Molecular pathology part 2

Question 1

Samples for molecular testing
Histological sample= ……..
Cytological samples = i……….. cells

sample preparation = means of …………

Answer 1

Samples for molecular testing
Histological sample= Tissue
Cytological samples = isolated cells

sample preparation = means of fixation

Question 2

Formalin fixed paraffin embedded (FFPE) vs fresh frozen tissues

FFPE
-  Routinely collected
Choice for histopathological diagnosis
-  Degradation of ....... material
Specimen processing:

nucleic acid fragmentation, DNA crosslinks, a basic sites leading to localized DNA den………. and strand breaks, and dea……….. leading to ….>…… mutation artefacts which impe…… downstream sequencing analysis.

Fresh tissue
- Not routinely collected

Choice for quick differentiation between ………… and ……… tumours in frozen sections

• Less d………… of nucleic material

less fragmented nucleic acids (longer fragments), enriched in double-stranded DNA molecules, less artefacts, better u…………. of coverage,

Answer 2

Formalin fixed paraffin embedded (FFPE) vs fresh frozen tissues

FFPE
-  Routinely collected
Choice for histopathological diagnosis
-  Degradation of nucleic material
Specimen processing:

nucleic acid fragmentation, DNA crosslinks, a basic sites leading to localized DNA denaturation and strand breaks, and deamination leading to C>T mutation artefacts which impede downstream sequencing analysis.

Fresh tissue
- Not routinely collected

Choice for quick differentiation between benign and malignant tumours in frozen sections

• Less degradation of nucleic material

less fragmented nucleic acids (longer fragments), enriched in double-stranded DNA molecules, less artefacts, better uniformity of coverage,

Question 3

Fundamental factors in pre-analytical workflows
Pre-fixation and specimen transport times. Excision of human tissue immediately triggers a ca……… of changes to mo……… expression. Whilst pathology laboratories and diagnosticians are hugely experienced in overcoming these changes using more traditional methods and analyses, the impact upon molecular pathology techniques is less well known. Hypoxic times, for example, directly alter ‘molecular’ targets such as RNA expression as well as ‘traditional’ variables like mitotic figure counts (Cross et al., 1990).

Fixation, typically using formaldehyde. As discussed in greater detail later, formaldehyde, typically as NBF, is a ‘mixed blessing’ as the cross-linking which sta....... and preserves tissue, also fragments ....... Fragmentation limits the ma......... le....... of DNA regions which can be targeted and amplified in the PCR reaction, the ‘amplicon’. Highly fragmented DNA is non-a..............., and the rate of PCR failure correlates directly with the severity of fragm............. Repeatability and reproducibility of molecular analyses cannot be guaranteed without adequate processing controls which include monitoring of fixation times. Both minimum (Qizilbash, 1982) and maximum (Nam et al., 2014) fixation times should be regulated, with consideration as to whether durations greater than 24hrs are necessary.

Physico-chemical influences. The temp........ and chemical components of the processing pathway both affect biomolecule sta...... and these must therefore be strictly regulated. Changes to processing times or methods, including ‘novel’ fixatives, or processes such as the use of h...... temperatures and/or microwave ra........., require thorough vali........ The effect upon molecular pathology analyses of ‘traditional’ histopathological techniques such as decalcification must be fully evaluated and understood.

Answer 3

Fundamental factors in pre-analytical workflows
Pre-fixation and specimen transport times. Excision of human tissue immediately triggers a cascade of changes to molecular expression. Whilst pathology laboratories and diagnosticians are hugely experienced in overcoming these changes using more traditional methods and analyses, the impact upon molecular pathology techniques is less well known. Hypoxic times, for example, directly alter ‘molecular’ targets such as RNA expression as well as ‘traditional’ variables like mitotic figure counts (Cross et al., 1990).

Fixation, typically using formaldehyde. As discussed in greater detail later, formaldehyde, typically as NBF, is a ‘mixed blessing’ as the cross-linking which stabilizes and preserves tissue, also fragments DNA. Fragmentation limits the maximum length of DNA regions which can be targeted and amplified in the PCR reaction, the ‘amplicon’. Highly fragmented DNA is non-amplifiable, and the rate of PCR failure correlates directly with the severity of fragmentation. Repeatability and reproducibility of molecular analyses cannot be guaranteed without adequate processing controls which include monitoring of fixation times. Both minimum (Qizilbash, 1982) and maximum (Nam et al., 2014) fixation times should be regulated, with consideration as to whether durations greater than 24hrs are necessary.

Physico-chemical influences. The temperature and chemical components of the processing pathway both affect biomolecule stability and these must therefore be strictly regulated. Changes to processing times or methods, including ‘novel’ fixatives, or processes such as the use of high temperatures and/or microwave radiation, require thorough validation. The effect upon molecular pathology analyses of ‘traditional’ histopathological techniques such as decalcification must be fully evaluated and understood.

Question 4

Frozen tissue > frozen ….. and L….. Sample >DNA/RNA/Protein extraction > sequencing/P…./cD….Array/ Epige……/ Pro……..

FFPE > LC…. sample and ma……. micro……… or FF…..curls > DNA/RNA/Protein extraction > sequencing/ PCR / cDNA Array /Epigenetics / Proteomics

Answer 4

Frozen tissue > frozen curls and LCM Sample >DNA/RNA/Protein > sequencing/PCR/cDNA Array/ Epigenetics/ Proteomics

FFPE > LCM sample and manual microsection or FFPE curls > DNA/RNA/Protein extraction > sequencing/ PCR / cDNA Array /Epigenetics / Proteomics

Question 5

Purity is determined by visual assessment

• Pathologists
• Trained Biomedical scientists

will estimate %age of tumour cell in the FPPE section on h&E slide

Answer 5

Pathologists

Trained Biomedical Scientists

will estimate %age of tumour cell in the FFPE section on H&E slide.

Question 6

Sample for DNA/RNA extraction

FFPE blocks must be cut on a m……….. following molecular standard operating procedures: clean all equipment (microtome, forceps, brush) with DNA/RNA removal cleaning agents, change and clean the w……. …….. after every block change the blade after every block, dry ……in a clean rack under e…….. hood

Answer 6

Sample for DNA/RNA extraction

FFPE blocks must be cut on a microtome following molecular standard operating procedures: clean all equipment (microtome, forceps, brush) with DNA/RNA removal cleaning agents, change and clean the water bath after every block change the blade after every block, dry slides in a clean rack under extraction hood

Question 7

Hypochlorite solutions (with 1-2% active h………. to decrease DNA ……………. in the laboratories

Answer 7

Hypochlorite solutions (with 1-2% active hypochlorite to decrease DNA contamination in the laboratories

Question 8

Examples of Mircodissection techniques for DNA/RNA extraction

Manual microdissection put in dewax solution or dry tube

The lase capture microdissection process

1) Place cap on t………
2) Pulse la…….. at ta…….. cells
3) ………… cap with ad……… target cells
4) E……… molecules from …….. cell

Put in …….. buffer sample storage

Answer 8

Examples of Mircodissection techniques for DNA/RNA extraction

Manual microdissection put in dewax solution or dry tube

The lase capture microdissection process

1) Place cap on tissue
2) Pulse laser at target cells
3) Remove cap with adhered target cells
4) Extract molecules from target cell

Put in lysis buffer sample storage