Huggett: If dPCR started same time as qPCR - feels that dPCR would surpass it. But how will dPCR be calibrated? (B/c it needs it) #TRICON
2:25pm March 8th 2016 via Hootsuite
Huggett: Use and sharing ot calibrators are a headache - dPCR can improve performance and design of secondary ref materials #TRICON
2:24pm March 8th 2016 via Hootsuite
Huggett: No calibration material used; the EQA results were 'fantastic'. dPCR can enable new applications, and improve existing ones #TRICON
2:23pm March 8th 2016 via Hootsuite
Huggett: Clinical virology labs - all within 0.8 logs, only meas. ref materials. "This is fantastic" - but still 0.5 log to improve #TRICON
2:21pm March 8th 2016 via Hootsuite
Huggett: Metrology for infectious disease: Charite Hosp for CMV detection, main EQA schemes. #TRICON
2:20pm March 8th 2016 via Hootsuite
Huggett: Can use as spike-in as extractio control; 40-54% extraction efficiencies. #TRICON
2:18pm March 8th 2016 via Hootsuite
Huggett: UK strategy board, mentions ctDNA standard from HDX, measured using dPCR, can assign uncertainty #TRICON
Huggett: Doing a 21-lab interlab QX100/200 study; 'I can say it is extremely good', results published in a few months. #TRICON
2:17pm March 8th 2016 via Hootsuite
Huggett: "Not possible using any other molecular method today" - within 10 or 20% of each other. #TRICON
2:16pm March 8th 2016 via Hootsuite
Huggett: 2750 vs 3000 molecules on Fluidigm. Formulatrix Consellation; QS3D, QX200, RDT: "Essentially the same' #TRICON
2:15pm March 8th 2016 via Hootsuite
Huggett: Looked at dsDNA bidning, 5' nuclease (TaqMan), Scorpion, all for digital PCR Minute differences, w/o calibration #TRICON
2:14pm March 8th 2016 via Hootsuite
Huggett: Consortium called SITrace, uses KRAS mutation as a model system. Potential for ctDNA non-invasive monitoring #TRICON
2:10pm March 8th 2016 via Hootsuite
Huggett: Full analytical workflow - in the biological matrix, or purified, or purified amplicon #TRICON
Huggett: Used to gude - ref material to 2nd-ary ref, to tertiary. Can dPCR be above 2ndary reference standards? #TRICON
2:09pm March 8th 2016 via Hootsuite
Huggett: ISO 17511:2003 - For IVD devices, in clinical chemistry is common. For molecular in clinical dx - rare #TRICON
2:07pm March 8th 2016 via Hootsuite
Huggett: LGC goes across metrology.Points made here reflect '14 review https://t.co/cg6NySNTBw #TRICON
2:06pm March 8th 2016 via Hootsuite
Huggett: NB: I interviewed Jim here at Behind the Bench @servingscience https://t.co/qJrce9TPx4 #TRICON
2:05pm March 8th 2016 via Hootsuite
Jim Huggett (Laboratory of Gov't Chemists UK) The role of digital PCR in revolutionizing the standardization of molecular medicine #TRICON
2:03pm March 8th 2016 via Hootsuite
Wang:Q:Biggest impediment to diagnostic for miRNA? A:Still a lot of potential, but need to understand fundamentals of pkging #TRICON
2:01pm March 8th 2016 via Hootsuite
Wang: Fasting, exercise, affect miRNA in circulation changes. Tissue injury too (he notes 'this is why I do not exercise') #TRICON
1:58pm March 8th 2016 via Hootsuite
Wang: Even sex-specific differences. #TRICON '13 ref https://t.co/P4d2tM2ps5
1:57pm March 8th 2016 via Hootsuite
Wang: Points to 2011 Clin Chem ref https://t.co/ukaBKci9e5 Also 4C and -20C storage, difference noted #TRICON
1:55pm March 8th 2016 via Hootsuite
Wang: Shows data from different library kits, sample type (serum vs plasma), cent. speed and hemolysis #TRICON
1:53pm March 8th 2016 via Hootsuite
Wang: Thus there is inconsistency and irreproducibility of miRNA data in circulation #TRICON
Wang: On top of this, RNA editing has an effect. Also library prep kit (NEB vs ILMN) show different profile levels (oops) #TRICON
1:52pm March 8th 2016 via Hootsuite
Wang: Sequence difference (length 17 vs 22nt and single base differences between species). Also pri- and pre-forms, and isomIRs too #TRICON
1:50pm March 8th 2016 via Hootsuite
Wang:Shows chart for measurement platforms - qPCR, microarray or NGS; NGS is now the most cost-effective. #TRICON
1:47pm March 8th 2016 via Hootsuite
Wang: About 2K miRNAs, and may be an ideal biomarker - specific, sensitive, predictive, robust, translatable, non-invasive. #TRICON
1:45pm March 8th 2016 via Hootsuite
Kai Wang (Inst System Biol) Standardization of measuring circulating RNA #TRICON
1:41pm March 8th 2016 via Hootsuite
Alikian:A2: May be redesign of the assay, or add'l processing. Not a contamination problem, it has to do w/assay or sample #TRICON
1:38pm March 8th 2016 via Hootsuite
Alikian:Q:Cp between qPCR vs dPCR? A: ABL is the same between; more BCR-ABL1 at the MMR level up. 0.01% or 0.001% FPs show. #TRICON
1:37pm March 8th 2016 via Hootsuite
Alikian:Q:Which primer/probe set? A:Identical for each platform, agreed upon by a EU consortium #TRICON
1:36pm March 8th 2016 via Hootsuite
Alikian: They look for MRD for clinical decision-making, impt to use the most sensitive method. #TRICON
1:34pm March 8th 2016 via Hootsuite
Alikian: One RDT is 9 replicates on the other platforms. Clinical assays a bit noisy, some FPs in neg samples #TRICON
1:33pm March 8th 2016 via Hootsuite
Alikian: Recommends that cost and cost/rcn would come into play. Balance of sensitivity. 'All platforms behave in similar manner' #TRICON
1:32pm March 8th 2016 via Hootsuite
Alikian: Across the platforms, 5 logs for QS3D and BioRad, 'Sensitivity of at least 5 log for RainDrop' so it appears a bit better #TRICON
1:31pm March 8th 2016 via Hootsuite
Alikian: FP signals from each platform will determine sensitivity of particular platform. Goes onto analysis of pt samples #TRICON
1:30pm March 8th 2016 via Hootsuite
Alikian: Below 3 logs - 0.1%, 0.01%, 0.001%; box-whisker shows some FP's in negatives in all 3 platforms #TRICON
1:26pm March 8th 2016 via Hootsuite
Alikian: Used this 2.3x as a correction factor. Then run across 3 platforms. Data: 3log reduction, almost no difference cross-plat #TRICON
1:25pm March 8th 2016 via Hootsuite
Alikian: Had to show 'in house plasmid bias': run reference material against in-house synthetic; found consistent bias 2.3x higher #TRICON
1:24pm March 8th 2016 via Hootsuite
Alikian: Have done duplex and uniplex reactions; found duplex is much more precise. #TRICON
1:23pm March 8th 2016 via Hootsuite
Alikian:EU measurement Inst produced secondary WHO ref material; used TMO QS3D (QuantStudio 3D), can go down to 4-8 copies #TRICON
Alikian: MMR 5-50 copies; CMR 0.01% ratio on IS 5-15 copies; 0.001% ratio on IS 1-5 copies. #TRICON
1:22pm March 8th 2016 via Hootsuite
Alikian: Slide of four platforms FLDM, TMO, BIO and RDT. FLDM ruled out due to too low number of partitions #TRICON
1:21pm March 8th 2016 via Hootsuite
Alikian: digital PCR - absolute quant, minimal std req's, improved sensitivity and precision. #TRICON
1:20pm March 8th 2016 via Hootsuite
Alikian: Shows spread of Ct at 10, 1 molecule detection. Each lab will have Ct assignments; differences in results is a lot of work #TRICON
1:19pm March 8th 2016 via Hootsuite
Alikian: Discontinue drug -for quality of life, and cost of drug; but qPCR has limitations (std curves, compromised sens.) #TRICON
1:18pm March 8th 2016 via Hootsuite
Alikian.Milestones: 100% is baseline, 1-log is CHR, 3 log is 'Major Molecular Resp'; >4 log is good #TRICON
1:17pm March 8th 2016 via Hootsuite
Alikian:Every 3 mo monitoring using RT-qPCR. BCR-ABL1/AB1 x 100 = % of fusion transcripts. Very effective - <10% good prognosis #TRICON
1:16pm March 8th 2016 via Hootsuite
Alikian: Oncogenes all dependent on the TK activity; effective in the clinic. Normal life expectancy. Marker is xcript of fusion #TRICON
1:15pm March 8th 2016 via Hootsuite
