Techniques

Question 1

Additive fixatives

Answer 1

Cross-linking, e.g. formaldehyde or glutaraldehyde - bind amino acids within proteins

Question 2

Formaldehyde mechanism

Answer 2

Formaldehyde + water = methylene glycol (MG) -> methylene bridges between protein groups such as amines

Question 3

Non-additive fixatives

Answer 3

Precipitating, e.g. (m)ethanol, acetone - remove water from ECM to precipitate protein structure

Question 4

Additive vs non-additive fixatives

Answer 4

Additive cannot be used for enzyme histochemistry as they denature enzymes, precipitating fixatives have better antigen/enzyme preservation but may cause shrinking/swelling
Glutaraldehyde has less penetration than neutral formalin/NBF but excellent ultrastructure preservation for EM

Question 5

Specimen categories

Answer 5

Cat. A - Simple transfer/biopsy
Cat. B - Representative sampling across a lesion/area (simple lipoma, unremarkable tonsils, prostatic chippings)
Cat. C - Complex targeted sampling in select areas
Cat. D/E - Complex resections including multi-organ malignant lesions

Question 6

Fixatives for bone/tooth samples

Answer 6

Fixation is essential - damage from acid is 4x greater in unfixed tissue
Teeth - 15% formic acid prefferred
Bone marrow - Zenker formol
Routine - Formal-saline
Bone - Can use Mullers fluid with {3% formic acid + formaldehyde} as decalcifying agent

Question 7

Acidic decalcification of bone/tooth samples

Answer 7

Acid hydrolysis - 10 % HNO3, 10% Formal nitric acid (FNA), 10 % Formic acid, 8% Potassium formate (KF) + 8% Formic acid (FA)
Forms soluble calcium salts (and oxalates?) from insoluble phosphates and carbonates
Faster but more damaging
Weak acids like formic, acetic, picric, or Gooding & Stewart solution may be optimal
For conc. nitric acid (fastest fixative), add urea to remove yellow discolouration due to formation of nitrous acid in the tissue
Formic acid + 10% formalin solution can be used to simultaneously fix and decalcify small bone pieces or needle biopsies

Question 8

Other (non-acid) decalcification techniques

Answer 8

EDTA chelation to form complexes with calcium - slower but less damaging when used alone, preserves nucleic acids and polysaccharides
EDTA, electrolysis (carbon cathode, bone on platinum anode, acidic electrolyte), or ion exchange resin can be used to remove solvated Ca ions from the decalc. solution - speeds up the decalc. process, preserves cellular detail better, and solutions no longer have to be changed daily

Question 9

Tests for decalcification

Answer 9

Physical - Bending, needling and probing at the cervical area of tooth [or bone?] using a fine needle or probe every day
Chemical - Calcium oxalate test - Precipitation of insoluble Ca (OH)2or calcium oxalate in decalcifying solution whenever solution is changed
Radiographic - “Radiolucency”

Question 10

Slide preparation for bone samples

Answer 10

Chrome-gelatine slides used, and floating water bath may be set to higher temperature as bone tends to crinkle when cut
Non-decalcified sections (mineralised:unmineralized bone ratio in metabolic bone disorders) - Can use adhesive tape method + Von-kossa technique, block impregnation method or resin embedded sections

Question 11

Tape transfer method (bone)

Answer 11

“Tape transfer systemscan allow you to remove good sections when your tissue is very friable, such as when fixation or processing are faulty. Apply special tape to the exposed surface of your embedded tissue and make one section; then carefully place the tape, tissue side down, onto a charged slide and cure the tape with UV light before immersing the slide in solvent to remove tape.”

Question 12

Von kossa technique, and block impregantion (bone)

Answer 12

Calcium phosphate + Silver nitrate -> Silver phosphate + calcium nitrate
Silver phosphate -> Metallic silver (Light)
Block impregnation - Modified von Kossa, with a reducer solution (sodium hypophosphite, sodium hydroxide, dis. water)
Mineralized bone - Black
Osteoid seams - Red

Question 13

HER2 Assessment

Answer 13

Presence - IHC
Overexpression - FISH - Organge probe for CEP17 housekeeping gene, green probe for HER2
Ratio >2.2 = HER2 amplified

Question 14

Cryotomy

Answer 14

OCT (optimal cutting temperature) embedding medium used after freezing - A mix of water-soluble glycols & resins, also helps to bond the sample to the cryotome chuck
Cryotomy sections are not ribboned, and a water bath is not used - instead the cryostat roll-bar induces an electrostatic charge in the sections, allowing them to be picked up onto glass slides one-by-one (they ‘jump’ onto the slides)

Question 15

Staining - Dewaxing and de/rehydration

Answer 15

De-wax (hot plate -> xylene -> rehydrate (graded IMS/alcohol -> water)) -> Stain -> Dehydrate (alcohol) -> Clear (xylene) -> Resinous mounting media (usually DPX)

Question 16

IHC - Avidin/biotin + HRP method

Answer 16

All IHC antibodies have to be optimised for concentration/titre and incubation time
Secondary antibody with biotin added alongside avidin - complexes with biotin, and HRP binds to the complex
HRP converts soluble DAB to a brown insoluble form
CuSO4 can be used to enhance the brown colour
Endogenous peroxidases have to be blocked first as well to prevent non-specific staining - for example, saturating amounts of peroxide can be used (like a solution of 3% H2O2 in methanol)
AEC can be used as alternative red chromogen

Question 17

EM - Applications

Answer 17

Needed for ~70% renal pathology diagnoses to visualise glomeruli and BMs in detail
BM thickness must be measured by EM for diagnosis of thin BM diseases
Also required for muscle/skin/peripheral nerve pathologies
Can be used to confirm and classify epidermolysis bullosa

Question 18

Epidermolysis bullosa - Classified via EM

Answer 18

EB simplex - Separation above the basement membrane, in the cytoplasm of the lower basal cell layer
Junctional EB - Separation in the lamina lucida within the basement membrane
Dystrophic EB - Separation below basement membrane, in anchoring fibrils of the upper dermis

Question 19

Enzyme histochemistry - Dehydrogensase technique

Answer 19

Substrates (e.g. sodium succinate or sodium L-lactate) oxidized after cleavage by the dehydrogenase -> Stoichiometric colour indicator tetranitrotetrazolium chloride blue (TNBT) reduced to blue/black formazan

Question 20

Enzyme histochemistry - Esterases and phosphatases

Answer 20

Uses diazonium salts - Naphthyl salt with ester or phosphate group gets this group cleaved off by the enzyme, and then the rest of the (now oxidized) naphthyl salt couples to the diazo-salt to produce a coloured compound.

Question 21

Enzymes which survive FFPE and their functions

Answer 21

Chloroacetate esterase - Myelopoiesis (production of leukocytes in blood) - Bad prognosis in PMLeukaemia if low activity in BM smears
Tartrate-resistant phosphatase - Osteoclastogenesis - Visualization of osteoclasts in bone biopsies and osteopetrosis (disorders of abnormally high bone density) after bone marrow transplantation

Question 22

Enzyme histochemistry - Requirements (cryotomy, thickness)

Answer 22

Formalin fixation stops EH reactions - So can be used to make EH-stained slides permanent, but also means that cryotomy is required in most cases - Frozen with dry ice or -25C isopentane (avoids cracking), then can be stored at -25C without loss of enzyme activity

Sections should be cut at 15 um thickness to overcome minimal enzyme activity required to start reactions - after thawing, spreading, and drying, it becomes 4 um thick

Question 23

Enzyme histochemistry - ALP applications

Answer 23

Liver injury - ALP eliminated in bile, so increase/accumulation -> Impaired secretion, likely toxic lesions infectious hep. or congestion (test alongside SDH/LDH)

Also represents tissue barrier functions in:
Brain capillaries - Focally lower activity shows damage to BBB, e.g. in boxer’s trauma/CTE or contusio cerebri (lower in cerebellar purkinje cells and brain capillaries)
Duodenal enterocyte brush borders - Used to diagnose coeliac disease - High in young adults, low in post-weaning children - Malabsorption syndromes show ALP loss in EBB, reappearance = ‘compensated’ syndrome
Prox. kidney tubule brush borders - Absorption of metabolites from 1’ary urine -> Sensitive to toxins and ischemia

Question 24

Enzyme histochemistry - SDH/LDH applications

Answer 24

Cancer - Focal decrease in LDH after radiotherapy for breast cancer, low SDH/LDH activity shows low vitality in tumour tissue, so effective therapy (because tumours exhibit aerobic glycolysis)
Craniocerebral trauma and transient ischemia hard to prove with H&E, but easily shown with LDH and SDH activity decrease in Purkinje cells of the cerebellum
ALP and SDH show vitality in kidney tissues after injury
Also - Ischaemia in peracute MI

Question 25

Enzyme histochemistry - AChE applications

Answer 25

Gold standard for diagnosing Hirschsprung’s disease (congenital colonic denervation -> chronic constipation) - Via increased AChE activity in parasympathetic nerve fibres of muscularis and lamina propria mucosae

Question 26

Enzyme histochemistry - Other enzyme applications

Answer 26

Inc. in lysosomal acid phosphatase -> Early marker in ischemic tissue lesions
LDH, SDH - Muscle neuropathology

Question 27

Compare the different types of in situ hybridization

Answer 27

FISH allows HER2 amplification to be distinguished from polysomy 17, and for deletion and rearrangement to be detected at the same time - CISH does not, but otherwise comparable results
However, CISH-stained slides are permanent and FISH is not - though images can be captured for later analysis
mRNA ISH is also possible, and may be cheaper, easier and less toxic - BUT limited by poor RNA stability

Question 28

In situ hybridization - Unique requirements

Answer 28

Requires heat induced or protease-mediated decrosslinking of DNA, after the deparraffinization/rehydration step
Hybridization buffers allow significant shortening of hybridization time

Question 29

Types of fish probes (ADD DETAIL LATER)

Answer 29

Locus-specific, dual fusion (two targets - e.g. normal and with deletion), or break apart (differently labelled 3’ and 5’ ends)

Question 30

IHC - Examples of CD markers

Answer 30

CD2 on T cells and NK cells
CD9 on endothelial/epithelial cells and macrophages
CD10 on stem cells and epithelial cells
CD16 NK/stem/macrophage/granulocyte
CD19 b cell, dendritic, stem (mainly B cell)

Question 31

FISH - Clinical value in cancers

Answer 31

Lung cancer (ALK, ROS1) / HER2 breast cancer / CCNE1 ovarian cancer - Prognostic value, but not diagnostic (must use other tests as well)
Glioma (1p19q co-deletion) / EWS (EWSR1) / Synovial sarcoma (SS18) - Prognostic AND diagnostic