Stains and diagnosis

Question 1

Congo red

Answer 1

Stains amyloid red

Question 2

Alcian blue

Answer 2

Stains acidic mucins blue

Question 3

Perl’s Prussian blue

Answer 3

Histochemical stain for iron/haemosiderin

Question 4

Grocott

Answer 4

Fungi

Question 5

PAS

Answer 5

Fungi

Question 6

Giemsa

Answer 6

Differentially stains human and bacterial cells purple and pink respectively, platelets/erythrocytes pink and lymphocytes sky blue

Question 7

Ziehl-Neelson

Answer 7

Bacteria, especially tubercules

Question 8

Martius Scarlett Blue

Answer 8

Tinctorial stain for connective tissues - Red fibrin, Blue collagen, Yellow RBCs

Question 9

Bacteria diagnosis stains

Answer 9

H&E, Gram, Giemsa, Ziehl-Neelson

Question 10

Fungi stains

Answer 10

H&E, PAS, Grocott

Question 11

Argyrophil and argentaffin

Answer 11

Argyrophil cells are capable of being impregnated with silver, but need a reducing agent to reduce it to a visible metallic silver; argentaffin cells can be impregnated with silver AND reduce the silver

Question 12

Toluidine blue

Answer 12

Metachromatic (changes colour when bound to tissue), used for carcinoids, acidophilic

Question 13

H&E

Answer 13

Haematoxylin is basic and purplish/blue (basophilic - nucleus/ribosomes/nucleoprotein)
Eosin is reddish/pink and acidic/cationic (acidophilic structures - cytoplasm, cell walls, ECM)
Haematoxylin oxidised to haematein, combined with mordant to stain cells

Question 14

Liver stains - PAS/D

Answer 14

Glycogen - For glycogen storage diseases - Diastase (alpha-amylase) digests glycogen and starches to glucose. Periodic acid oxidizes glycols (and glucose??) to aldehydes, detected by Schiff reagent

Question 15

Liver stains - Perl’s

Answer 15

Haemosiderin - For haemochromatosis or haemosiderosis

Question 16

Liver stains - Orcein

Answer 16

HepB surface antigens, Elastin, Copper-bound proteins (Wilson’s)

Question 17

Liver stains - Rhodamine

Answer 17

Copper - For Wilson’s disease

Question 18

Liver stains - Masson Trichrome

Answer 18

Collagen - For fibrosis/cirrhosis

Question 19

Fontana Masson

Answer 19

Argenaffinic substances - Often melanin

Question 20

LFT panel

Answer 20

Bilirubin - jaundice, obstructions…
ALP - Bile duct system
AST, ALT, GGT - Damage or disease of hepatocytes
Albumin - made by liver

Question 21

Types of liver biopsy

Answer 21

Percutaneous - Through skin, usually w/ imaging/USG/MRI
Transvenous - Catheter via jugular vein, navigate to hepatic veins (clotting issues, ascites)
Laparoscopic/open wedge - Invasive, one large or multiple small incisions, greater visualisation

Question 22

Alcoholic liver disease (ALD) - stages

Answer 22

AFattyLD (steatosis) -> Alcoholic hepatitis (inflammation) -> Cirrhosis (scarring and fibrosis)

Question 23

Alcoholic liver disease (ALD) - ethanol mechanism

Answer 23

Ethanol -> ADH -> Acetaldehyde -> ROS -> OxStress -> Steatosis
Also - Inflammation -> Kupffer cells -> NADPH oxidase -> ROS -> OxStress -> Steatosis

Question 24

Alcoholic liver disease (ALD) - treatment options

Answer 24

Alcohol cessation, antioxidants, GFs, anti-caspase, anti-fibrotics, anti-inflammatories, TLR agonists

Question 25

Wilson's disease

Answer 25

Autosomal recessive ATP7B mutation increases circulating copper -> Chronic hepatitis and cirrhosis + RBC haemolysis Penicillamine and trientine are two chelating agents used to treat Wilson disease

Question 26

IHC markers - pulmonary, adenocarcinoma, squamous cell carcinoma

Answer 26

TTF1 shows pulmonary origin, adenocarcinoma is P63 -ve, squamous cell carcinoma is P63 +ve

Question 27

Druggable gene mutations associated with lung cancer, with assays

Answer 27

EGFR (FISH gold standard, can use IHC), ALK1 (IHC), PDL1 (IHC), ROS1 (IHC & FISH)

Question 28

IHC marker - breast

Answer 28

CK14

Question 29

Normal andrology values - semen volume, pH, sperm per ml/ejaculation, total motility, vitality, morphology

Answer 29

- semen volume min 1.5 ml - pH min 7.2 - sperm min 15 mil per ml - therefore total sperm min 39 mil per ejaculation (~ 15 x 1.5??) - total motility min 40% - vitality min 58% live spermatozoa - morphology min 4% normal forms of spermatozoa

Question 30

Breast cancer - Nottingham Prognostic Index

Answer 30

Nottingham Prognostic Index (NPI) = = [0.2 x S] + N + G Where: S is the size of the index lesion in centimetres N is the node status (0 nodes = 1, 1-3 nodes = 2, >3 nodes = 3) G is the grade of tumour (I to III - 1 to 3)

Question 31

Breast cancer - NPI - Grading criteria

Answer 31

Differentiation, variation in size/shape of tumour cells, mitotic counts

Question 32

CRC - Screening tests

Answer 32

Foecal Occult Blood (FOB) test for non-visible blood in stool - All men and women 60-74 Bowel scope test (colonoscopy) - All men and women over 55 FOB being replaced by FIT (Foecal Immunochemical Test) Younger individuals in high-risk groups also invited - Hereditary Non-Polyposis Colorectal Cancer (HNPCC), family history, IBD

Question 33

Masson Trichrome

Answer 33

Red keratin and muscle fibres, blue or green collagen and bone, light red or pink cytoplasm, and dark brown to black cell nuclei

Question 34

Gömöri trichrome

Answer 34

Histological stain used on muscle tissue, can be used to test for certain forms of mitochondrial myopathy - Stains nuclei black, muscle fibre and cytoplasm red, collagen blue

Question 35

Demonstrating nucleic acids

Answer 35

Nucleic acids can be demonstrated in histological sections using the special stain methyl green-thionine (or methyl-green pyronin) - RNA stains red; DNA stains blue-green For DNA - Feulgen reaction can also be used - Warm HCl, then Schiff's reagent - DNA turns red as its hydrolysis releases aldehydes which make red complexes(?) with Schiff

Question 36

IHC markers - SCLC vs NSCLC

Answer 36

TTF1 for lung origin Small cell lung cancer is CK7-, Non-small cell is CK7+ Both are CK20-

Question 37

IHC markers - CRC vs others

Answer 37

TTF- CK7- CK20+ CDX2+: Colorectal carcinoma CK20-:

Question 38

Ishak grading

Answer 38

For hepatitis/liver injury: Interface hepatitis 0-4 + Confluent necrosis 0-6 + Necrosis/apoptosis/lobular inflammation 0-4 + Portal inflammation 0-4 1-3 is minimal, 4-8 mild, 9-12 moderate, 13-18 severe

Question 39

EWSR1 - FISH vs RT-PCR

Answer 39

FISH analysis using a break-apart probe is highly sensitive in detecting EWSR1 rearrangements but does not identify its translocation partner, whereas RT-PCR analysis is a more specific test in identifying the EWSR1-FLI1 fusion gene but has suboptimal sensitivity (54%) in formalin-fixed, paraffin-embedded tissue

Question 40

SRBCTs - Diagnosis and fusions

Answer 40

Diagnostic IHC used for subtyping, and alongside RT-PCR for diagnosis IHC/RT-PCR - Expression of CD99 + FLI1 (EWS/pPNETs), myogenin (myogenic transcriptional regulator - RMS), keratin/EMA (SS), PAX5 (EWS/pPNET and RMS) FISH - Fusions - PAX3/7-FKHR (FKHR == FOXO1) for RMS, SYT-SSX for SS, EWS-FLI1 for EWS/pPNETs NOTE -- FLI1 is also expressed in many lymphomas, including lymphoblastic lymphoma, which is also CD99 positive, so further analysis and consideration is needed

Question 41

IHC for breast cancer - Scoring methods

Answer 41

H-score = SUM[ (1 x % cells weakly stained) + (2 x % cells moderately stained) + (3 x % cells strongly stained) ] Allred QuickScore = Proportion score + Intensity score Proportion of staining (0 - no staining, 1 - 1 nucleus, 2 - 1 to 10 nuclei, 3 - 11 to 33 nuclei, 4 - 34 to 66 nuclei, 5 - 66 to 100 nuclei) Intensity score (0 - negative, 1 - weak, 2 - intermediate, 3 - strong)

Question 42

Breast cancer NPI+

Answer 42

FFPE and IHC - Molecular classification via ER, PR, CK5/6/7/8, EGFR (AKA HER1), HER2/3/4, p53, Mucin 1 Then different NPI-like algorithms (size, shape, LN stage) for each class More personalized and better prognostic ability, but more time-consuming and requires more tests (cost)

Question 43

Breast cancer - SLNB

Answer 43

Sentinel lymph node biopsy - Radioactive tracer or blue dye injected near tumour site allows ID of the first lymph node draining the breast, and so the first lymph node the cancer is likely to spread to This is then biopsied, dissected, and assessed for presence of tumour cells to determine if the cancer has become invasive or not yet -> STAGING and prognosis BC, melanoma, penile cancer SNLs sectioned at levels throughout the node - less samples but still representative IHC can be used at the same time to help locate small deposits of metastatic tumour cells -> Broad-spectrum CKs - AE1/AE3 and CAM5.2

Question 44

Breast cancer - TDLU IHC

Answer 44

The outer basal layer (of myoepithelial cells) stains brown with CK14 IHC in normal breast, and not the inside. CD44 can also stain rare but vital breast stem cells within the double-walled structure of the TDLU.

Question 45

Breast cancer - Diagnostic techniques

Answer 45

X-rays - Densities, (micro)calcifications, age-related atrophy of glandular tissue (and replacement by adipose tissue) FNA - Cannot distinguish in situ vs invasive, not much prognostic info or functional measures (e.g. ER/PR/HER2 status) Needle core biopsy may be preferred > FNA Sentinel lymph node biopsy

Question 46

GI diagnostic techniques

Answer 46

Radiology, manometry (pressure), endoscopy (can take biopsies!), EUS (endoscopic ultrasound), scintigraphy (radiotracers) Endoscopy - white light, narrow band (filter to block long-wavelength, deep-penetrating red light, keep blue/green light -> better visualisation of superficial morphology), chromoendoscopy (stains and dyes used during endo)

Question 47

Colorectal adenocarcinoma IHC markers

Answer 47

TTF1 & CK7 - ; CK20 & CDX2 +

Question 48

MSI tests in CRC

Answer 48

PCR for length/number of a panel of 5 microsatellite regions (BAT25/26, etc..), IHC for MMR gene protein expression.