WbcBackgroundSignalChecker

URS & FS — User Requirements and Functional Specification

This document describes the controlled interface and behaviour of WbcBackgroundSignalChecker for WBC Background Signal Checker.

Domain limits and critical parameters

• • CD45+ Events: Number of leukocyte events in enriched CTC/EV fractions.
• • WBC background is the primary limiting factor of liquid biopsy sensitivity (not sequencing depth).
• • CTC Analysis: Assessment of enriched fraction purity from WBC contamination.
• • Exosomes/EVs: Control of WBC-derived vesicles in isolated preparation.
• Liquid biopsy sensitivity is limited not by detection technology but by biological noise.
• This is critically important for MRD monitoring reliability and early recurrence detection.
• ⚠️ CRITICAL:
• • Residual WBC ≤10⁵/mL: For cfDNA analysis. Exceedance = insufficient centrifugation.
• • WBC DNA ≤10%: Above this level, tumor signal is significantly diluted.
• • SNR ≥3.0: Below this threshold, tumor signal is statistically indistinguishable from WBC background.
• • Hemolysis ≤50: High index = WBC lysis = gDNA contamination.
• • CD45 Depletion ≥95%: When depletion is used, efficiency must be confirmed.
• • Four-tier classification (Acceptable / Elevated / High / Critical)
• Critical parameters:
• • Residual WBC: ≤ 1×10⁵ cells/mL (cfDNA)
• • WBC DNA Contribution: ≤ 10%
• • CD45+ Events: ≤ Assay-specific limit
• • Signal-to-Noise Ratio: ≥ 3.0

URS — User Requirements Specification

input.csv contract

SampleID,AssayType,BiofluidSource,ResidualWBC_per_mL,MaxResidualWBC_per_mL,WBC_DNA_Contribution_Percent,MaxWBC_DNA_Contribution_Percent,CD45_Positive_Events,MaxCD45_Events,TumorSignal_VAF_or_Count,WBC_Background_Noise,MinSignalToNoise_Ratio,Hemolysis_Index,MaxHemolysis_Index,CentrifugationSpeed_g,DoubleSpin_Performed,CD45_Depletion_Used,DepletionEfficiency_Percent,MinDepletionEfficiency_Percent
LB-WBC-001,cfDNA_NGS,Plasma,2500,100000,1.2,10.0,3,50,0.45,0.02,3.0,8,50,16000,1,true,99.2,95.0
LB-WBC-002,CTC_Enumeration,Whole Blood,8500000,100000,35.0,10.0,420,50,5,3.8,3.0,85,50,800,0,false,0,95.0
LB-WBC-003,Methylation,Plasma,45000,100000,8.5,10.0,28,50,0.15,0.06,3.0,62,50,16000,1,true,96.5,95.0

Input validation rules

FS — Functional Specification

Example output.json

{
  "utilityId": "wbcbackgroundsignalchecker",
  "utilityFolder": "WbcBackgroundSignalChecker",
  "package": "LiquidBiopsy",
  "overallStatus": "PASS|WARNING|FAIL",
  "sourceFile": "input.csv",
  "processedAtUtc": "2026-06-10T00:00:00Z",
  "checks": [
    {
      "parameter": "SampleID",
      "value": "LB-WBC-001",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-001"
    },
    {
      "parameter": "AssayType",
      "value": "cfDNA_NGS",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-002"
    },
    {
      "parameter": "BiofluidSource",
      "value": "Plasma",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-003"
    },
    {
      "parameter": "ResidualWBC_per_mL",
      "value": "2500",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-004"
    },
    {
      "parameter": "MaxResidualWBC_per_mL",
      "value": "100000",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-005"
    },
    {
      "parameter": "WBC_DNA_Contribution_Percent",
      "value": "1.2",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-006"
    },
    {
      "parameter": "MaxWBC_DNA_Contribution_Percent",
      "value": "10.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-007"
    },
    {
      "parameter": "CD45_Positive_Events",
      "value": "3",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-008"
    },
    {
      "parameter": "MaxCD45_Events",
      "value": "50",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-009"
    },
    {
      "parameter": "TumorSignal_VAF_or_Count",
      "value": "0.45",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-010"
    },
    {
      "parameter": "WBC_Background_Noise",
      "value": "0.02",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-011"
    },
    {
      "parameter": "MinSignalToNoise_Ratio",
      "value": "3.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-012"
    }
  ],
  "criticalFindings": [],
  "warnings": [],
  "audit": {
    "inputHash": "sha256:<calculated at runtime>",
    "rulesVersion": "<utility executable version>",
    "documentation": "WbcBackgroundSignalChecker.documentation.html"
  }
}

Traceability matrix

IQ/OQ/PQ test scenarios

QA/QC and change control

• Do not rename columns without updating validator, documentation and test set.
• Retain input.csv, output.json, executable version and checksum.
• Before production use, perform IQ/OQ/PQ or equivalent CSV/CSA verification.
• Critical limits shall be verified against the approved specification, registration dossier and local SOPs.
• The utility provides structured QC decision support; final release decision remains with QA/QP and approved procedures.