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Cytogenetics

Proficiency Testing

Proficiency Testing (PT), often referred to as External Quality Assessment (EQA), is the ultimate validation of a laboratory’s analytical accuracy. It involves the analysis of unknown samples provided by an external accrediting agency (such as the College of American Pathologists - CAP) to verify that the laboratory’s results match the “correct” answer and the consensus of peer laboratories. Under CLIA’88 regulations, participation in PT is mandatory for all non-waived testing, including high-complexity Cytogenetics

The Golden Rule: “Treat Like a Patient”

The fundamental principle of proficiency testing is that PT samples must be treated exactly like patient samples to test the routine workflow of the laboratory

  • Routine Workflow: The sample should be accessioned, processed, analyzed, and reported using the lab’s standard operating procedures (SOPs). It should not receive “special treatment” (e.g., being analyzed repeatedly by every senior laboratory scientist to ensure perfection)
  • Rotation of Staff: Testing duties must be rotated among all testing personnel. It is a compliance violation to have only the Lead Laboratory scientist or Supervisor perform all PT samples
  • Prohibition on Communication
    • Strict Rule: It is strictly forbidden to communicate with any other laboratory regarding PT samples before the submission deadline
    • Consequence: If two labs are caught comparing answers (collusion), both labs face the most severe penalty: immediate revocation of their CLIA certificate and a ban on testing for at least one year

The PT Process Cycle

The PT process follows a strict calendar cycle, typically occurring three times per year

  • Receipt: The lab receives a package containing the “unknowns.” In Cytogenetics, because shipping live cell cultures is logistically difficult and prone to failure, PT samples are often “Dry Challenges.”
    • Dry Challenge: Pre-fixed slides, cell suspensions, or (most commonly) digital images downloadable from the web
  • Analysis
    • Karyotyping: Laboratory scientistsanalyze digital images of metaphase spreads, construct a karyogram, and write the International System for Human Cytogenomic Nomenclature (ISCN) string
    • FISH: Laboratory scientistscount signals in digital images or on provided slides to determine amplification or deletion
    • Microarray (CMA): The lab downloads raw data files (e.g., CEL files) and processes them through their own software to detect Copy Number Variants (CNVs)
  • Reporting: Results are submitted to the agency (e.g., CAP) by a strict deadline
  • Evaluation: The agency grades the results against the Intended Response (the correct diagnosis) and the Peer Consensus (what >80% of other labs reported)

Grading & Scoring

Cytogenetics PT is generally graded on a pass/fail basis for the diagnosis and the ISCN nomenclature

  • The 80% Rule: To pass a specific “event” (one mailing), the laboratory must generally achieve a score of 80% or higher
  • Consensus: Grading is often based on peer group consensus. If 95% of labs identify a deletion on chromosome 5, and your lab identifies it as normal, your lab is penalized. If only 40% of labs find the abnormality (due to poor sample quality), the challenge may be “ungraded” and not count against the score
  • Nomenclature Errors: Even if the diagnosis is correct (e.g., “Down Syndrome identified”), the lab can lose points for incorrect ISCN formatting (e.g., writing 47,XY,+21 instead of 47,XY,+21 or using incorrect punctuation)

Performance Status & Sanctions

CLIA tracks performance over time. A single mistake is handled differently than a pattern of failure

  • Unsatisfactory Performance
    • Definition: Failing to achieve 80% on a single: testing event
    • Action: The lab must perform an internal investigation and document corrective action, but testing continues
  • Unsuccessful Performance (The “2 out of 3” Rule)
    • Definition: Failing two out of three: consecutive testing events (e.g., Failed Event 1, Passed Event 2, Failed Event 3)
    • Action: This is a critical regulatory failure. The laboratory may be required to Cease Testing: for that specific analyte (e.g., stop performing Amniotic Fluid analysis) until they can demonstrate reinstatement
    • Reinstatement: Usually requires passing two consecutive re-instatement PT events

Corrective Action for PT Failures

Every lost point on a PT survey requires a documented investigation, even if the overall score was a “Pass” (e.g., 90%)

  • Investigation Steps
    • Clerical Check: Did we get the right answer but type the code in wrong on the website? (Most common error)
    • Methodological Check: Did our probe fail to hybridize? Was the image quality on our monitor poor?
    • Interpretive Check: Did the laboratory scientist miss a subtle banding change?
  • Remediation
    • Retraining the laboratory scientist
    • Reviewing the “Critique” provided by CAP (which explains the intended answer)
    • Re-testing the sample (if material remains) to see if the error is reproducible

Alternative Assessment Protocols (AAP)

For some rare tests, no commercial PT program exists (e.g., a specific “home-brew” FISH probe for a rare sarcoma). The lab is still required to verify accuracy twice a year

  • Split-Sample Testing: Dividing a patient sample and sending half to another certified laboratory to ensure results match
  • Blind Internal Review: A previously analyzed sample is re-assigned to a laboratory scientist (blinded to the original result) to see if they generate the same result
  • Clinical Correlation: Retroactively checking if the genetic result matched the clinical outcome (less robust, but acceptable in limited cases)