Redefining Reference Standards

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Biopsy Process Control

Output from patient biopsies is subject to very high levels of variability. The quantity of DNA that can be obtained is dependent on a range of factors, including:

  • Tissue volume (size of biopsy, thickness of cut sections)
  • Tissue quality (necrotic vs. living)
  • Tumor:normal cell ratio
  • Mutant:wild-type gene ratio
  • Conservation method (FFPE vs. Frozen)
  • FFPE method (formalin concentration, formalin:tissue perfusion ratio, time to fixation)

Laboratories currently employ a range of reference materials in an attempt to address the heterogeneity seen in biopsies, however their accuracy is limited due to a range of issues, including copy number or allelic burden not being defined, or not being representative of real tumor samples. Horizon Diagnostics has overcome these limitations by developing an extensive menu of genetically standardized reference materials.While there are a variety of other reference materials available each one has its own limitations and below is a table to summarise these:

 Defined copy numberDefined Allele BurdenStoichiometric Multiplexing CapabilityRepresents Tumor Samples
Genome ComplexityCompromised DNA (FFPE)Tumor Cell Genetics
Synthetic oligos
Cell line mixtures
Primary tissue samples
Synthetic oligos spiked into cell DNA
HDx™ Reference Standards

To create its' HDx™ Reference Standards, Horizon Diagnostics engineers the mutation of interest (e.g. BRAF V600E) into a human cell line at the appropriate gene loci using its GENESIS™ gene engineering platform. This process introduces the desired mutation into the cell’s genome exactly as it would occur in a tumor, yielding one ‘mutant’ cell line and one matched ‘normal’ or parent cell line, differing only by the specific alteration precisely introduced into the genome.

The mutant cell line is then titrated against the unmodified parental cell line to achieve the desired ratio of mutant to wild-type alleles (e.g. B-Raf V600E at 5%). The allelic frequency is validated by digital PCR. 

FFPE Block Allelic Frequency
Below is data demonstrating Horizon Diagnostics' ability to manufacture FFPE blocks at defined allelic frequencies that are consistent throughout the block. Sections 1, 2, 3 are at the start middle and end of each block. 

Gene Mutation
B-Raf V600E 25% 5% 3.5% 1%
B-Raf V600K 50% 5% 1% --

 

Consistent allelic frequency throughout the FFPE Blocks

The blended cells are processed into formalin fixed paraffin embedded blocks, which are then cut into sections. A proprietary method is used for formatting the FFPE blocks which preserves DNA integrity and ensures identical DNA yield and mutant:wild-type allelic ratios across every section from each block.

The FFPE sections are rigorously validated using Droplet Digital PCR and are supplied in PCR tubes, ready to be incorporated directly into your genotyping assay 

Below is a summary of the typical workflow for the manufacture of our FFPE sections. To view more information on the QC steps Click Here

Process for manufacturing HDx™ FFPE Reference Standards 

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