Techniques in diagnostic pathology

Question 1

What are the different types of fixatives

Answer 1

formalin,bouin(mercury based), carnoy(alcohol based), 3%glutaraldehyde

Question 2

What step do you do after fixation for non frozen tissue?

Answer 2

Embed tissue in wax, cut from wax and dewax with xylene, deparaffinze and rehydrate with alcohol and stain

Question 3

When do you administer adjuvant therapy?

Answer 3

When incomplete resection, vascular invasion, advanced stage

Question 4

What’s the difference between ptx and pt0

Answer 4

PTX can’t be assessed histologically

pt0- no histological evidence of primary tumour

Question 5

Difference between V1 and V2

Answer 5

venous invasion microscopic and venous invasion macroscopic

Question 6

How to deal with frozen tissue

Answer 6

Put OCT

Question 7

What factors do we evaluate when looking at an invasive carcinoma?

Answer 7

Histological type, grade, tumour size, lymph node stage, vascular grade

Question 8

What does histological grade asses? And how is it done?

Answer 8

degree of differentiation

By looking at morphological characteristics such as size, nuclear pleomorphism, mititic count, tubule formation

Question 9

Does histological grade correlate to patient survival?

Answer 9

yes

Question 10

What methods are often used to study tissues and is each one associated iwth fresh, fixed tissue or both?

Answer 10

PCR-FFPE(300-500bp) or fresh tissue
Pyrosequencing
RT-PCR-FFPE(100BP) or fresh tissue
FISH-fresh or fixed
spectral karyotype-fresh
flow cytometry- fresh
expression profiling-both
CGH-both, but needs amplification step for FFPE tissue
Western blotting-fresh
MS-fresh
metabolomics-fresh

Question 11

Why is immunocytochemistry important?

Answer 11

Evaluating subtypes of tumour
differentiation of tumours
secretory products
identification of metastasis
prognostic markers

Question 12

How is antigen retrieval done?

Answer 12

Unmasking epitopes by formaldehyde fixation
digestion with proteolytic enzymes
microwave energy
cook in heat and pressure

Question 13

What are the problems with ICH?

Answer 13

antigens might crosslink, not be specific, they can be lost, cross reactivity, appropriate concentration of antibody has to be used, entrapment of normal tissue in tumour cells
release of soluble proteins

Question 14

Give examples of IHC diagnostic markers

Answer 14

CK20-squamous cell&adenocarcinoma
PAS-mucin
CEA-present in carcinoma
CK7-Lung.breast, ovary cancer, ductal, glandular,transitional epithelium

Question 15

Give the therapy most appropraite to the following breats cancer patients and prognosis
ER,PR +/+ 
ER-, 
ER+,
ER low

Answer 15

ER,PR +/+ better response
ER-, poor prognosis, give chemotherapy
ER+, endocrine therapy, better prognosis
ER low both endocrine and chemotherapy

Question 16

What does CISH do? And why is is it better than FISH?

Answer 16

Allows direct comparison between morphology and gene amplification. Doesn’t need fluorescent microscopy and is quick

Question 17

What’s electron microscopy for?

Answer 17

soft tissue tumours, muscle disease, peripheral nerve tumours, renal pathology, tumour differentiation, paedriatic tumours,diagnostic ultrastructural features

Question 18

Whta’s immunofluorescence and confocal microscopy used for?

Answer 18

To see if proteins are being co-expressed, interacting, metabolism, cell membrane kinetics,

Question 19

What does PAS, mason fontana, oil red O, PAS, alcian blue

Answer 19

PAS-detection of mucin in poorly differentiated tumours
Mason fontana-detection of melanin in metastatic deposits
Oil red O-lipid and specifc markers differentiating between tumours
PAS, alcian blue- metaplasia in GIST

Question 20

What is intra-operative smears

Answer 20

swap taken from CBS fluid or spine to assess lesions by seeing nuclei abnormality, blood vessel sizes, necrosis

Question 21

What are the different methods of doing immunohistochemistry analysis?

Answer 21

Peroxidase antiperoxidase immune complex
peroxidase enzyme AB conjugate
labelled antigen 
labelled bridge
biotinylated primary AB
Biotinylated peroxidase
avidin biotinylated peroxidase complex

Question 22

How is spindle cell sarcoma diagnosed using IHC? which markers do they look for?

Answer 22

Vementin-IF of mesenchymal cells
LCA-leukemia common antigen
AE1-cytokeratin
CD31-endothelial cells
CD34-soft tissue

Question 23

What are the charactersitics of spindle cell lesions? What is it diagnosed as?

Answer 23

Proliferation rate is low, express BCL-2, cd34, CD99 but not EMA,SMA(smooth muscle actin),s100, hmb45(found in melanoma)
solitary fibrous tumour

Question 24

Markers for lymphoma

Answer 24

CD3-T-cells
CD20-B-cells
BCL-2-follicular lymphoma
MIB-1- cell proliferative marker

Question 25

What does cytogenetics study normally?

Answer 25

pedriatic tumours, hematological malignancies, clonality studies

Question 26

What kind of probes are used for FISH?

Answer 26

centromere, telomere probes, chromosome paints, microdeletion probes, gene amplification, translocation junction

Question 27

What kind of information do we get from FISH?

Answer 27

Microdeletions, copy number of chromosomes and genes, DM&HSR, translocation

Question 28

How is neuroblastoma diagnosed?

Answer 28

Loss of material from 1P which has tumour suppressors
amplification of n-myc(>10) located at 2p24
gain of 17q
overexpression of BCL2

Question 29

What are the characteristics of ewing sarcoma?

Answer 29

t(11;22)q(24:12)
Fli1-EWS fusion gene
fli-DNA binding domain
EWS-promoter region
11q24- DNA binding domain+transcription activator
22Q12-RNA bidning domain and promoter region

Question 30

How is molecular studies done usually?

Answer 30

PCR, RNA extraction, proetin extraction