DdpcrMutationQuantificationChecker

Ddpcr Mutation Quantification

Liquid Biopsy жидкостная биопсия cfDNA ctDNA CTC exosomes NGS qPCR

Utility description: Ddpcr Mutation Quantification

Ddpcr Mutation Quantification Checker — ddPCR Mutation Quantification Validation

ℹ️ Utility performs comprehensive verification of somatic mutation absolute quantification results according to MIQE-dPCR, CLSI MM26 and liquid biopsy standards:
• Statistical Reliability: Assessment of positive droplet count and 95% confidence interval width.
• NTC Purity: Control of false-positive signal absence in negative controls.
• Reproducibility: Verification of technical replicate consistency (CV%).
• LOD/LOQ Classification: Clear separation into reliably quantifiable, detected below LOQ, and not detected results.
• Poisson Compliance: Verification of Poisson distribution applicability to data.

⚠️ IMPORTANT:
• With <3 positive droplets, result is in stochastic uncertainty zone.
• Any NTC contamination renders sample positive results unreliable.
• Below-LOQ results MUST NOT be used for fold-change calculation or therapy response monitoring.

Usage:
DdpcrMutationQuantificationChecker.exe → demo mode (console output)
DdpcrMutationQuantificationChecker.exe input.csv output.json → evaluate your data

Input format:
SampleID,MutationTarget,PositiveDroplets,NegativeDroplets,TotalAcceptedDroplets,MeasuredConcentration_copies_uL,DilutionFactor,FinalConcentration_copies_mL,VAF_Percent,LOD_copies_mL,LOQ_copies_mL,NTC_PositiveDroplets,MaxNTC_Positives,ReplicateCV_Percent,MaxReplicateCV_Percent,LowerCI_95,UpperCI_95

Example:
LB-001,EGFR_L858R,145,19855,20000,7.3,1.0,7300,2.8,50,200,0,2,8.5,25,6100,8600

📍 Scope of Application (Usage Where):
• Liquid Biopsy: Minimal residual disease (MRD) and therapy response monitoring.
• Clinical Oncology: Detection of resistance mutations (EGFR T790M, KRAS G12C).
• Companion Diagnostics Development: Validation of test analytical characteristics.
• Regulatory Submissions: Documentation of ddPCR assay analytical validity.

— WHY IS THIS NEEDED?
ddPCR operates at sensitivity limit where every positive signal matters.
Without strict statistical verification, it is easy to mistake noise for mutation or overestimate rare event precision.
Automated validation guarantees that clinical decisions are based on statistically sound data.

⚠️ CRITICAL:
• NTC Contamination: >Max positives = INVALID. All results questionable.
• Below LOQ: Qualitative result only. Do not use for trends.
• Replicate CV: >25% with >10 positive droplets indicates pipetting or homogenization issue.
• CI Width: Width >50% of value = low confidence, requires more droplets or sample concentration.
• Zero Positives: "Not Detected" is valid only with sufficient total droplets (>10,000).

Key features:
• Four-status result classification
• Automatic 95% CI calculation and verification
• Integrated NTC and replicate checking
• Support for multiple mutation targets
• Compliance with MIQE-dPCR and CLSI MM26

Critical parameters:
• Positive Droplets: ≥3 for reliable detection
• NTC Positives: ≤ Max allowed
• Replicate CV: ≤ 25%
• Concentration vs LOQ: Determines reporting category
• 95% CI Width: ≤ 50% of measured value

💡 Usage tips:
1. LOQ Establishment: Establish LOQ based on dilution series with CV≤25% and ≥95% detection rate.
2. NTC Design: Use minimum 3 NTC wells per plate for reliable background control.
3. Replicates: Minimum 2 technical replicates per sample.
4. Reporting: For below-LOQ results, report "Detected, <LOQ" instead of specific number.
5. Trend Monitoring: In MRD monitoring, use only above-LOQ results for quantitative conclusions.

⚠️ Note: This utility is a statistical validation tool. It does not replace oncologist clinical interpretation but guarantees that ddPCR numerical data is statistically reliable and suitable for decision making.

input.csv

SampleID,MutationTarget,PositiveDroplets,NegativeDroplets,TotalAcceptedDroplets,MeasuredConcentration_copies_uL,DilutionFactor,FinalConcentration_copies_mL,VAF_Percent,LOD_copies_mL,LOQ_copies_mL,NTC_PositiveDroplets,MaxNTC_Positives,ReplicateCV_Percent,MaxReplicateCV_Percent,LowerCI_95,UpperCI_95
LB-2026-PT-001,EGFR L858R,145,19855,20000,7.3,1.0,7300,2.8,50,200,0,2,8.5,25.0,6100,8600
LB-2026-PT-002,KRAS G12D,3,19997,20000,0.15,1.0,150,0.04,50,200,0,2,45.0,25.0,30,420
LB-2026-PT-003,BRAF V600E,28,19972,20000,1.4,1.0,1400,0.5,50,200,5,2,38.0,25.0,900,1950
LB-2026-PT-004,PIK3CA H1047R,0,20000,20000,0.0,1.0,0,0.0,50,200,0,2,0.0,25.0,0,0

URS & FS — User Requirements and Functional Specification

This document describes the controlled interface and behaviour of DdpcrMutationQuantificationChecker for Ddpcr Mutation Quantification Checker.

Domain limits URS input.csv FS IQ/OQ/PQ

Domain limits and critical parameters

Key fragments from the source description are shown below. Before production use, limits must be verified against the approved specification, registration dossier and local SOPs.

• NTC Purity: Control of false-positive signal absence in negative controls.
• With <3 positive droplets, result is in stochastic uncertainty zone.
• Below-LOQ results MUST NOT be used for fold-change calculation or therapy response monitoring.
ddPCR operates at sensitivity limit where every positive signal matters.
⚠️ CRITICAL:
• NTC Contamination: >Max positives = INVALID. All results questionable.
• Replicate CV: >25% with >10 positive droplets indicates pipetting or homogenization issue.
• CI Width: Width >50% of value = low confidence, requires more droplets or sample concentration.
• Zero Positives: "Not Detected" is valid only with sufficient total droplets (>10,000).
Critical parameters:
• Positive Droplets: ≥3 for reliable detection
• NTC Positives: ≤ Max allowed
• Replicate CV: ≤ 25%
• 95% CI Width: ≤ 50% of measured value
1. LOQ Establishment: Establish LOQ based on dilution series with CV≤25% and ≥95% detection rate.
4. Reporting: For below-LOQ results, report "Detected, <LOQ" instead of specific number.

URS — User Requirements Specification

ID	Requirement	Criticality	Acceptance criterion
URS-001	The utility shall accept an input.csv file for Ddpcr Mutation Quantification Checker with headers defined in the data contract.	High	The file is processed without manual header editing.
URS-002	The utility shall perform deterministic QC evaluation without machine learning and without probabilistic conformance decisions.	High	Identical input data, rule version and configuration produce reproducible results.
URS-003	The utility shall validate mandatory fields, data types, ranges, units and domain plausibility.	High	Schema, conversion and range errors are explicitly reported.
URS-004	The utility shall apply domain limits and rules from the description, approved specification, registration dossier and local SOPs.	High	Each check has PASS/WARNING/FAIL and a clear message.
URS-005	The utility shall generate output.json with machine-readable results, source values, warnings, failures and critical findings.	High	JSON is suitable for LIMS/ELN/MES integration and QA/QC review.
URS-006	The utility shall preserve traceability between batch/sample, input file, applied rules and final status.	High	Output contains identifiers, checked parameters and audit metadata.
URS-007	The documentation shall support IQ/OQ/PQ, CSV/CSA and review by internal QA or inspectors.	Medium	URS, FS, input/output contract and test scenarios are supplied with the utility.
URS-008	The utility shall be used as a QC decision-support tool and not as a substitute for approved specifications and QA/QP release decision.	Medium	Documentation states change control and limit-verification expectations.

input.csv contract

#	Field	Type	Sample	Purpose
1	`SampleID`	string / controlled vocabulary	`LB-2026-PT-001`	Sample or laboratory specimen identifier.
2	`MutationTarget`	string / controlled vocabulary	`EGFR L858R`	Controlled input parameter for deterministic QC rules.
3	`PositiveDroplets`	decimal	`145`	Controlled input parameter for deterministic QC rules.
4	`NegativeDroplets`	decimal	`19855`	Controlled input parameter for deterministic QC rules.
5	`TotalAcceptedDroplets`	decimal	`20000`	Controlled input parameter for deterministic QC rules.
6	`MeasuredConcentration_copies_uL`	decimal	`7.3`	Component ratio; structural or formulation CQA.
7	`DilutionFactor`	decimal	`1.0`	Controlled input parameter for deterministic QC rules.
8	`FinalConcentration_copies_mL`	decimal	`7300`	Volume/dose used for load, limit or release calculation.
9	`VAF_Percent`	decimal	`2.8`	Controlled input parameter for deterministic QC rules.
10	`LOD_copies_mL`	decimal	`50`	Volume/dose used for load, limit or release calculation.
11	`LOQ_copies_mL`	decimal	`200`	Volume/dose used for load, limit or release calculation.
12	`NTC_PositiveDroplets`	decimal	`0`	Controlled input parameter for deterministic QC rules.
13	`MaxNTC_Positives`	decimal	`2`	Controlled input parameter for deterministic QC rules.
14	`ReplicateCV_Percent`	decimal	`8.5`	Controlled input parameter for deterministic QC rules.
15	`MaxReplicateCV_Percent`	decimal	`25.0`	Controlled input parameter for deterministic QC rules.
16	`LowerCI_95`	decimal	`6100`	Controlled input parameter for deterministic QC rules.
17	`UpperCI_95`	decimal	`8600`	Controlled input parameter for deterministic QC rules.

SampleID,MutationTarget,PositiveDroplets,NegativeDroplets,TotalAcceptedDroplets,MeasuredConcentration_copies_uL,DilutionFactor,FinalConcentration_copies_mL,VAF_Percent,LOD_copies_mL,LOQ_copies_mL,NTC_PositiveDroplets,MaxNTC_Positives,ReplicateCV_Percent,MaxReplicateCV_Percent,LowerCI_95,UpperCI_95
LB-2026-PT-001,EGFR L858R,145,19855,20000,7.3,1.0,7300,2.8,50,200,0,2,8.5,25.0,6100,8600
LB-2026-PT-002,KRAS G12D,3,19997,20000,0.15,1.0,150,0.04,50,200,0,2,45.0,25.0,30,420
LB-2026-PT-003,BRAF V600E,28,19972,20000,1.4,1.0,1400,0.5,50,200,5,2,38.0,25.0,900,1950

Input validation rules

ID	Field	Rule	Criticality
VR-001	`SampleID`	The field shall match an approved dictionary or accepted string representation.	High
VR-002	`MutationTarget`	The field shall match an approved dictionary or accepted string representation.	High
VR-003	`PositiveDroplets`	The field shall match an approved dictionary or accepted string representation.	High
VR-004	`NegativeDroplets`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-005	`TotalAcceptedDroplets`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-006	`MeasuredConcentration_copies_uL`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-007	`DilutionFactor`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-008	`FinalConcentration_copies_mL`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-009	`VAF_Percent`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-010	`LOD_copies_mL`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-011	`LOQ_copies_mL`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-012	`NTC_PositiveDroplets`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-013	`MaxNTC_Positives`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-014	`ReplicateCV_Percent`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-015	`MaxReplicateCV_Percent`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-016	`LowerCI_95`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-017	`UpperCI_95`	The field shall match an approved dictionary or accepted string representation.	Medium

FS — Functional Specification

ID	Function	Implementation
FS-001	CLI execution	Support execution modes: demo mode without arguments and production mode input.csv output.json.
FS-002	CSV import	Read input.csv in UTF-8/CSV-compatible format and validate header and expected columns.
FS-003	Schema validation	Check mandatory fields, column count, unknown key fields and empty mandatory values.
FS-004	Type conversion	Convert numeric, flag and text values; invalid format is recorded as a row-level error.
FS-005	Domain rule engine	Apply rules for Ddpcr Mutation Quantification Checker, including critical limits from the description and approved specification.
FS-006	Status aggregation	Produce final status: FAIL for critical failure, WARNING for non-critical deviation, PASS for conformance.
FS-007	JSON export	Write output.json with detailed checks, source values, warnings, failures and critical findings.
FS-008	Audit support	Keep result structure suitable for review, deviation investigation and calculation reproduction.
FS-009	Integration contract	Support the scenario LIMS/ELN/MES → input.csv → utility → output.json → portal/admin review.
FS-010	Error handling	Return explicit messages for missing file, empty CSV, invalid schema, output write failure and invalid format.

Example output.json

{
  "utilityId": "ddpcrmutationquantificationchecker",
  "utilityFolder": "DdpcrMutationQuantificationChecker",
  "package": "LiquidBiopsy",
  "overallStatus": "PASS|WARNING|FAIL",
  "sourceFile": "input.csv",
  "processedAtUtc": "2026-06-10T00:00:00Z",
  "checks": [
    {
      "parameter": "SampleID",
      "value": "LB-2026-PT-001",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-001"
    },
    {
      "parameter": "MutationTarget",
      "value": "EGFR L858R",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-002"
    },
    {
      "parameter": "PositiveDroplets",
      "value": "145",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-003"
    },
    {
      "parameter": "NegativeDroplets",
      "value": "19855",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-004"
    },
    {
      "parameter": "TotalAcceptedDroplets",
      "value": "20000",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-005"
    },
    {
      "parameter": "MeasuredConcentration_copies_uL",
      "value": "7.3",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-006"
    },
    {
      "parameter": "DilutionFactor",
      "value": "1.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-007"
    },
    {
      "parameter": "FinalConcentration_copies_mL",
      "value": "7300",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-008"
    },
    {
      "parameter": "VAF_Percent",
      "value": "2.8",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-009"
    },
    {
      "parameter": "LOD_copies_mL",
      "value": "50",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-010"
    },
    {
      "parameter": "LOQ_copies_mL",
      "value": "200",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-011"
    },
    {
      "parameter": "NTC_PositiveDroplets",
      "value": "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": "DdpcrMutationQuantificationChecker.documentation.html"
  }
}

Traceability matrix

URS	FS	Test	Evidence
URS-001	FS-001, FS-002	OQ-001	Verify execution and import of valid input.csv.
URS-002	FS-005, FS-006	OQ-004	Repeat the same dataset and compare output.json.
URS-003	FS-003, FS-004, FS-010	OQ-002, OQ-003	Verify missing columns and invalid types.
URS-004	FS-005, FS-006	OQ-004, PQ-001	Verify critical deviations on real/boundary data.
URS-005	FS-007, FS-009	OQ-005	Verify JSON schema and downstream-system suitability.
URS-006	FS-008	OQ-006	Verify identifiers and audit metadata.
URS-007	FS-008, FS-010	IQ-001, OQ-007	Verify documentation completeness and control evidence.
URS-008	FS-005, FS-008	PQ-002	Verify review workflow and no replacement of QA decision.

IQ/OQ/PQ test scenarios

ID	Scenario	Expected result
IQ-001	Verify executable, input.csv, documentation and checksum availability.	Delivery set is complete; version is recorded.
OQ-001	Valid sample row from input.csv.	PASS or acceptable WARNING according to rules.
OQ-002	Remove a mandatory CSV column.	Schema error or FAIL with missing-column reference.
OQ-003	Place a non-numeric value into a numeric field.	Type-conversion error with row/field reference.
OQ-004	Set a critical parameter outside the limit.	FAIL and critical finding.
OQ-005	Verify output.json structure.	All mandatory sections are present and JSON is valid.
OQ-006	Verify batch/sample traceability.	Input and result identifiers match.
PQ-001	Verify 3–5 real user batches/samples.	Result is confirmed by QC/QA review.
PQ-002	Verify deviation workflow and manual QA decision.	Utility supports review but does not replace approved decision.

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.

Included in packages

Liquid Biopsy QC Suite

QC and pre-analytical control package for liquid biopsy workflows: cfDNA/ctDNA, CTC, EV/exosomes, methylation, NGS/qPCR/ddPCR, sample quality, contamination, sensitivity and reporting checks.

Open