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Molecular Biology

Result Calculation, Interpretation, & Reporting

The final phase of the molecular workflow is the translation of raw data (fluorescence, band intensity, or sequence reads) into a clinically actionable result. This stage requires the laboratory scientist to move beyond wet-bench technique and apply statistical and clinical judgment. Quality Assurance (QA) in this phase ensures that the calculated values are accurate, the interpretation considers all control data, and the final report is clear, standardized, and legally defensible

Result Calculation: Quantitative Analysis

In quantitative assays (e.g., HIV-1, HCV, CMV Viral Loads), the laboratory must calculate the exact number of target molecules present. This is almost exclusively achieved using Real-Time PCR (qPCR) and the Standard Curve method

  • The Crossing Threshold (Ct)
    • The raw data point in qPCR is the Ct value (also known as Cp or Cq). This is the cycle number at which the fluorescence of the sample exceeds the background threshold
    • Relationship: There is an inverse relationship between the Ct value and the starting concentration. High viral loads amplify early (Low Ct, e.g., 15), while low viral loads amplify late (High Ct, e.g., 35)
  • The Standard Curve
    • To convert Ct to Concentration, the lab runs a set of standards with known concentrations (e.g., \(10^3, 10^4, 10^5\) copies/mL)
    • Regression Analysis: The instrument plots the Ct values (\(y\)-axis) against the Logarithm of the Concentration (\(x\)-axis). This generates a linear regression line: \(y = mx + b\)
    • Calculation: The patient’s Ct is plugged into this equation to solve for \(x\) (Concentration)
  • Reaction Efficiency (\(E\))
    • The slope of the standard curve indicates the efficiency of the PCR. Ideally, the amount of DNA doubles every cycle (\(100\%\) efficiency)
    • Ideal Slope: \(-3.32\) indicates \(100\%\) efficiency
    • QA Criteria: Slopes outside the range of \(-3.1\) to \(-3.6\) indicate a compromised reaction (e.g., pipetting errors in standards or expired reagents), and the calculated results may be inaccurate
  • Dilution Factors
    • If a sample is too concentrated (above the linear range) and requires manual dilution, the software result must be multiplied by the dilution factor
    • \(\text{Final Result} = \text{Instrument Result} \times \text{Dilution Factor}\)

Result Interpretation: Reading the Signals

The instrument software provides a preliminary call, but the scientist is responsible for the final interpretation. This involves reviewing the curve shape, the controls, and the validity of the data

  • Qualitative Interpretation (Detected vs. Not Detected)
    • True Positive: The sample must display a sigmoidal (S-shaped) amplification curve that crosses the threshold. The Ct value must be within the valid range (e.g., \(< 40\) cycles)
    • True Negative: No amplification curve is seen for the target, BUT the Internal Control (IC) must amplify. If the IC fails, the result is not “Negative”; it is “Invalid”
    • The “Gray Zone” (Weak Positives): A sample with a very late Ct (e.g., 38 or 39) requires scrutiny. The scientist must check the curve shape. If it is jagged, linear, or drifting, it is likely background noise (artifact) rather than true amplification
  • Quantitative Interpretation (Ranges)
    • Within the AMR: Results falling within the Analytical Measurement Range (e.g., 100 to 10,000,000 IU/mL) are reported as the specific number calculated
    • Above the AMR: Result is “\(> 10,000,000\).” To get a specific number, the sample must be diluted and re-run
    • Detected, but Below the LoD: If the assay detects the virus (curve is present), but the calculated value is lower than the Limit of Detection (e.g., calculated at 15 IU/mL when LoD is 20 IU/mL), the result is interpreted as “Detected, < 20 IU/mL.” This informs the doctor that the virus is present, but at a level too low to accurately quantify
  • Genotyping/Mutation Analysis (Allelic Discrimination)
    • For assays detecting mutations (e.g., Factor V Leiden), results are interpreted based on fluorescence clusters
    • Homozygous Wild Type: High signal in Channel A (Normal), No signal in Channel B (Mutant)
    • Homozygous Mutant: No signal in Channel A, High signal in Channel B
    • Heterozygous: Intermediate signals in both channels

Reporting Protocols

The laboratory report is the legal medical record. It must be standardized to prevent ambiguity. Molecular reporting often differs from chemistry reporting due to the complexity of the methodologies

  • Nomenclature and Language
    • “Detected / Not Detected”: This is preferred over “Positive / Negative” in molecular diagnostics, as it strictly describes the analytical finding (presence of DNA) rather than a clinical diagnosis
    • Units of Measure: Quantitative results must specify units
      • Copies/mL: The physical number of DNA/RNA molecules
      • International Units (IU/mL): A standardized unit normalized against World Health Organization (WHO) standards. This allows results to be compared between different labs using different manufacturers. Always prefer IU/mL when available
    • Logarithmic Transformation: Physicians often monitor “Log Changes.” Reports may include the Log10 value alongside the absolute number (e.g., \(100,000 \text{ IU/mL} = 5.0 \text{ Log IU/mL}\))
  • Required Elements of the Report
    • Methodology: The report must state the method used (e.g., “Real-Time PCR”) and the specific limits of the assay (e.g., “Limit of Detection: 20 IU/mL”)
    • Disclaimer (ASR/LDT): If the test uses Analyte Specific Reagents (ASR) or is a Laboratory Developed Test (LDT), a federally mandated disclaimer must be appended: “This test was developed and its performance characteristics determined by [Lab Name]. It has not been cleared or approved by the FDA.”
  • Critical Values
    • Certain molecular results are considered “Critical” and require immediate notification to the provider (documented read-back)
    • Examples: Positive CSF (Meningitis) panel, Positive Blood Culture Identification (Sepsis), or detection of Select Agents (Bioterrorism organisms like Francisella tularensis)
  • Clinical Significance Comments
    • Molecular reports often include interpretive comments to aid the clinician
    • Example (Hepatitis C): “A change in viral load of less than 0.5 Log10 is not considered biologically significant.”
    • Example (Pharmacogenetics): “Patient is homozygous for CYP2C19*2. This genotype is associated with poor metabolism of Plavix.”