OPTIMIZATION AND VALIDATION OF THE LIGHTSCANNER® ASSAY FOR THE MCAD

DEFICIENCY DISEASE

Sadhaasivam KamalanaadhanMST Program – Biotechnology Track

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COMPANY BACKGROUND

Founded in 1984.

Enterprise of the University of Utah – Department of Pathology.

Currently employs more than 2600 employees.

Among nation’s leading clinical and pathological reference laboratories.

INTERNSHIP OBJECTIVES

Optimize and validate the Lightscanner® Assay for the MCAD deficiency disease.

Prepare an internal database of MCAD mutations.

Learn the process of pricing an assay.

PROJECT BACKGROUND MCAD – Medium-chain acyl-CoA dehydrogenase deficiency

(ACADM gene) Autosomal Recessive hereditary disease. Most common mutation

being c.985A>G.

Impairs beta-oxidation of medium-chain fatty acids.

1/15,000 live births in the USA.

Symptoms include lethargy, vomiting, hypoglycemic coma.

Treatable through diet management.

Fatal if not diagnosed early.

ASSAY WORKFLOW

TECHNICAL BACKGROUND Scanning assay is based

on Hi-Res Melting® for mutation scanning. DNA is denatured or

‘melted’. Special dye whose

fluorescence decreases as DNA becomes single stranded – real time melt curves

Lightscanner® detects differences between melt curves of different DNA.

Source: http://www.path.utah.edu/news/hi-res-dna-melting-analysis/image/image_view_fullscreen

Source: http://en.wikipedia.org/wiki/File:STD_HRM_plot.JPG

OPTIMIZATION OF ASSAY – PCR CONDITIONS ACADM gene has 12 exons.

Exon specific primers designed by Idaho Technology Inc.

Amplify exons with a mix of Lightscanner® Master Mix, primers and mineral oil overlay.

Thermocycling conditions were optimized using a touchdown PCR protocol on the BioRad iCycler® (Ramp rate 3.3°C/second)

AGAROSE GEL ELECTROPHORESIS

OPTIMIZATION OF ASSAY - LIGHTSCANNER®

Melting temperature range set between 70°C – 95°C.

Normalized melt curves to yield tight fitting of curves.

DNA extraction methods affect the melt curve analysis.

Lack of mineral oil, presence of bubbles etc. affect performance.

VALIDATION - PRECISION

Defined as the reproducibility between replicate measurements. Within Runs – How well does instrument perform when

same sample is tested repeatedly? Between Runs – How well does instrument perform when

same samples are tested between different days, instrument, operating conditions etc.?

CM#1 – Wild type ACC#1 – Patient sample Day1: CM#1 vs. ACC#1 Day2: CM#1 vs. ACC #1 Day3: CM#1 vs. ACC#1, ACC#1, ACC#1

(triplicates of Acc#1 vs. CM#1)Within

Between

Exons 4 & 5 – within runs

Problematic exons 8 &12 – Within Runs

Instrument Calls

Operator Calls

n – negative, p – positive, ?n – questionable negative

VALIDATION - ACCURACY

Concordance of results using two different assays. Analytical Sensitivity –

How sensitive is the assay? True Positives/(True Positives+ False Negatives)

Analytical Specificity – How specific is the assay? True Negatives/(True Negatives + False positives)

SETUP OF ACCURACY RUNS

25 de-identified samples KS#1 to KS#25 run over 4 days vs. Wild-type CM#1 Day4: CM#1 vs. KS#1 to KS#6 Day5: CM#1 vs. KS#7 to KS#12 Day6: CM#1 vs. KS#13 to KS#18 Day7: CM#1 vs. KS#19 to KS#25

Operator vs. Instrument Calls

Compared with existing sequencing data.

OPERATOR ASSISTANCE REQUIRED

Instrument Scores

Operator Scores

OVERALL – INSTRUMENT VS. OPERATOR

Instrument Operator

Exon Results

Can I ever become

independent?

Someday maybe you

will!

INSTRUMENT (DEJECTED)

OPERATOR(REASSURING)

VALIDATION SUMMARY FOR EXONS

DISCUSSION AND RECOMMENDATIONS

Operator judgment is needed to increase sensitivity of the assay. Problematic when there are large amount of samples to be

analyzed

Further optimization for the assay is needed. Software settings optimization Redesigning primers Consistency in DNA extraction methods

Assay can be used along with sequencing.

INTERNSHIP OBJECTIVES

Optimize and validate the Lightscanner® Assay for the MCAD deficiency disease.

Prepare an internal database of MCAD mutations.

Learn the process of pricing an assay.

MUTATION DATABASE

Why the need for an internal database? Huge amount of literature sources, databases for mutations

available. Condensing the data helps the clinical lab.

About the database. Created in MS-Excel after streamlining mutations from

various sources such as HGMD, NCBI etc. Contains MCAD gene sequence, nucleotide and codon

position of mutations and links to publications /references. Mutations categorized into Missense/Nonsense, Splice

mutations, deletions and insertions.

INTERNSHIP OBJECTIVES

Optimize and validate the Lightscanner® Assay for the MCAD deficiency disease.

Prepare an internal database of MCAD mutations.

Learn the process of pricing an assay.

PRICING COMPONENTS

COST ACCOUNTING

Documents the actual cost of performing the test.

Helps maintain inventory.

Two types of costs involved. Direct Costs - Cost of reagents, supplies, man-hours, royalties

Factored into a pre-made spreadsheet.

Indirect Costs - Costs associated with running the lab – Refrigeration, Electricity, General maintenance etc. Determined by the accounting department.

COMPETITOR ANALYSIS

Lets us know what competition offers for the same test in terms of price, features etc.

Helps ARUP to be competitive. ARUP hotline – R&D to Clinical Lab.

Spreadsheet containing following details Background of disease and the test. Name of Lab/Company performing the test. Internal test code and CPT code. Name of the test and the methodology used for testing. Price of the test offered by competition plus comments.

SAMPLE COMPETITOR ANALYSIS REPORT

(Black = Input; Blue = Formulas)

Description Driver $

Direct & Indirect Testing Costs per BU 50.00 Variable Overhead Costs 15.0% 7.50 Fixed Overhead Costs 0.0% - Net Margin Markup Factor 12.0% 6.00

Minimum Price, Excluding Add-On's 27.0% 63.50

Add-On's:Logistics 3.00 Royalties 66.50 16.63 20.0%

Minimum Price, Including Add-On's 83.13

If test is subject to a royalty, insert royalty % here.

PRICING SPREADSHEET - EXAMPLE

CLIENT PRICING

CONCLUSION – THE STORY OF AN ASSAY

THANK YOU