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

• Discuss various elements of collecting,

processing and storing samples used for

biomarker analysis- DNA, RNA, or cellular

based assays

Goals

Disclaimer: Views expressed herein are sole responsibility of the author

and not that of her employer.2

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• 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?

Sample Collection Considerations

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• 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

Sample Collection

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• 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

Sampling Options

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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.

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Solid Tissue

• Micro arrays FFPE Tissue Blocks

• Downstream testing 7

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• 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

– Method of de-calcification (bone marrow core)

Tissue Fixation

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Details of the breakdown of the different fixatives.Fixative Method of fixation Contents

B5 Denaturing5.4% Mercuric Chloride (w/v), 1.1% Sodium Acetate (w/v), 4%

Formaldehyde (v/v), Water

Bouin’sDenaturing<comma>

cross-linking25% 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)

Cross-linking 10% of 37% formaldehyde solution, in a neutral pH

Paraformaldehyde (PFA)

Cross-linking Generally, 4% w/v of paraformaldehyde to Water/PBS

Zenker’s Denaturing5% Mercuric Chloride (w/v), 2.5% Potassium Dichromate (w/v), 5%

Glacial acetic acid (v/v), Water

Various fixatives

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• 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.

Fixative Selection

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• FFPE samples pose a major challenge for molecular pathologists.

• Nucleic acids are heavily modified and trapped by extensive protein-

nucleic 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

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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

Nucleic Acid Preparations

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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

Nucleic Acid Isolation Considerations

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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 816 bp, 450 bp ,700 bp

Z7 2400 bp 361 bp

Nucleic Acid Size

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• 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™) OD260/280 ratio, OD260/230 ratio

– Qubit®

– Picogreen assay

– qPCR assay

Nucleic Acid Quality

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• 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 (10-200 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

Requirements of Molecular Assays

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• 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

Challenges to Nucleic Acid isolations

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• 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

Protein Based Assays

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• 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?

Sample Storage Considerations

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• 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.

Sample Storage

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Pre-analytical steps (sample collection,

storage and processing) are critical for

accurate downstream biomarker

assessment.

Conclusion

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