Assay for hemoglobin a (HBA) detection and genotyping
Inventors
Walker, Andrew • Robinson, Matt
Assignees
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Publication Number
US-12224041-B2
Publication Date
2025-02-11
Expiration Date
Abstract
Disclosed herein are techniques for implementation of a Hemoglobin A (HBA) assay and an HBA genotyping decision tree matrix in clinical testing. Particularly, aspects are directed to obtaining raw data from the HBA assay performed on a plurality of samples, calculating a first set of probe ratios for each sample based on the raw data, identifying a number of reference samples to be combined as a synthetic reference sample based on the first set of probe ratios, calculating a second set of probe ratios for each sample of the plurality of samples based on the raw data and the synthetic reference sample, and determining, by the decision tree matrix, an HBA genotype for each sample based on the second set of probe ratios for each sample and copy number calling thresholds for sample probe/reference probe ratios associated with each probe of the plurality of probes.
Core Innovation
The invention relates to a clinical Hemoglobin A (HBA) genotyping system that performs an HBA assay on a plurality of samples to obtain raw probe copy-number data for probes targeting an α-globin gene cluster region. Raw probe data are converted to probe ratios using a selected reference sample, where the reference sample is selected based on variability among probes and an absence of a copy number variation.
A synthetic reference is then generated by combining multiple low-variability, CNV-negative samples, and second probe ratios are calculated relative to the synthetic reference. The system uses a decision tree matrix and copy number calling thresholds associated with each probe to determine an HBA genotype for each sample based on the second probe ratios.
The resulting classifications assign samples to normal, polymorphic, or CNV states, and further sub-classify CNVs into particular targeted deletions or duplications, including α13.7, α14.2, SEA, FIL/THAI, MED, α20.5, HS-40, and Constant Spring. The system also determines zygosity and compound heterozygotes or duplications, including handling an α13.7A/α14.2C indeterminate phasing scenario.
A confirmation testing step is used for a subset of samples that are determined to have one or more particular copy number variations, using multiplex assay methodology and gel electrophoresis or Sanger sequencing. The described approach includes quality control and performance evaluation metrics, including analytical sensitivity, specificity, reproducibility, and concordance with manual review.
Claims Coverage
The independent claim coverage describes a computer-implemented HBA genotyping workflow with reference refinement using multiple CNV-negative samples, probe ratio calculation, decision-tree classification using per-probe thresholds, genotype assignment, and confirmatory testing for a subset. Dependent claims add variability/QC gating and detailed CNV subclassification into normal, CNV, or polymorphic states with named α-globin deletion and duplication types.
Synthetic reference generation from multiple reference samples
Identifying multiple reference samples to be combined as a synthetic reference sample based on probe ratios, and generating the synthetic reference sample as a concatenation of the reference samples in the list.
Probe ratio calculation relative to reference and synthetic reference
Calculating a first set of probe ratios based on the raw data and the reference sample, and calculating a second set of probe ratios based on the raw data and the synthetic reference sample.
Decision tree matrix genotype determination using per-probe copy-number calling thresholds
Inputting the second set of probe ratios into a decision tree matrix; determining an HBA genotype based on the second set of probe ratios and copy number calling thresholds associated with each probe; and assigning one or more particular copy number variations based on the HBA genotype.
Confirmation testing for a subset of samples
Performing a confirmation test by multiplex assay methodology and gel electrophoresis or Sanger sequencing for a subset of samples determined to have one or more particular copy number variations.
Variability metric-based acceptance gating using a predetermined threshold
Classifying each sample as failing if any variability metric exceeds a predetermined threshold based on control probe variability, and for samples that do not fail, calculating probe ratios for test probes relative to the reference.
Normal, CNV, and polymorphic classification with CNV sub-classification
Classifying samples as normal, having a copy number variation, or polymorphic based on aberrant probe ratio patterns, and further sub-classifying CNV samples as large targeted deletion, duplication, or other.
Zygosity determination and detailed CNV deletion/duplication subclassification for named α-globin types
Subclassifying samples with large targeted deletions into heterozygous versus homozygous deletions and subclassifying those deletions or related states for specific deletions and duplications including SEA, FIL/THAI, MED, α20.5, α13.7, and α14.2, or classifying remaining samples as other.
HBA assay using an oligonucleotide pool targeting an α-globin gene cluster region with capillary electrophoresis
Performing an HBA assay using an oligonucleotide pool targeting an α-globin gene cluster region, hybridizing the nucleic acid with the pool to generate probes, and resolving the probes by capillary electrophoresis to produce raw data comprising HBA copy number data.
Overall, the claims cover a computer-implemented HBA genotyping approach that refines reference selection via a synthetic reference, computes probe ratios, applies a decision tree matrix with per-probe copy-number calling thresholds to call HBA genotypes and assign particular copy number variations, and confirms a subset by multiplex assay with gel electrophoresis or Sanger sequencing. Dependent claims further specify variability-based gating, normal/CNV/polymorphic and detailed CNV subclassification including zygosity and named α-globin deletion and duplication types.
Stated Advantages
Reported analytical sensitivity for targeted calls with manual-review fallbacks.
Reported analytical specificity for negatives.
Reported reproducibility and concordance with manual review.
Documented Applications
Clinical Hemoglobin A (HBA) genotyping of samples using an HBA assay with probe-based copy number data, including classification into normal, polymorphic, or CNV and assignment of particular targeted deletion and duplication types.
Confirmatory testing for a subset of samples determined to have particular copy number variations using multiplex assay methodology with gel electrophoresis or Sanger sequencing.
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