Redefining Reference Standards

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Quality Controlled

HDx™ Reference Standards are fully characterized to guarantee the quality, specificity and consistency of each batch. The production workflow is precisely monitored and controlled, from cell line engineering (where appropriate) through to Reference Standard production.  Explore the workflow diagram below to learn more about what we do at each step.

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"Wild type cell line"
Clonal wild type cell line
Clonal mutant
cell line
Reference Standard Manufacturing
Cell Line Validation Single Cell Dilute Genomic DNA Digital PCR Analysis GENESIS™ Gene-Editing FISH Slides FFPE Sections FFPE Block

Horizon Diagnostics' Manufacturing and Quality Control Steps

Single Cell Dilute


Single Cell Dilution

HDx works with highly characterized wild type cell lines, which have been single cell diluted to ensure a homogenous clonal population ready for gene engineering

 

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Genomic DNA


Genomic DNA Validation

A broad range of molecular assays are used to monitor batch consistency of Horizon Diagnostics reference standards. For our DNA range these cover:

Reference Standard Test

Assay

Allelic frequency

Droplet Digital PCR™

Gene copy number

Droplet digital PCR™

Genotype

Sanger sequencing of locus specific PCR

Quality

Agarose gel electrophoresis, Quantitatitive PCR of GAPDH locus

Quantification

Spectophotometric assay

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Digital PCR Analysis


At Horizon Diagnostics we have adopted Digital PCR into our manufacturing workflow and developed >150 mutation specific assays.

For each digital PCR assay a dilution curve (50%, 20%, 10%, 5% and 1%) has been analysed. Data below shows data for our B-Raf V600K DNA and corresponding FFPE Reference Standards.

Example of Digital PCR Analysis of B-Raf V600E Reference standards

For the FFPE standards we routinely analyse extracted DNA from multiple locations (e.g. beginning, middle, end) from the FFPE block to monitor block homogeneity. 

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GENESIS™ Gene-Editing


Horizon Diagnostics uses its proprietary GENESIS™ cell line engineering platform to incorporate cancer relevant mutations at the endogenous gene loci.

Gene-editing capabilities include:

  • Point mutations/SNPs
  • Gene deletions (1bp - 100kb’s)
  • Translocations
  • Amplifications
  • Bar-coding

Click here for background information on GENESIS™

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FISH Slides


FISH Slide Validation

A broad range of molecular assays are used to monitor batch consistency of Horizon Diagnostics reference standards. Our FISH reference slides are fully characterised using microscopic inspection and summarised below:

Reference Standard Test

Assay

Chromosomal Aberration

Specific FISH assay

Cell Number

Microscopic inspection

Spread Quality

Microscopic inspection following DAPI staining

Fusion-specific FISH assay

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FFPE Sections


FFPE Section Validation

A broad range of molecular assays are used to monitor batch consistency of Horizon Diagnostics reference standards. We analyse the FFPE sections directly together with DNA extracted from these samples. These tests are summarised below:

Reference Standard Test

Assay

Allelic frequency

Droplet Digital PCR™

FFPE block homogeneity

H+E Staining

FFPE block cell number

Aperio® Digital section scan

Genotype

Sanger sequencing of locus specific PCR

Quality

Quantitatitive PCR of GAPDH locus

Quantification

Quantifluor™ assay

Confirm gene copy number

Droplet digital PCR™

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FFPE Block


FFPE Block Validation

At Horizon Diagnostics we have developed a proprietary FFPE preparation method to ensure FFPE block consistency. Each section we provide has a theoretical DNA quantity of >1μg and due to the nature of formalin fixation we expect a DNA yield of 400ng – 700ng depending on the extraction method used.

For maximum recovery we use the automated PromegaMaxwell® 16 FFPE Plus LEV DNA Purification Kit.

For each FFPE block we analyse DNA extraction from multiple sections and perform an Aperio®digital scan to verify cell numbers per section. Below is data from DNA extracted from 10 sections each from 8 independent FFPE blocks together with the corresponding H+E stains.

We pride ourselves on the consistency and homogeneity of our FFPE blocks and our FFPE preparation abilities are the best in the world.

H+E staining & DNA Extraction demonstrates the homogeneity and consistency of our FFPE technology

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Cell line validation


Cell line validation confirms the homogenous wild type (parental) cell line pre‐engineering and
isogenic mutant cell line post‐engineering, to ensure the final mutant cell line is identical to the
original wild type cell line except for the specific mutation incorporated. The validation tests
performed are summarized below:

Cell Validation Test

Assay

Cell line ID

STR and/or SNP6.0

Cell engineering integration

gDNA locus specific PCR & Sanger

Sequencing

Gene expression of modified allele

cDNA-PCR & Sanger Sequencing

Clonality

Droplet Digital PCR™, gDNA PCR & Sanger

Sequencing

Gene copy number

Droplet Digital PCR™

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