CtDnaSampleStabilityChecker

CtDna Sample Stability

Liquid Biopsy жидкостная биопсия cfDNA ctDNA CTC exosomes NGS qPCR

Utility description: CtDna Sample Stability

CtDna Sample Stability Checker — ctDNA Sample Stability Assessment

ℹ️ Utility performs comprehensive pre-analytical stability assessment for liquid biopsy samples according to CLSI MM26 and tube manufacturer guidelines:
• Time to Processing: Control of "collection → centrifugation" interval accounting for stabilization tube type.
• Temperature Regime: Verification of storage and transportation conditions compliance.
• Freeze-Thaw Cycles: Accounting for thermal stress leading to DNA fragmentation.
• Hemolysis and Fragmentation: Assessment of leukocyte lysis markers contaminating sample with genomic DNA.

⚠️ IMPORTANT:
• In EDTA tubes, leukocytes begin lysing within 2–4 hours at room temperature, diluting ctDNA with genomic DNA.
• High hemolysis level correlates with PCR inhibition and reduced NGS sensitivity.
• Fragmentation (short/long fragment ratio) is a direct indicator of sample degradation.

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

Input format:
SampleID,TubeType,CollectionTime,ProcessingTime,MaxTimeToProcessing_Hours,StorageTemp_C,MaxStorageTemp_C,FreezeThawCycles,MaxFreezeThawCycles,HemolysisIndex,MaxHemolysisIndex,CfDNA_Yield_ng_mL,ExpectedMinYield_ng_mL,FragmentationRatio,MaxFragmentationRatio

Example:
CTDNA-001,Streck,2026-06-08 08:00,2026-06-09 10:00,48,22,30,0,1,12,50,18.5,5.0,0.15,0.3

📍 Scope of Application (Usage Where):
• Liquid Biopsy Laboratories: Incoming sample control before cfDNA extraction.
• Clinical Trials: Monitoring of sample collection protocol compliance in multi-center trials.
• Biobanking: Retrospective assessment of archival samples suitability for molecular analysis.
• Test Development: Validation of pre-analytical conditions for new ctDNA panels.

— WHY IS THIS NEEDED?
ctDNA concentration in plasma is fractions of a percent of total cfDNA.
Any pre-analytical error (processing delay, overheating, re-freezing) increases genomic DNA background and masks tumor signal.
Automated verification prevents expensive sequencing of unsuitable samples and reduces false-negative result risk.

⚠️ CRITICAL:
• Tube-Specific Limits: EDTA ≤2–4h, Streck ≤7 days, PAXgene ≤3 days at RT. Do not apply one tube's limits to another.
• Hemolysis Index: >50–100 units = high risk of inhibition and gDNA contamination.
• Fragmentation Ratio: Elevated ratio indicates leukocyte lysis even with normal cfDNA yield.
• Freeze-Thaw: Each cycle increases fragmentation; ≤1 cycle recommended for ctDNA.

Key features:
• Adaptive limits by tube type
• Five-parameter stability assessment
• Integration of physical and molecular degradation markers
• Three-tier classification (Stable / At Risk / Unstable)
• Compliance with CLSI MM26-A2

Critical parameters:
• Time to Processing: ≤ Tube-specific limit
• Storage Temperature: ≤ Max allowed
• Freeze-Thaw Cycles: ≤ Max allowed
• Hemolysis Index: ≤ Max threshold
• Fragmentation Ratio: ≤ Max ratio

💡 Usage tips:
1. Tube Type: Always specify correct type — all limits depend on it.
2. Double Centrifugation: Mandatory for complete leukocyte removal; record both step times.
3. Aliquoting: Aliquot plasma immediately after processing to minimize freeze-thaw cycles.
4. Documentation: Record exact time of each pre-analytical step in LIS.
5. Trends: Monitor % unstable samples by site to identify systemic logistics issues.

⚠️ Note: This utility assesses pre-analytical stability. It does not replace post-extraction cfDNA QC (yield, size profile) but guarantees that source material was collected and processed correctly.

input.csv

SampleID,TubeType,CollectionTime,ProcessingTime,MaxTimeToProcessing_Hours,StorageTemp_C,MaxStorageTemp_C,FreezeThawCycles,MaxFreezeThawCycles,HemolysisIndex,MaxHemolysisIndex,CfDNA_Yield_ng_mL,ExpectedMinYield_ng_mL,FragmentationRatio,MaxFragmentationRatio
CTDNA-2026-001,Streck,2026-06-08 08:00:00,2026-06-09 10:00:00,48.0,22.0,30.0,0,1,12,50,18.5,5.0,0.15,0.3
CTDNA-2026-002,EDTA,2026-06-08 08:00:00,2026-06-08 14:00:00,2.0,22.0,25.0,2,1,85,50,45.0,5.0,0.55,0.3
CTDNA-2026-003,PAXgene,2026-06-08 09:00:00,2026-06-10 09:00:00,72.0,4.0,25.0,0,2,8,50,22.0,5.0,0.12,0.3

URS & FS — User Requirements and Functional Specification

This document describes the controlled interface and behaviour of CtDnaSampleStabilityChecker for CtDna Sample Stability 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.

⚠️ CRITICAL:
• Tube-Specific Limits: EDTA ≤2–4h, Streck ≤7 days, PAXgene ≤3 days at RT. Do not apply one tube's limits to another.
• Hemolysis Index: >50–100 units = high risk of inhibition and gDNA contamination.
• Freeze-Thaw: Each cycle increases fragmentation; ≤1 cycle recommended for ctDNA.
• Adaptive limits by tube type
Critical parameters:
• Time to Processing: ≤ Tube-specific limit
• Storage Temperature: ≤ Max allowed
• Freeze-Thaw Cycles: ≤ Max allowed
• Hemolysis Index: ≤ Max threshold
• Fragmentation Ratio: ≤ Max ratio
1. Tube Type: Always specify correct type — all limits depend on it.

URS — User Requirements Specification

ID	Requirement	Criticality	Acceptance criterion
URS-001	The utility shall accept an input.csv file for CtDna Sample Stability 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	`CTDNA-2026-001`	Sample or laboratory specimen identifier.
2	`TubeType`	string / controlled vocabulary	`Streck`	Controlled input parameter for deterministic QC rules.
3	`CollectionTime`	string / controlled vocabulary	`2026-06-08 08:00:00`	Time parameter for process, incubation, storage or analytical workflow.
4	`ProcessingTime`	string / controlled vocabulary	`2026-06-09 10:00:00`	Time parameter for process, incubation, storage or analytical workflow.
5	`MaxTimeToProcessing_Hours`	integer / decimal	`48.0`	Time parameter for process, incubation, storage or analytical workflow.
6	`StorageTemp_C`	decimal	`22.0`	Temperature profile/MKT; storage, shipping and stability parameter.
7	`MaxStorageTemp_C`	decimal	`30.0`	Temperature profile/MKT; storage, shipping and stability parameter.
8	`FreezeThawCycles`	integer / decimal	`0`	Controlled input parameter for deterministic QC rules.
9	`MaxFreezeThawCycles`	integer / decimal	`1`	Controlled input parameter for deterministic QC rules.
10	`HemolysisIndex`	decimal	`12`	Controlled input parameter for deterministic QC rules.
11	`MaxHemolysisIndex`	decimal	`50`	Controlled input parameter for deterministic QC rules.
12	`CfDNA_Yield_ng_mL`	decimal	`18.5`	Volume/dose used for load, limit or release calculation.
13	`ExpectedMinYield_ng_mL`	decimal	`5.0`	Volume/dose used for load, limit or release calculation.
14	`FragmentationRatio`	decimal	`0.15`	Component ratio; structural or formulation CQA.
15	`MaxFragmentationRatio`	decimal	`0.3`	Component ratio; structural or formulation CQA.

SampleID,TubeType,CollectionTime,ProcessingTime,MaxTimeToProcessing_Hours,StorageTemp_C,MaxStorageTemp_C,FreezeThawCycles,MaxFreezeThawCycles,HemolysisIndex,MaxHemolysisIndex,CfDNA_Yield_ng_mL,ExpectedMinYield_ng_mL,FragmentationRatio,MaxFragmentationRatio
CTDNA-2026-001,Streck,2026-06-08 08:00:00,2026-06-09 10:00:00,48.0,22.0,30.0,0,1,12,50,18.5,5.0,0.15,0.3
CTDNA-2026-002,EDTA,2026-06-08 08:00:00,2026-06-08 14:00:00,2.0,22.0,25.0,2,1,85,50,45.0,5.0,0.55,0.3
CTDNA-2026-003,PAXgene,2026-06-08 09:00:00,2026-06-10 09:00:00,72.0,4.0,25.0,0,2,8,50,22.0,5.0,0.12,0.3

Input validation rules

ID	Field	Rule	Criticality
VR-001	`SampleID`	The field shall match an approved dictionary or accepted string representation.	High
VR-002	`TubeType`	The field shall match an approved dictionary or accepted string representation.	High
VR-003	`CollectionTime`	The field shall match an approved dictionary or accepted string representation.	High
VR-004	`ProcessingTime`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-005	`MaxTimeToProcessing_Hours`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-006	`StorageTemp_C`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-007	`MaxStorageTemp_C`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-008	`FreezeThawCycles`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-009	`MaxFreezeThawCycles`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-010	`HemolysisIndex`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-011	`MaxHemolysisIndex`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-012	`CfDNA_Yield_ng_mL`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-013	`ExpectedMinYield_ng_mL`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-014	`FragmentationRatio`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-015	`MaxFragmentationRatio`	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 CtDna Sample Stability 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": "ctdnasamplestabilitychecker",
  "utilityFolder": "CtDnaSampleStabilityChecker",
  "package": "LiquidBiopsy",
  "overallStatus": "PASS|WARNING|FAIL",
  "sourceFile": "input.csv",
  "processedAtUtc": "2026-06-10T00:00:00Z",
  "checks": [
    {
      "parameter": "SampleID",
      "value": "CTDNA-2026-001",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-001"
    },
    {
      "parameter": "TubeType",
      "value": "Streck",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-002"
    },
    {
      "parameter": "CollectionTime",
      "value": "2026-06-08 08:00:00",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-003"
    },
    {
      "parameter": "ProcessingTime",
      "value": "2026-06-09 10:00:00",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-004"
    },
    {
      "parameter": "MaxTimeToProcessing_Hours",
      "value": "48.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-005"
    },
    {
      "parameter": "StorageTemp_C",
      "value": "22.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-006"
    },
    {
      "parameter": "MaxStorageTemp_C",
      "value": "30.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-007"
    },
    {
      "parameter": "FreezeThawCycles",
      "value": "0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-008"
    },
    {
      "parameter": "MaxFreezeThawCycles",
      "value": "1",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-009"
    },
    {
      "parameter": "HemolysisIndex",
      "value": "12",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-010"
    },
    {
      "parameter": "MaxHemolysisIndex",
      "value": "50",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-011"
    },
    {
      "parameter": "CfDNA_Yield_ng_mL",
      "value": "18.5",
      "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": "CtDnaSampleStabilityChecker.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