Cytogenetic And Molecular Investigations Flashcards
CLPDs
Chronic lymphoproliferative disorders
Single abnormal lymphocyte
Genetic changes -> accumulation of cell -> clonal population
Clonality/antigen receptor rearrangements used for…
Used to determine clonality (reactive or clonal)
Malignant cells => identical rearrangement -> clonal
In benign lesions each cell carries its own rearrangement and are referred to as polyclonal
Material for antigen receptor PCR
Performed using DNA from:
Peripheral blood
BM
Nodal aspirates
Surgical biopsy
Serous effusions
Formaldehyde fixed, paraffin embedded
Chromosomal translocations result in either
Fusion of two genes -> fusion protein BCR-ABL in CML
Relocation of gene under influence of new promoter causing dysregulation of gene expression e.g. IgHV and Bcl-1 in mantle-cell lymphoma
When are DNA-PCR and RT-PCR used?
DNA-PCR can be used for detection of translocations when breakpoints tightly clustered t(14;18)
RT-PCR used when breakpoints scattered in large introns t(9;22)
Explain the importance of molecular techniques in assessment of malignant lymphoma and leukaemia
Provide important information & early markers of relapse in MRD monitoring
MRD
Minimal residue disease
High percentage of patients with malignancy relapse due to residual tumour cells
Patients in clinical remission 10^10 malignant cells
Detection involves PCR with single set of primers
RT-PCR
Real time PCR
Internal oligonucleotide probe with reporter and quenching activities
Reporter dye cleaved by tau polymerase during primer extension
Cleavage separates reporter from quencher dye increasing reporter dye signal
Extent of fluorescence directly proportional to amount of PCR product
Using CML as an example carry out cytogenetics testing and molecular investigations
Morphology
Peripheral blood
BM
Immunotyping
Cytogenetics
Philadelphia chromosome
FISH
BCR:ABL dual fusion probe t(9;22)
RT-PCR
Detect BCR-ABL1 mRNA
