    4. DIn fresh 3:1 methanol-acetic acid fixative

    Answer and explanation in LearnFree account