Stains and diagnosis Flashcards
Congo red
Stains amyloid red
Alcian blue
Stains acidic mucins blue
Perl’s Prussian blue
Histochemical stain for iron/haemosiderin
Grocott
Fungi
PAS
Fungi
Giemsa
Differentially stains human and bacterial cells purple and pink respectively, platelets/erythrocytes pink and lymphocytes sky blue
Ziehl-Neelson
Bacteria, especially tubercules
Martius Scarlett Blue
Tinctorial stain for connective tissues - Red fibrin, Blue collagen, Yellow RBCs
Bacteria diagnosis stains
H&E, Gram, Giemsa, Ziehl-Neelson
Fungi stains
H&E, PAS, Grocott
Argyrophil and argentaffin
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
Toluidine blue
Metachromatic (changes colour when bound to tissue), used for carcinoids, acidophilic
H&E
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
Liver stains - PAS/D
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
Liver stains - Perl’s
Haemosiderin - For haemochromatosis or haemosiderosis
Liver stains - Orcein
HepB surface antigens, Elastin, Copper-bound proteins (Wilson’s)
Liver stains - Rhodamine
Copper - For Wilson’s disease
Liver stains - Masson Trichrome
Collagen - For fibrosis/cirrhosis
Fontana Masson
Argenaffinic substances - Often melanin
LFT panel
Bilirubin - jaundice, obstructions…
ALP - Bile duct system
AST, ALT, GGT - Damage or disease of hepatocytes
Albumin - made by liver
Types of liver biopsy
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
Alcoholic liver disease (ALD) - stages
AFattyLD (steatosis) -> Alcoholic hepatitis (inflammation) -> Cirrhosis (scarring and fibrosis)
Alcoholic liver disease (ALD) - ethanol mechanism
Ethanol -> ADH -> Acetaldehyde -> ROS -> OxStress -> Steatosis
Also - Inflammation -> Kupffer cells -> NADPH oxidase -> ROS -> OxStress -> Steatosis
Alcoholic liver disease (ALD) - treatment options
Alcohol cessation, antioxidants, GFs, anti-caspase, anti-fibrotics, anti-inflammatories, TLR agonists
Wilson’s disease
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
IHC markers - pulmonary, adenocarcinoma, squamous cell carcinoma
TTF1 shows pulmonary origin, adenocarcinoma is P63 -ve, squamous cell carcinoma is P63 +ve
Druggable gene mutations associated with lung cancer, with assays
EGFR (FISH gold standard, can use IHC), ALK1 (IHC), PDL1 (IHC), ROS1 (IHC & FISH)
IHC marker - breast
CK14
Normal andrology values - semen volume, pH, sperm per ml/ejaculation, total motility, vitality, morphology
- 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
Breast cancer - Nottingham Prognostic Index
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)
Breast cancer - NPI - Grading criteria
Differentiation, variation in size/shape of tumour cells, mitotic counts
CRC - Screening tests
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
Masson Trichrome
Red keratin and muscle fibres, blue or green collagen and bone, light red or pink cytoplasm, and dark brown to black cell nuclei
Gömöri trichrome
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
Demonstrating nucleic acids
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
IHC markers - SCLC vs NSCLC
TTF1 for lung origin
Small cell lung cancer is CK7-, Non-small cell is CK7+
Both are CK20-
IHC markers - CRC vs others
TTF- CK7-
CK20+ CDX2+: Colorectal carcinoma
CK20-:
Ishak grading
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
EWSR1 - FISH vs RT-PCR
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
SRBCTs - Diagnosis and fusions
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
IHC for breast cancer - Scoring methods
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)
Breast cancer NPI+
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)
Breast cancer - SLNB
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
Breast cancer - TDLU IHC
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.
Breast cancer - Diagnostic techniques
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
GI diagnostic techniques
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)
Colorectal adenocarcinoma IHC markers
TTF1 & CK7 - ; CK20 & CDX2 +
MSI tests in CRC
PCR for length/number of a panel of 5 microsatellite regions (BAT25/26, etc..), IHC for MMR gene protein expression.
