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BSI PD CEN/TS 17981-2:2023

BSI PD CEN/TS 17981-2:2023

$198.66

In vitro diagnostic Next Generation Sequencing (NGS) workflows – Human RNA examination

Published By Publication Date Number of Pages
BSI 2023 68
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This document specifies requirements and gives recommendations for next generation sequencing (NGS) workflows for in vitro diagnostics and biomedical research. This document covers the pre-examination processes, human RNA isolation, sequencing library preparation, sequencing, sequence analysis and reporting of the examination of sequences for diagnostic purposes from isolated RNA from, e.g. formalin-fixed and paraffin embedded tissues, fresh frozen tissues, fine needle aspirates (FNA), whole blood, circulating tumour cells (CTCs), exosomes and other extracellular vesicles, and circulating cell free RNA from plasma. NOTE 1 Typical applications include, but are not limited to, NGS for oncology and clinical genetics, certain single-cell analyses. This document is applicable to molecular in vitro diagnostic examinations including laboratory developed tests performed by medical laboratories, molecular pathology laboratories and molecular genetic laboratories. This document is also applicable to laboratory customers, in vitro diagnostics developers and manufacturers, biobanks, institutions, and organisations performing biomedical research. This document is not applicable for in situ sequencing, forensic sequencing, sequencing of pathogens or microorganisms and microbiome analysis. NOTE 2 International, national or regional regulations or requirements or multiples of them can also apply to specific topics covered in this document.

PDF Catalog

PDF Pages PDF Title
2 undefined
6 European foreword
7 Introduction
8 1 Scope
2 Normative references
9 3 Terms and definitions
22 4 General requirements
4.1 General
23 4.2 Examination design
27 4.3 Examination development
4.4 Examination performance verification and validation
31 Table 1 — Examples for intended uses and corresponding clinical performance characteristics
32 4.5 Technical examination performance characteristics
33 5 Pre-examination processes for examination development
5.1 General
34 Table 2 — Analyte specific requirements for pre-examination processes
35 5.2 Human RNA isolation
5.2.1 General
5.2.2 Isolation from formalin fixed and paraffin embedded (FFPE) tissue
5.2.3 Isolation from fresh frozen tissue
5.2.4 Isolation from fine needle aspirates (FNA)
5.2.5 Isolation from whole blood
5.2.5.1 Venous whole blood
5.2.5.2 Circulating tumour cells (CTCs)
5.2.5.3 Extracellular vesicles (EVs)
36 5.2.6 Isolation of circulating cell free RNA from plasma
5.3 RNA sample quality and quantity evaluation
38 6 Examination processes for examination development
6.1 Sequencing library preparation for examination development
6.1.1 General
6.1.2 Sequencing library preparation steps
6.1.2.1 General
39 6.1.2.2 Enrichment
6.1.2.3 Fragmentation
6.1.2.4 Quality assessment of the fragmented RNA sample
6.1.2.5 cDNA synthesis
6.1.2.6 End repair
40 6.1.2.7 Base modifications
6.1.2.8 Adaptor ligation
6.1.2.9 Barcoding/indexing
6.1.2.10 Size selection
41 6.1.2.11 Pre-/Amplification
6.1.2.12 Cleaning
6.1.2.13 Sequencing library quantification and qualification
42 6.1.3 RNA sequencing (RNA-Seq)
6.1.3.1 General
43 6.1.3.2 Requirements and recommendations for RNA-Seq
6.1.3.3 Extracellular vesicle (EV) panels
44 Table 3 — Methods used for EV RNA sequencing
45 6.2 Sequencing examination development
6.2.1 General
6.2.2 Techniques
46 6.2.3 Sequencing quality control
6.3 Data analysis requirements for examination development
47 6.4 Quality control (QC) requirements for examination development
6.4.1 General
6.4.2 RNA Sequencing
6.4.2.1 General
6.4.2.2 RNA-Seq
48 6.4.2.3 Extracellular vesicles
7 Requirements for the development of the examination reporting tool
7.1 General
49 7.2 Report attributes
7.3 Report content
50 8 Implementation of the in vitro diagnostic NGS workflow into routine practice
51 9 Reporting and interpretation of results
52 10 Quality assurance procedures
10.1 General
10.2 Performance monitoring, optimization of the examination and interlaboratory comparison
53 Annex A (normative) in vitro diagnostic NGS workflow for single-cell analyses
A.1 General information and requirements on single-cell analyses
54 A.2 Pre-examination processes for examination development
A.2.1 General information of applicable procedures
A.2.2 Requirements for CTCs from blood specimen collection to CTC isolation
A.2.3 Requirements for fresh frozen/FFPE human tissue from specimen collection to isolation of single cells
A.2.3.1 Collection to storage of the tissue specimen or sample
A.2.3.2 Dissociation of tissue sections into a single cell suspension
55 A.2.3.3 Quality control of the single cell suspension
A.2.3.4 Storage of the single cell suspension
A.2.3.5 Enrichment of the cell suspension
56 A.2.3.6 Single-cell isolation
A.2.4 Human RNA isolation
A.2.4.1 Regardless of the origin of single cells (e.g. single cell from tissue or CTCs) and the enrichment/isolation procedure used to sort them, RNA is not usually isolated.
A.2.4.2 For CTCs, 5.2.5.2 shall be followed depending on the chosen CTC detection procedure (i.e. direct or indirect). For storage of isolated RNA, CEN/TS 17390-1 applies.
A.2.4.3 Isolated single cells from tissue shall not be processed for RNA isolation due to the low RNA quantity. Instead, the isolated single cells shall either be:
57 A.2.5 RNA sample quality evaluation
A.3 Examination phase for examination development
A.3.1 Sequencing library preparation for examination development for CTCs and single cells from tissues
A.3.2 Sequencing examination development for CTCs and single cells from tissues
A.3.2.1 General
58 A.3.2.2 Sequencing QC
A.3.3 Data analysis requirements for examination development for CTCs and single cells from tissues
59 A.3.4 QC requirements for examination development
A.4 Implementation of the in vitro diagnostic NGS workflow into routine practice
A.5 Reporting and interpretation of results
A.6 Quality assurance procedures
60 Annex B (normative) Exemplary in vitro diagnostic NGS workflow for spatial transcriptomics
61 Annex C (informative) in vitro diagnostic NGS workflow scheme for the examination of RNA
Figure C.1 — in vitro diagnostic NGS workflow scheme for the examination of RNA
62 Bibliography

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BSI PD CEN/TS 17981-2:2023
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