Viva Qs Flashcards
Microorganism stains
(4)
Ziehl Neelson
Gram Tworts
Warthin and Starry
Grocotts methenamine silver stain
Pigment stains
(4)
Haemosiderin/Iron -> Perl’s prussian blue
Amyloid -> Congo Red
Melanin -> masson fontana
Calcium -> Von Kossa
Silver stains
(4)
Grocotts methenamine silver stain for fungi
Masson fontana for melanin
Warthrin and Starry for Spirochetes e.g. Helicobacter
Gordon and Sweets Reticulin Stain
Trichrome stains
Masson Trichrome for muscle + CT
Gomori Trichrome for muscle + CT
Martius Scarlet Blue for fibrin
Carbohydrate stains
(4)
Periodic Acid Shiff -> carbohydrates + glycogen
Diastase periodic acid-schiff -> carbohydrates - glycogen
Alcian blue -> acidic mucins
Alcian Blue/PAS
Step by step of processing
Formalin fixation
Alcohol dehydration
Clearing in xylene
Impregnation with parafin wax
What would you do if your QC for specials failed but test worked?
Check if correct material has been used
Check if block has been cut through
Check processing of control block
Repeat test
What would you do if QC for specials failed and test failed
Check reagents
Check method -> correct incubation etc
Check processing
Check if correct control used
Check if block cut through
Repeat
Undifferentiated panel
Melanoma: S100, HMB45, Vimentin
Lymphoma: CD45, Vimentin
Sarcoma: Desmin, Vimentin
Carcinoma: EMA, AE1/AE3, CAM5.2
NE: Neuron Specific Enolase
Breast Cancer Panel
Estrogen Receptor
Progesterone Receptor
Her2
Ki67
PDL-1
DCIS: p63/SMMS
What would you do if IHC control failed?
Check reagents -> have they been stored correctly, freeze/thaw cycles for antibodies etc
Might need longer incubation of primary antibody/higher concentration
Insufficient/incorrect form of antigen retrieval
Incorrect antibodies used
Primary and secondary antibodies not compatible
Antibody denatured
Secondary antibody exposed to light
Insufficient deparaffinisation
PBS contaminated with bacteria -> damage the phosphate groups on the protein of interest -> use fresh PBS
What would a lot of background staining in IHC mean
Insufficient blocking -> endogenous antigen present
Primary antibody concentration too high
Non specific binding of secondary antibody
Too much amplification
Too much substrate applied
What is antibody optomisation for IHC
ascertain which, if any, form of antigen retrieval is necessary to unmask the antigen (heat or enzyme mediated)
Ascertains which dilution gives appropriate staining
Ascertains incubation time and temperatures
Ascertains which is the most suitable clone for IHC
Peroxidase blocking
▪ Some enzymes used in chromogenic detection methods such as peroxidase are also naturally occurring in some tissues -> if not blocked this will cause interference
▪ Peroxidase activity can be seen in tissues such as the kidney, liver and tissue containing rbcs such as vasculature
▪ Can test the tissue for endogenous peroxidase by incubating it in DAB -> if a brown colour is observed then a blocking step is required
Blocking involves incubating the tissue with Hydrogen peroxide
EMAR vs HIER
EMAR = proteinase K proteolytic enzyme
HIER = heat for epitope retrieval in microwave
% Receptor expression for breast cancer
- Estrogen receptors are highly expressed in over 70% of breast cancer
Her2 is over expressed in over 20% of breast cancers
DDISH
○ Dual-colour dual-hapten in-situ hybridisation
○ Used for the quantitative detection of amplification of the Her-2 gene (human epidermal growth factor receptor 2) using a dual colour chromogenic CISH as well as SISH
Looks at the Her2 and C17 genes
Used for breast cancer and gastric cancer patients for whom Herceptin treatment is being considered
○ Carried out using a cocktail of probes – a HER2 probe and a control chromosome 17 probe
This allows for the enumeration of both Her2 positive tumour nuclei and normal cells
ISH
- A technique used to detect specific nucleic acid sequences of RNA or DNA within cells or a tissue.
- Makes use of labelled probes to hybridise a known target mRNA or DNA sequence within the tissue.
- The labelled probes are then detected using an antibody specific to the label
Through this the probes can be used to detect the expression of a particular gene of interest as well as the location of its mRNA
Types of ISH
FISH -> fluorescent
CISH -> chromogenic
SISH -> silver
DDISH -> dual-colour dual-hapten
Examples of ISH
DDISH is done for any Her-2 IHCs which are 2+
FISH is done for EBV -> linked to sporadic Burkitt lymphoma in HIV patients
Looking at getting ISHf or Kappa and Lamdba chains
Molecular testing
BRAF = melanoma
MSI = colorectal
NRAS = melanoma + colorectal
KRAS = lung, colorectal + pancreatic
Cytology stains:
PAP and MGG
Cytology samples
Mostly CSFs, BALs and cyst fluid
CSF (looking for tumour mets)
FNAs
Bronchial aspirates, washings, lavages
EBUS
Cyst fluid
Thyroid aspirates (from ?HIV in infectious disease clinic)
Joint fluid -> query goitre
Ascites, pleural etc
Thin Prep
Creates a monolayer of cells for PAP stain
ThinPrep 2000
Sample mixed -> laser determines concentration of cells -> quantifies how much of the sample is needed -> sample taken up through filter -> any cells present don’t make it through filter -> filter pressed to charged slide -> cells transferred to slide -> slide placed in IMS for staining
