ExosomeSampleQcChecker

Exosome Sample QC

Liquid Biopsy жидкостная биопсия cfDNA ctDNA CTC exosomes NGS qPCR

Utility description: Exosome Sample QC

Exosome Sample QC Checker — Exosome/EV Quality Control

ℹ️ Utility performs comprehensive exosome/EV characterization according to MISEV2023 (ISEV) guidelines:
• Physical Characterization: Particle concentration, mean diameter, polydispersity index (PDI).
• Purity: Particle-to-protein ratio — key indicator of non-vesicular contaminant co-isolation.
• Molecular Identification: Presence of ≥2 positive exosome markers (CD63, CD81, TSG101).
• Contamination Exclusion: Absence of intracellular/organelle markers (Calnexin, GM130).
• Integrity: Assessment of vesicle membrane preservation.

⚠️ IMPORTANT:
• Term "exosomes" applies ONLY upon confirmation of endosomal origin (markers + size).
• Precipitation method often yields high protein and lipoprotein contamination.
• Calnexin+ indicates cell lysis and intracellular vesicle contamination.

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

Input format:
SampleID,IsolationMethod,BiofluidSource,ParticleConcentration_per_mL,MinParticleConcentration,MeanDiameter_nm,MinDiameter_nm,MaxDiameter_nm,PDI,MaxPDI,ProteinConcentration_ug_mL,ParticlesPerUgProtein,MinParticlesPerUgProtein,PositiveMarker_CD63,PositiveMarker_CD81,PositiveMarker_TSG101,NegativeMarker_Calnexin,NegativeMarker_GM130,MembraneIntegrity_Percent,MinMembraneIntegrity

Example:
EXO-001,SEC,Plasma,2.5E10,1E8,95,30,150,0.18,0.3,120,2.1E8,1E8,true,true,true,false,false,92,70

📍 Scope of Application (Usage Where):
• Liquid Biopsy Research: EV QC before RNA/protein extraction.
• EV Therapeutics Development: Characterization of exosome-based pharmaceutical products.
• Standardization: Ensuring inter-laboratory reproducibility per MISEV2023.
• Data Publication: Mandatory QC data provision for ISEV journals.

— WHY IS THIS NEEDED?
EV research field suffers from reproducibility crisis due to lack of standardization.
Different isolation methods yield fundamentally different vesicle populations.
Without strict multi-parameter characterization, it is impossible to distinguish exosomes from microvesicles, lipoproteins, or protein aggregates.
MISEV2023 establishes minimum requirements, and this utility automates their verification.

⚠️ CRITICAL:
• Size 30-150 nm: Outside this range — not exosomes (possibly MVs or apoptotic bodies).
• Positive Markers ≥2/3: Mandatory MISEV2023 requirement for identification.
• Negative Markers = ABSENT: Calnexin/GM130+ = contamination, sample is impure.
• Purity Ratio ≥1×10⁸: Low ratio indicates protein predominance over vesicles.
• PDI ≤0.3: High PDI means heterogeneous mixture requiring additional purification.

Key features:
• Seven-parameter MISEV2023 assessment
• Automatic positive/negative marker verification
• Integration of physical and molecular criteria
• PASS / WARNING / FAIL classification
• Support for various isolation methods and biofluids

Critical parameters:
• Mean Diameter: 30–150 nm
• PDI: ≤ 0.3
• Purity Ratio: ≥ 1×10⁸ particles/µg protein
• Positive Markers: ≥ 2 of 3 (CD63, CD81, TSG101)
• Negative Markers: Absent (Calnexin, GM130)
• Membrane Integrity: ≥ 70%

💡 Usage tips:
1. Isolation Method: SEC and immunoaffinity yield cleaner exosomes than precipitation.
2. NTA vs TRPS: Use orthogonal methods to confirm concentration and size.
3. Western Blot: Always include positive and negative markers in every WB.
4. Biofluid: Plasma preferred over serum (fewer coagulation artifacts).
5. Documentation: Report ALL MISEV2023 parameters in publications and reports.

⚠️ Note: This utility implements minimum MISEV2023 requirements. Therapeutic applications may require additional tests (sterility, endotoxins, functional activity).

input.csv

SampleID,IsolationMethod,BiofluidSource,ParticleConcentration_per_mL,MinParticleConcentration,MeanDiameter_nm,MinDiameter_nm,MaxDiameter_nm,PDI,MaxPDI,ProteinConcentration_ug_mL,ParticlesPerUgProtein,MinParticlesPerUgProtein,PositiveMarker_CD63,PositiveMarker_CD81,PositiveMarker_TSG101,NegativeMarker_Calnexin,NegativeMarker_GM130,MembraneIntegrity_Percent,MinMembraneIntegrity
EXO-2026-001,SEC,Plasma,2.5E10,1E8,95,30,150,0.18,0.3,120,2.1E8,1E8,true,true,true,false,false,92,70
EXO-2026-002,Precipitation,Serum,5.0E9,1E8,185,30,150,0.45,0.3,850,5.9E6,1E8,true,false,true,true,false,65,70
EXO-2026-003,Immunoaffinity,Urine,1.2E9,1E8,110,30,150,0.22,0.3,45,2.7E7,1E8,true,true,false,false,false,88,70

URS & FS — User Requirements and Functional Specification

This document describes the controlled interface and behaviour of ExosomeSampleQcChecker for Exosome Sample QC 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.

• Purity: Particle-to-protein ratio — key indicator of non-vesicular contaminant co-isolation.
• Molecular Identification: Presence of ≥2 positive exosome markers (CD63, CD81, TSG101).
SampleID,IsolationMethod,BiofluidSource,ParticleConcentration_per_mL,MinParticleConcentration,MeanDiameter_nm,MinDiameter_nm,MaxDiameter_nm,PDI,MaxPDI,ProteinConcentration_ug_mL,ParticlesPerUgProtein,MinParticlesPerUgProtein,PositiveMarker_CD63,PositiveMarker_CD81,PositiveMarker_TSG101,NegativeMarker_Calnexin,NegativeMarker_GM130,MembraneIntegrity_Percent,MinMembraneIntegrity
Different isolation methods yield fundamentally different vesicle populations.
⚠️ CRITICAL:
• Positive Markers ≥2/3: Mandatory MISEV2023 requirement for identification.
• Purity Ratio ≥1×10⁸: Low ratio indicates protein predominance over vesicles.
• PDI ≤0.3: High PDI means heterogeneous mixture requiring additional purification.
• Support for various isolation methods and biofluids
Critical parameters:
• PDI: ≤ 0.3
• Purity Ratio: ≥ 1×10⁸ particles/µg protein
• Positive Markers: ≥ 2 of 3 (CD63, CD81, TSG101)
• Membrane Integrity: ≥ 70%
1. Isolation Method: SEC and immunoaffinity yield cleaner exosomes than precipitation.
⚠️ Note: This utility implements minimum MISEV2023 requirements. Therapeutic applications may require additional tests (sterility, endotoxins, functional activity).

URS — User Requirements Specification

ID	Requirement	Criticality	Acceptance criterion
URS-001	The utility shall accept an input.csv file for Exosome Sample QC 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	`EXO-2026-001`	Sample or laboratory specimen identifier.
2	`IsolationMethod`	string / controlled vocabulary	`SEC`	Method or process approach; controlled reference value.
3	`BiofluidSource`	string / controlled vocabulary	`Plasma`	Controlled input parameter for deterministic QC rules.
4	`ParticleConcentration_per_mL`	decimal	`2.5E10`	Volume/dose used for load, limit or release calculation.
5	`MinParticleConcentration`	integer / decimal	`1E8`	Component ratio; structural or formulation CQA.
6	`MeanDiameter_nm`	decimal	`95`	Controlled input parameter for deterministic QC rules.
7	`MinDiameter_nm`	decimal	`30`	Controlled input parameter for deterministic QC rules.
8	`MaxDiameter_nm`	decimal	`150`	Controlled input parameter for deterministic QC rules.
9	`PDI`	decimal	`0.18`	Controlled input parameter for deterministic QC rules.
10	`MaxPDI`	decimal	`0.3`	Controlled input parameter for deterministic QC rules.
11	`ProteinConcentration_ug_mL`	decimal	`120`	Volume/dose used for load, limit or release calculation.
12	`ParticlesPerUgProtein`	integer / decimal	`2.1E8`	Biological/molecular component controlled as a CQA.
13	`MinParticlesPerUgProtein`	integer / decimal	`1E8`	Biological/molecular component controlled as a CQA.
14	`PositiveMarker_CD63`	string / controlled vocabulary	`true`	Controlled input parameter for deterministic QC rules.
15	`PositiveMarker_CD81`	string / controlled vocabulary	`true`	Controlled input parameter for deterministic QC rules.
16	`PositiveMarker_TSG101`	string / controlled vocabulary	`true`	Controlled input parameter for deterministic QC rules.
17	`NegativeMarker_Calnexin`	string / controlled vocabulary	`false`	Controlled input parameter for deterministic QC rules.
18	`NegativeMarker_GM130`	string / controlled vocabulary	`false`	Controlled input parameter for deterministic QC rules.
19	`MembraneIntegrity_Percent`	decimal	`92`	Controlled input parameter for deterministic QC rules.
20	`MinMembraneIntegrity`	decimal	`70`	Controlled input parameter for deterministic QC rules.

SampleID,IsolationMethod,BiofluidSource,ParticleConcentration_per_mL,MinParticleConcentration,MeanDiameter_nm,MinDiameter_nm,MaxDiameter_nm,PDI,MaxPDI,ProteinConcentration_ug_mL,ParticlesPerUgProtein,MinParticlesPerUgProtein,PositiveMarker_CD63,PositiveMarker_CD81,PositiveMarker_TSG101,NegativeMarker_Calnexin,NegativeMarker_GM130,MembraneIntegrity_Percent,MinMembraneIntegrity
EXO-2026-001,SEC,Plasma,2.5E10,1E8,95,30,150,0.18,0.3,120,2.1E8,1E8,true,true,true,false,false,92,70
EXO-2026-002,Precipitation,Serum,5.0E9,1E8,185,30,150,0.45,0.3,850,5.9E6,1E8,true,false,true,true,false,65,70
EXO-2026-003,Immunoaffinity,Urine,1.2E9,1E8,110,30,150,0.22,0.3,45,2.7E7,1E8,true,true,false,false,false,88,70

Input validation rules

ID	Field	Rule	Criticality
VR-001	`SampleID`	The field shall match an approved dictionary or accepted string representation.	High
VR-002	`IsolationMethod`	The field shall match an approved dictionary or accepted string representation.	High
VR-003	`BiofluidSource`	The field shall match an approved dictionary or accepted string representation.	High
VR-004	`ParticleConcentration_per_mL`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-005	`MinParticleConcentration`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-006	`MeanDiameter_nm`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-007	`MinDiameter_nm`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-008	`MaxDiameter_nm`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-009	`PDI`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-010	`MaxPDI`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-011	`ProteinConcentration_ug_mL`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-012	`ParticlesPerUgProtein`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-013	`MinParticlesPerUgProtein`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-014	`PositiveMarker_CD63`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-015	`PositiveMarker_CD81`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-016	`PositiveMarker_TSG101`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-017	`NegativeMarker_Calnexin`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-018	`NegativeMarker_GM130`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-019	`MembraneIntegrity_Percent`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-020	`MinMembraneIntegrity`	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 Exosome Sample QC 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": "exosomesampleqcchecker",
  "utilityFolder": "ExosomeSampleQcChecker",
  "package": "LiquidBiopsy",
  "overallStatus": "PASS|WARNING|FAIL",
  "sourceFile": "input.csv",
  "processedAtUtc": "2026-06-10T00:00:00Z",
  "checks": [
    {
      "parameter": "SampleID",
      "value": "EXO-2026-001",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-001"
    },
    {
      "parameter": "IsolationMethod",
      "value": "SEC",
      "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": "ParticleConcentration_per_mL",
      "value": "2.5E10",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-004"
    },
    {
      "parameter": "MinParticleConcentration",
      "value": "1E8",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-005"
    },
    {
      "parameter": "MeanDiameter_nm",
      "value": "95",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-006"
    },
    {
      "parameter": "MinDiameter_nm",
      "value": "30",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-007"
    },
    {
      "parameter": "MaxDiameter_nm",
      "value": "150",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-008"
    },
    {
      "parameter": "PDI",
      "value": "0.18",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-009"
    },
    {
      "parameter": "MaxPDI",
      "value": "0.3",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-010"
    },
    {
      "parameter": "ProteinConcentration_ug_mL",
      "value": "120",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-011"
    },
    {
      "parameter": "ParticlesPerUgProtein",
      "value": "2.1E8",
      "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": "ExosomeSampleQcChecker.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