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CLIA CCC: Molecular Diagnostics and Cytogenetics

CLIA CCC · Work through your exam, one topic at a time.

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Molecular Diagnostics and Cytogenetics

What this domain tests

This domain covers nucleic acid extraction, amplification and sequencing, cytogenetic and microarray methods, variant interpretation, and the controls that keep molecular results trustworthy.

Methods and quality

  • Extraction quality: an A260/280 near 1.8 indicates acceptable DNA purity; heme and heparin inhibit amplification.
  • PCR: in real-time PCR the cycle threshold is inversely proportional to starting target; digital PCR gives absolute quantification; multiplexing requires validated primer interactions.
  • Contamination control: no-template controls, unidirectional workflow, and uracil-N-glycosylase systems for carryover.
  • Sequencing: Sanger for single variants and confirmations; next-generation sequencing for panels and exomes. Analytical sensitivity depends on depth; variant allele fraction = variant reads ÷ total reads.
  • Variant classification follows ACMG/AMP categories (pathogenic, likely pathogenic, uncertain significance, likely benign, benign); HGVS governs nomenclature.

Cytogenetics

  • G-banded karyotype resolution is roughly 5–10 Mb and detects balanced rearrangements that microarrays miss.
  • FISH is targeted (about 100–200 kb) and needs known probes; chromosomal microarray detects copy-number gains and losses but not balanced events.
  • ISCN nomenclature describes karyotypes; do not mix it with HGVS.

Worked example

A tumor NGS report lists a variant at 3% variant allele fraction with 800× depth. Reasoning: 3% of 800 reads is about 24 variant reads, well above typical minimal read thresholds, so the call is technically credible. However, a low fraction can reflect tumor heterogeneity, low tumor cellularity, or clonal hematopoiesis. Compare with a blood or normal tissue sample, confirm the assay's validated limit of detection (often around 5%), and avoid reporting a germline implication from tumor-only testing.

Traps that cost marks

  • Reporting a variant of uncertain significance as actionable.
  • Ignoring clonal hematopoiesis in a plasma or tumor-only result.
  • Contamination-driven false positives when no-template controls fail.
  • Over-reading low-level variants below the validated limit of detection.
  • Using microarray to exclude a balanced translocation.