ExtracellularVesicleIsolationChecker

Extracellular Vesicle Isolation

Liquid Biopsy жидкостная биопсия cfDNA ctDNA CTC exosomes NGS qPCR

Utility description: Extracellular Vesicle Isolation

Extracellular Vesicle Isolation Checker — EV Isolation Process Quality Control

ℹ️ Utility performs comprehensive assessment of EV isolation process according to MISEV2023 and ISEV standards:
• Particle Yield: Number of isolated vesicles per mL input material — method efficiency indicator.
• Purity Ratio: Particle-to-protein ratio — key isolation specificity metric.
• Contaminant Removal: Assessment of albumin, lipoprotein, and nucleic acid removal.
• Reproducibility: Coefficient of variation between technical replicates.
• Method Validation: Confirmation of method applicability to specific biofluid type.

⚠️ IMPORTANT:
• No universal "best" isolation method exists — choice depends on downstream application.
• Precipitation yields high output but low purity; SEC — vice versa.
• Proteomics requires purity ≥1×10⁸ particles/µg protein; RNA-seq allows lower.
• Lipoproteins (especially LDL/HDL) are primary contaminants in plasma/serum.

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

Input format:
SampleID,IsolationMethod,BiofluidSource,InputVolume_mL,ParticleYield_per_mL_input,MinExpectedYield,ProteinContamination_ug_per_mL_input,MaxProteinContamination,ParticlesPerUgProtein,MinParticlesPerUgProtein,AlbuminRemoval_Percent,MinAlbuminRemoval,LipoproteinContamination_Score,MaxLipoproteinScore,NucleicAcidContamination_ng_mL,MaxNucleicAcidContamination,ReplicateCV_Percent,MaxReplicateCV,MethodValidatedForSource

Example:
EV-001,SEC,Plasma,1.0,3.2E10,1E8,85,500,3.8E8,1E8,98.5,95,1.5,3.0,12,50,12,25,true

📍 Scope of Application (Usage Where):
• Liquid Biopsy Research: Selection and validation of EV isolation method.
• EV Diagnostic Development: Standardization of sample preparation.
• Data Publication: Mandatory provision of isolation QC per MISEV2023.
• Inter-Laboratory Comparisons: Harmonization of isolation protocols.

— WHY IS THIS NEEDED?
Isolation quality determines all downstream results.
Protein contamination masks true EV proteome; lipoproteins mimic EV signals in NTA.
Without objective purity assessment, biological signal cannot be distinguished from method artifact.
This utility formalizes MISEV2023 criteria into automated verification.

⚠️ CRITICAL:
• Purity Ratio ≥1×10⁸: Baseline threshold for proteomics/RNA-seq.
• Albumin Removal ≥95%: Below this level, preparation is contaminated with plasma proteins.
• Lipoprotein Score ≤3: High score indicates need for additional purification.
• Method Validation: Use of unvalidated method/source pair = unacceptable.
• Replicate CV ≤25%: High variability requires protocol optimization.

Key features:
• Six-parameter isolation quality assessment
• Adaptive thresholds per biofluid type and method
• Integration of physical and biochemical metrics
• ACCEPTABLE / SUBOPTIMAL / UNACCEPTABLE classification
• Compliance with MISEV2023 and ISEV guidelines

Critical parameters:
• Particle Yield: ≥ Min Expected
• Purity Ratio: ≥ 1×10⁸ particles/µg protein
• Albumin Removal: ≥ 95%
• Lipoprotein Score: ≤ 3.0
• Replicate CV: ≤ 25%
• Method Validated: True

💡 Usage tips:
1. Method Selection: Use SEC or IA for proteomics; UC acceptable for RNA-seq.
2. Orthogonal Verification: Confirm NTA with TEM or TRPS data.
3. Blank Controls: Always include isolation blanks (PBS instead of sample) for background assessment.
4. Standardization: Use same method throughout entire study.
5. Documentation: Report ALL isolation parameters in publication Methods section.

⚠️ Note: This utility assesses the isolation process, not vesicle characteristics themselves. For complete EV characterization, use ExosomeSampleQcChecker after isolation completion.

input.csv

SampleID,IsolationMethod,BiofluidSource,InputVolume_mL,ParticleYield_per_mL_input,MinExpectedYield,ProteinContamination_ug_per_mL_input,MaxProteinContamination,ParticlesPerUgProtein,MinParticlesPerUgProtein,AlbuminRemoval_Percent,MinAlbuminRemoval,LipoproteinContamination_Score,MaxLipoproteinScore,NucleicAcidContamination_ng_mL,MaxNucleicAcidContamination,ReplicateCV_Percent,MaxReplicateCV,MethodValidatedForSource
EV-ISO-2026-001,SEC,Plasma,1.0,3.2E10,1E8,85,500,3.8E8,1E8,98.5,95.0,1.5,3.0,12,50,12.0,25.0,true
EV-ISO-2026-002,Precipitation,Serum,0.5,8.5E10,1E8,1200,500,7.1E7,1E8,72.0,95.0,7.5,3.0,180,50,35.0,25.0,false
EV-ISO-2026-003,Immunoaffinity,Urine,5.0,1.5E9,1E8,25,500,6.0E7,1E8,99.2,95.0,0.5,3.0,8,50,18.0,25.0,true

URS & FS — User Requirements and Functional Specification

This document describes the controlled interface and behaviour of ExtracellularVesicleIsolationChecker for Extracellular Vesicle Isolation 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.

Extracellular Vesicle Isolation Checker — EV Isolation Process Quality Control
ℹ️ Utility performs comprehensive assessment of EV isolation process according to MISEV2023 and ISEV standards:
• Particle Yield: Number of isolated vesicles per mL input material — method efficiency indicator.
• Purity Ratio: Particle-to-protein ratio — key isolation specificity metric.
• No universal "best" isolation method exists — choice depends on downstream application.
• Precipitation yields high output but low purity; SEC — vice versa.
• Proteomics requires purity ≥1×10⁸ particles/µg protein; RNA-seq allows lower.
ExtracellularVesicleIsolationChecker.exe → demo mode (console output)
ExtracellularVesicleIsolationChecker.exe input.csv output.json → evaluate your data
SampleID,IsolationMethod,BiofluidSource,InputVolume_mL,ParticleYield_per_mL_input,MinExpectedYield,ProteinContamination_ug_per_mL_input,MaxProteinContamination,ParticlesPerUgProtein,MinParticlesPerUgProtein,AlbuminRemoval_Percent,MinAlbuminRemoval,LipoproteinContamination_Score,MaxLipoproteinScore,NucleicAcidContamination_ng_mL,MaxNucleicAcidContamination,ReplicateCV_Percent,MaxReplicateCV,MethodValidatedForSource
• Liquid Biopsy Research: Selection and validation of EV isolation method.
• Data Publication: Mandatory provision of isolation QC per MISEV2023.
• Inter-Laboratory Comparisons: Harmonization of isolation protocols.
Isolation quality determines all downstream results.
Without objective purity assessment, biological signal cannot be distinguished from method artifact.
⚠️ CRITICAL:
• Purity Ratio ≥1×10⁸: Baseline threshold for proteomics/RNA-seq.
• Albumin Removal ≥95%: Below this level, preparation is contaminated with plasma proteins.

URS — User Requirements Specification

ID	Requirement	Criticality	Acceptance criterion
URS-001	The utility shall accept an input.csv file for Extracellular Vesicle Isolation 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	`EV-ISO-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	`InputVolume_mL`	decimal	`1.0`	Volume/dose used for load, limit or release calculation.
5	`ParticleYield_per_mL_input`	decimal	`3.2E10`	Volume/dose used for load, limit or release calculation.
6	`MinExpectedYield`	decimal	`1E8`	Controlled input parameter for deterministic QC rules.
7	`ProteinContamination_ug_per_mL_input`	decimal	`85`	Volume/dose used for load, limit or release calculation.
8	`MaxProteinContamination`	integer / decimal	`500`	Biological/molecular component controlled as a CQA.
9	`ParticlesPerUgProtein`	integer / decimal	`3.8E8`	Biological/molecular component controlled as a CQA.
10	`MinParticlesPerUgProtein`	integer / decimal	`1E8`	Biological/molecular component controlled as a CQA.
11	`AlbuminRemoval_Percent`	decimal	`98.5`	Controlled input parameter for deterministic QC rules.
12	`MinAlbuminRemoval`	decimal	`95.0`	Controlled input parameter for deterministic QC rules.
13	`LipoproteinContamination_Score`	decimal	`1.5`	Biological/molecular component controlled as a CQA.
14	`MaxLipoproteinScore`	decimal	`3.0`	Biological/molecular component controlled as a CQA.
15	`NucleicAcidContamination_ng_mL`	decimal	`12`	Volume/dose used for load, limit or release calculation.
16	`MaxNucleicAcidContamination`	string / controlled vocabulary	`50`	Controlled input parameter for deterministic QC rules.
17	`ReplicateCV_Percent`	decimal	`12.0`	Controlled input parameter for deterministic QC rules.
18	`MaxReplicateCV`	decimal	`25.0`	Controlled input parameter for deterministic QC rules.
19	`MethodValidatedForSource`	string / controlled vocabulary	`true`	Method or process approach; controlled reference value.

SampleID,IsolationMethod,BiofluidSource,InputVolume_mL,ParticleYield_per_mL_input,MinExpectedYield,ProteinContamination_ug_per_mL_input,MaxProteinContamination,ParticlesPerUgProtein,MinParticlesPerUgProtein,AlbuminRemoval_Percent,MinAlbuminRemoval,LipoproteinContamination_Score,MaxLipoproteinScore,NucleicAcidContamination_ng_mL,MaxNucleicAcidContamination,ReplicateCV_Percent,MaxReplicateCV,MethodValidatedForSource
EV-ISO-2026-001,SEC,Plasma,1.0,3.2E10,1E8,85,500,3.8E8,1E8,98.5,95.0,1.5,3.0,12,50,12.0,25.0,true
EV-ISO-2026-002,Precipitation,Serum,0.5,8.5E10,1E8,1200,500,7.1E7,1E8,72.0,95.0,7.5,3.0,180,50,35.0,25.0,false
EV-ISO-2026-003,Immunoaffinity,Urine,5.0,1.5E9,1E8,25,500,6.0E7,1E8,99.2,95.0,0.5,3.0,8,50,18.0,25.0,true

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	`InputVolume_mL`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-005	`ParticleYield_per_mL_input`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-006	`MinExpectedYield`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-007	`ProteinContamination_ug_per_mL_input`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-008	`MaxProteinContamination`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-009	`ParticlesPerUgProtein`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-010	`MinParticlesPerUgProtein`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-011	`AlbuminRemoval_Percent`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-012	`MinAlbuminRemoval`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-013	`LipoproteinContamination_Score`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-014	`MaxLipoproteinScore`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-015	`NucleicAcidContamination_ng_mL`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-016	`MaxNucleicAcidContamination`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-017	`ReplicateCV_Percent`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-018	`MaxReplicateCV`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-019	`MethodValidatedForSource`	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 Extracellular Vesicle Isolation 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": "extracellularvesicleisolationchecker",
  "utilityFolder": "ExtracellularVesicleIsolationChecker",
  "package": "LiquidBiopsy",
  "overallStatus": "PASS|WARNING|FAIL",
  "sourceFile": "input.csv",
  "processedAtUtc": "2026-06-10T00:00:00Z",
  "checks": [
    {
      "parameter": "SampleID",
      "value": "EV-ISO-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": "InputVolume_mL",
      "value": "1.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-004"
    },
    {
      "parameter": "ParticleYield_per_mL_input",
      "value": "3.2E10",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-005"
    },
    {
      "parameter": "MinExpectedYield",
      "value": "1E8",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-006"
    },
    {
      "parameter": "ProteinContamination_ug_per_mL_input",
      "value": "85",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-007"
    },
    {
      "parameter": "MaxProteinContamination",
      "value": "500",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-008"
    },
    {
      "parameter": "ParticlesPerUgProtein",
      "value": "3.8E8",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-009"
    },
    {
      "parameter": "MinParticlesPerUgProtein",
      "value": "1E8",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-010"
    },
    {
      "parameter": "AlbuminRemoval_Percent",
      "value": "98.5",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-011"
    },
    {
      "parameter": "MinAlbuminRemoval",
      "value": "95.0",
      "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": "ExtracellularVesicleIsolationChecker.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