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Molecular Haematology

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  1. Molecular Haematology Alberto Catalanoemail: catalano.alberto@gmail.com 10 Mar 2011

  2. Outline of topics • Molecular biology refresher • Molecular biology in haematology • Quality control issues • Laboratory layout and equipment • Case studies • Our wish list

  3. Molecular biology • DNA or RNA: when and why • Genes and gene structure • Methodology • Nucleic acid extraction • DNA electrophoresis • Polymerase chain reaction (PCR) • Quantitative PCR • High resolution melting (HRM) • DNA sequencing

  4. Chromosomes are DNA

  5. Sugar + Phosphate + Base Sugar + Phosphate form the backbone DNA: R = H RNA: R = OH

  6. Base-pairing Purines Pyrimidines

  7. DNA or RNA: when and why • One chromosome = 1 dsDNA molecule • Autosome pair = 2 dsDNA molecules • Mitochondrial DNA (many copies/cell) • DNA is more stable for analysis • DNases are easily heat denatured • DNA autolysis is minimal under normal pH & temp • RNA is less stable • RNases are ubiquitous & difficult to remove • RNA autolysis in mildly acidic pH

  8. The three bones from Vindija from which Neandertal DNA was sequenced. 38,310yo  44,450yo

  9. DNA or RNA: when and why • Gene level: • DNA there whether expressed or not • RNA copies depend on level of expression in cell • Different cell types: Different expression levels • Messenger RNA is an “edited” version of DNA • Shorter • Without introns

  10. … because of large introns and variable genomic breakpoints • mRNA lacks the introns present in gDNA • Fusion genes are more easily amplified from a shorter sequence • Breakpoint clustering and multiple breakpoint clusters • Sizes of PCR products indicate the types of breakpoints present • However, RNA is less stable than DNA

  11. Gene structure Promoter Transcription start site Exons Poly adenylation site Open reading frame AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA 7-Methylguanosine cap Poly A tail ORF

  12. Alternative splicing One gene : many mRNA transcripts: many protein isoforms DNA Primary RNA transcript Skipped exon Skipped exon RNA splicing Altered reading frame

  13. Genomic Breakpoints in BCR-ABL

  14. Sample preparation • Purity of sample • Hypotonic lysis of blood or bone marrow • Ficoll density fractionation • Speed & sample turnover • RNA stabilization • DNA extraction • Quick for robust assays (e.g. blood boiling) • High purity DNA for troublesome assays

  15. RNA Stabilization • Guanidine isothiocyanate • Trizol reagent • mono-phasic solution of phenol and guanidine isothiocyanate • RNA-Later • Ammonium sulphate protein precipitation • Stabilises RNA at ambient temperature • Suitable for transport with minimal packaging (post)

  16. RNA-based assays • RNA purification from Trizol lysed cells • Synthesis of copy DNA (cDNA) from RNA • Gene-specific PCR of: • Target gene (leukaemia specific fusion gene) • Control gene (“housekeeping” gene) to assess the quality of the RNA • For quantitative assays: • Result = fusion gene copies / control gene copies

  17. RNA preparation from cells using Trizol® reagent

  18. Inherited disorders • Thalassaemias • Hereditary Haemochromatosis • Factor V Leiden, MTHFR (Ala677Val), Prothrombin gene 20210 mutation

  19. Clonal disease • Clonal markers • X chromosome inactivation (HUMARA assay) • Gene rearrangements • Antigen receptor genes • T-cell receptor • Immunoglobulin • Abnormal fusion genes • Mutations • Loss of heterozygosity & uniparental disomy (acquired)

  20. Qualitative nested PCRFIP1L1-PDGFRA for CEL a b Patient samples Patient samples 10-3 10-4 10-3 10-4 M - N 1 2 3 4 5 6 + + W M - N 1 2 3 4 5 6 + + W Assays run in duplicate Slight differences due to stochastic factors e.g. Pt # 4 Controls to give an estimate of assay sensitivity

  21. Transferrin receptor TFRCRNA quality control Patient samples M - N 1 2 3 4 5 6 Positive TFRC indicates RNA quality is acceptable

  22. ABL qPCR RNA quality control

  23. ABL qPCR RNA quality control

  24. Reduction of T315I mutation 16/2/2011 ~100% T315I 25/1/2011 10%-30% T315I

  25. Residual disease monitoring • Detection of fusion gene or clonal TCR or IgH • Highly sensitive • Very early detection of molecular relapse • Quantitation of fusion gene • Allows monitoring of treatment effect • Detection of relapse • Chimerism in post-transplant patients

  26. Sensitivity of TCRg PCR analysis

  27. BCR-ABL qPCR

  28. BCR-ABL IS calculation BCR-ABL copies (average of 2 cDNA) IS= X laboratory correction factor BCR copies (average of 2 cDNA) Correction factor checked annually against reference laboratory(IMVS Royal Adelaide Hospital)

  29. Laboratory layout and equipment Measures to ensure quality

  30. Laboratory Automation Reducing manual handling reduces chances of sample mix-up

  31. Promega Maxwell 16 Automated DNA extraction systemUses magnetic bead cartridges containing lysis and wash reagents

  32. CAS-1200 Robot

  33. Real time PCR instrumentation Rotor Gene™ 6000 (Corbett Research) 36 / 72/ 100 well rotor format Thermal uniformity ±0.01°C, Resolution ±0.02°C, HRM data acquisition (read) rate: 20 reads for each 0.02°C increment 5-20 µl Capacity 15 minutes per run (after amplification) HRM, real time PCR and allelic discrimination (5 colours) Rotor Gene™ 3000 (Corbett Research) 36 / 72 well rotor format 5-20 µl Capacity Real time PCR and allelic discrimination (4 colours)

  34. LABORATORY FACILITIES • Laboratories configured to minimise the risk of contamination of samples and reagents by amplified material or other samples in the laboratory • Minimum Standards • Additional Standards for Nested PCR

  35. Minimum Standards • area for the extraction of nucleic acids from samples and for the addition of sample DNA • dedicated clean area for the preparation of reagents (including dispensing of the master mix) • a dedicated, contained area for amplification and product detection

  36. Reagent preparation Samplepreparation Templateaddition Detection Amplification Workflow

  37. Laboratory Layout 1 Sample preparation Reagent preparation 2 Template addition Amplification 3 Detection

  38. Laboratory Layout 1 Sample preparation Reagent preparation 2 Template addition Amplification Detection

  39. Quality assurance Undertaking a volume of testing that is sufficient to maintain the knowledge, experience and expertise of staff Benefits of centralisation versus those of developing local expertise and autonomy Associations or collaborations between diagnostic laboratories and research laboratories are encouraged for small volume testing

  40. RCPA QAP programme JAK2 V617F BCR-ABL PML-RARA DNA Chimerism Factor V Leiden, Prothrombin 20210, MTHFR (A677V) BCL1, BCL2, TCR, IGH Thal a, Thal b Haemochromatosis Cys282Tyr, His63Asp

  41. Inter-laboratory sample exchanges Informal regular exchanges of samples with other laboratories Blinded Comparison of sensitivities Not generally surveyed by RCPA QAP For establishment of new methods

  42. Contamination • specimen collection or transport • handling or testing in the testing or referring laboratory before nucleic acid detection • during: • extraction of nucleic acids from the sample • amplification • product detection • by contamination from the reagents used for the test

  43. Contamination Sources positive samples (cross contamination); amplified nucleic acid (e.g. contamination of stock reagents or equipment, or in aerosol droplets);

  44. Measures to Control Contamination • the competency of staff at performing laboratory tasks • the routine use of controls to detect contamination • Splitting samples • Uracil-N-glycosylase (UNG) • the design of the laboratory

  45. Uracil-N-glycosylase • If dUTP is used instead of TTP in PCR • Uracil-N-glycosylase (UNG) cleaves contaminating PCR products prior to PCR • Real template lacks dU and therefore is not degraded • Prevents amplification of minor amounts of contaminating DNA • Cannot prevent gross contamination

  46. dUTP incorporation | | | | | | | | | | | | ||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| UNG treatment UNG ||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||

  47. Nested PCR • Products from the 1st round of PCR are used as templates for 2nd round of PCR • Requires 4th isolated area • Laboratory • Class 2 biosafety cabinet within area 2 • Uracil-N-glycosylase (UNG) : • 1st round: + UNG , - dUTP • 2nd round: - UNG , + dUTP

  48. Sample processing • Hypotonic lysis: • BCR-ABL, AML-ETO, CBFB-MYH11, FIP1L1-PDGFRA, JAK2 • Ficoll purification of mononuclear cells: • PML-RARA • Ficoll purification of granulocytes: • For low level JAK2 V617F • Granulocyte and T-cell isolation: • DNA chimerism • DNA-based assays: JAK2, chimerism • RNA-based assays: all others