Sample Considerations for Biomarker Analysis

Transcription

1 Sample Considerations for Biomarker Analysis Jelveh Lameh, Ph.D. Executive Director, Head, BioPharma Services Laboratory Genoptix, a Novartis company May 18, 2016 AAPS National Biotechnology Conference

2 Goals Discuss various elements of collecting, processing and storing samples used for biomarker analysis- DNA, RNA, or cellular based assays Disclaimer: Views expressed herein are sole responsibility of the author and not that of her employer. 2

3 Sample Collection Considerations What is the purpose of the intended study? Which biomolecular class(es) is of interest? What sample source will be used and what are its characteristics? Which analytical technique(s) will be used? What are the capabilities of the analytical technique, and what are the criteria for optimal overall detectability? What is dynamic range of the biomarker being assessed? What is the limit of detection of individual biomarkers? Level of purity for analysis of a single biomarker species? What is the contaminant tolerance? How important is overall biomarker integrity? 3

4 Sample Collection For effective utilization of biomarkers, the measured concentration of the analyte in patient samples tested must be as close as possible to the actual analyte concentration at the time of sample collection. Requires optimal preservation of biomarker in specimens from collection to testing. Factors influencing results of biomarker assessment: Type of biological specimen (tissue, cells, serum, plasma, blood, etc.) Sample collection, processing and storage at the clinical site Shipment and transportation of samples from the clinical site to the bioanalytical facility Handling and storage of these samples at the bioanalytical laboratory: Freeze-Thaw Stability Short Term (Bench-Top) Stability Long Term Stability 4

5 Sampling Options Urine, Sputum, Feces and vomitus Saliva Sweat Hair sample Biopsy Normal tissue structure is preserved, while a sample for cytopathology is prepared primarily for the examination of individual cells. e.g., bone marrow biopsy, brain biopsy, skin biopsy and liver biopsy Tissue, excision Removal of solid or soft tissue samples Puncture followed by aspiration Sampling of tissues and body fluids. e.g. thoracocentesis (pleural fluid), amniocentesis (amniotic fluid), lumbar puncture Blood collection Scraping or swiping Pap smear test, cells are scraped off uterine cervix with a special spatula and brush Epithelial cells obtained by swabbing the inside of a cheek in a mouth with a swab 5

6 Liquid Samples Multiple collection tubes for blood and bone marrow, saliva Select the right tube for the downstream analysis EDTA, Citrate- molecular assays Heparin- flow cytometry, molecular assays? Paxgene RNA assays- long term storage Saliva collection kits Etc. 6

7 Micro arrays Solid Tissue FFPE Tissue Blocks Downstream testing 7

8 Fixation prevents the autolysis and degradation of the tissue and tissue components Maintains tissue integrity for anatomic and microscopic assessment following sectioning Issues to consider: Type of fixative Processing prior to fixation Time to fixation Time in fixative Rate of penetration of fixative Temperature during fixation Tissue Fixation Method of de-calcification (bone marrow core) 8

9 Details of the breakdown of the different fixatives. Fixative Method of fixation Contents B5 Various fixatives Denaturing 5.4% Mercuric Chloride (w/v), 1.1% Sodium Acetate (w/v), 4% Formaldehyde (v/v), Water Bouin s Denaturing<comma> cross-linking 25% of 37% formaldehyde solution, 70% picric acid, 5% acetic acid Carnoy s Denaturing 60% ethanol, 30% chloroform, 10% Glacial acetic acid Glutaraldehyde Cross-linking Generally, 2% v/v of glutaraldehyde to water/pbs Methacarn Denaturing 60% methanol, 30% chloroform, 10% Glacial acetic acid Neutral buffered formalin (NBF) Paraformaldehyde (PFA) Zenker s Cross-linking Cross-linking Denaturing 10% of 37% formaldehyde solution, in a neutral ph Generally, 4% w/v of paraformaldehyde to Water/PBS 5% Mercuric Chloride (w/v), 2.5% Potassium Dichromate (w/v), 5% Glacial acetic acid (v/v), Water 9

10 Fixative Selection Fixation is a vital part of the overall life of a tissue sample and cannot be oversimplified. The type of fixative used, whether cross-linking or dehydrating, will in some way compromise the sample s morphology, RNA/DNA extraction ability, protein evaluation or histochemical staining of the tissue and therefore the fixation regime used can be tailored to the end-result. Neutral-buffered formaldehyde remains the fixative of choice in the majority of histological laboratories. While other fixation regimes may increase the read length of RNA, the utilization of alternative technologies and extraction techniques may circumvent this. Similarly, while some laboratories report an improvement in the detection of epitopes with alternative fixation regimes, an equal or greater number do not. 10

11 FFPE samples pose a major challenge for molecular pathologists. Nucleic acids are heavily modified and trapped by extensive proteinnucleic acid and protein-protein cross linking. Protease digestion is used to release microgram amounts of DNA and RNA from FFPE samples. Purified nucleic acids, although highly fragmented, are suitable for a variety of downstream genomic and gene expression analyses: Polymerase chain reaction (PCR) Quantitative reverse transcription PCR (qrt-pcr) Microarray, array comparative genomic hybridization (CGH), microrna, and methylation profiling Deep sequencing, NGS FFPE Consideration 11

12 Nucleic Acid Preparations Whole blood, serum, plasma, CSF, ascites, semen, saliva, amniotic fluid, and lymph Lysis buffer is typically added directly to cell-free body fluids Whole blood samples, lysis is often a two-step process, RBC lysis, WBC collection/lysis and further processing Formalin Fixed Paraffin-Embedded (FFPE) tissue Tumor region dissected (macro or micro) Deparaffinized using an organic solvent such as xylene Some methods use mineral oil to improve quality of isolated DNA Proteins and nucleases are digested by proteinase K Tissue lysed and processed 12

13 Nucleic Acid Isolation Considerations 1. Automated vs manual 2. Bead vs column based 3. Maximize DNA recovery 4. Removal of inhibitors 5. Removal or inhibition of nucleases 6. Maximize the quality of DNA 7. Double strand vs. single strand 13

14 Nucleic Acid Size Fixative Read lengths DNA Read lengths RNA HOPE 600 bp 300 bp Methacarn 1900 bp 850 bp, 463 bp PAXgene 571 bp 712 bp RCL2 600 bp,> 850 bp 377 bp, 463 bp UMFIX/RTP 1.4 kb, 450 bp Z bp 361 bp 816 bp, 450 bp,700 bp 14

15 Degraded nucleic acids or the presence of contaminants result in: Extra products generated during PCR analysis potential false positives Reduced activity of nucleic acid restriction and modifying enzymes failed assays Short read lengths during sequencing failed assays Particular isolation method is selected based on: Performance of the product Time, effort, and monetary expense for the isolation Whether the nucleic acid is to be used for one or several downstream applications Purity and quality requirements for the most stringent of those applications Assessment of nucleic acid quality: Spectroscopy ( Nanodrop ) OD 260/280 ratio, OD 260/230 ratio Qubit Picogreen assay qpcr assay Nucleic Acid Quality 15

16 Requirements of Molecular Assays Molecular biological applications vary in their requirements for quantity and quality of samples: DNA based assays: PCR requires as little as 1-5 ng DNA, smaller amplicons- more tolerant to fragmented DNA NGS, requires more DNA ( ng), more sensitive to fragmented DNA RNA bases assays: Northern blot analysis is fairly tolerant of contaminants, but is not as tolerant to partially degraded RNA. RT-PCR, it is highly sensitive to contaminants such as phenol, salts, and ethanol. Microarray analysis, contaminants may interfere with enzymatic labeling reactions or increase background signals. Produce highly enriched, un-degraded RNA, free from DNA and proteins Protect RNA from degradation by RNases 16

17 Challenges to Nucleic Acid isolations Extremely small sample sizes (sometimes just a few cells) Presence of contaminants that may interfere with analysis Degradation that starts as soon as the sample is collected; particularly relevant when preparing RNA Samples that are difficult to disrupt (e.g., skin) Isolation of more than one molecule (e.g., genomic DNA, RNA, protein) from a single sample (e.g., cfdna applications) Detection of low-abundant RNA transcripts Detection of viral nucleic acids in biological fluids Isolation of small (< 200 nt) RNA 17

18 Protein Based Assays Stability of proteins critical to immuno-histochemical localization and assessment of expression level of specific proteins in FFPE tissue Same fixation parameters apply as with nucleic based assays The extent that pre-analytical conditions affect protein levels varies with the analyte Phosphoproteins more challenging than other proteins Sample preservation critical for cell based assays, e.g., flow cytometry Newer technologies like Nanostring enable assessment of protein expression along with RNA and DNA assessment 18

19 Sample Storage Considerations Is archiving of samples needed, and for how long? From what sources (e.g., human blood, plant tissue) will you collect samples? How many samples will you collect in one day? How many each year? What is the sample size? Will sample collection take place outside the laboratory? Are facilities available for refrigeration/freezing? Is the sample considered a biohazard? Will the sample be transported off-site? Which molecule(s) (e.g., DNA, RNA, protein) will be isolated from the samples? What is the intended downstream application for the molecule of interest? 19

20 Sample Storage For short-term storage, conditions vary by sample type. Blood samples can be stored at room temp for up to 7 days before isolating DNA, while tissue samples and pelleted cells may be stored at -80 C for several weeks or months. Cells may be frozen directly, or by using a stabilizing agent. Most fresh tissues should be flash frozen in liquid nitrogen for best results. Biological samples that require refrigeration or freezing also require wet ice/cold pack or dry ice for shipping. Human samples must be labeled as potential biohazards. Shipping of these samples to less accessible regions may be limited to certain days of the week, and the shipper must be willing to deal with biohazardous materials. 20

21 Conclusion Pre-analytical steps (sample collection, storage and processing) are critical for accurate downstream biomarker assessment. 21