NWIP on 'Molecular in-vitro diagnostic examinations - Specifications for pre-examination processes for blood - ccfdna'

Transcription

1 CEN/TC 140 N 911 CEN/TC 140 In vitro diagnostic medical devices of Secretary: bernd.boesler@din.de Secretariat: DIN NWIP on 'Molecular in-vitro diagnostic examinations - Specifications for pre-examination processes for blood - ccfdna' Date of document Expected action Comment Due Date Background Dear Members of CEN/TC 140, Because of higher efficiency for the use of the included information, CEN/TC 140/WG 3 recommends to divide the content of the CEN/TS Molecular in-vitro diagnostic examinations Specifications for pre-examination processes for selected types of sample into a series of four different Technical Specifications one for each class of specimen type, blood, plasma, tissue, metabolomics. Each series will contain separately published parts under the generic specimen type according to specific methods. CEN/TC 140/WG 3 prepared nine NWI proposals to be circulated in the CEN/TC 140. The decision will be taken during the next CEN/TC 140 meeting on 23rd October 2013 in Berlin. All members are more than welcome to give their comments on each NWIP. These comments will be taken into considderation in the discussion during th next CEN/TC 140 meeting.

2 Form N Proposal for a new work item Title: Molecular in-vitro diagnostic examinations Specifications for pre-examination processes for blood ccfdna Proposer: CEN/TC 140/WG 3 Information to be supplied by the proposer of the NWI A1 Subject A1.1 Scope: This Technical Specification recommends the handling, documentation and processing of blood specimens intended for circulating cell free DNA (ccfdna) analysis during the pre-analytical phase before a molecular assay is performed. This Technical Specification is applicable to molecular in-vitro diagnostic examinations (e.g. in-vitro diagnostic laboratories, laboratory customers, in-vitro diagnostics developers and manufacturers, institutions and companies performing biomedical research, biobanks, and regulation authorities). A1.2 Keywords (Descriptors) characterising the scope (multiple ticks are possible and/or necessary) - Product - System/ Workflow - Service Interface - Requirements - Characteristics - Guidance - Test method - Terminology etc. - EN - CEN/TS - CEN/TR - other (e.g. CEN Guide) A2 Market relevance A2.1 Frame conditions Subject of mandate from EC or EFTA: Reference of mandate Transposition of International Standard: Reference of IS Adoption of draft provided by European professional body: Name of organization + Reference of document Other: Please specify: Working document prepared by CEN/TC 140/WG 3 A2.2 General market needs Safety Environment Consumers Economy Barriers to trade Other: Improvement of diagnostic analysis tests results A2.3 Special aspects (problems or difficulties to be solved by the standard, impacts and benefits to be expected from the standard; please describe shortly): Blood ccf DNA can change significantly after collection (e.g. ccfdna fragmentation, ccfdna quantity, release of genomic DNA from white blood cells). Special measures have to be taken to secure good quality blood samples for ccfdna analysis. A2.4 Urgency high medium low Version 1 September 2004

3 Form N A3 Resources and timeframe - First working draft(s) available *) - Suitable source document(s) available *) - Pre-normative research necessary - Strong interest of stakeholders in terms of financing expected - Active participation of stakeholders expected - Expertise available - External (e.g. EC) financing expected - Timely consensus expected *) To be added to the proposal A4 Participation - Proposer prepared to participate actively - Proposer prepared to run secretariat - Proposer prepared to take over convenoror project leadership - Special liaison proposed: A5 Name: Function: Organisation: Signature... Date: Version 1 September 2004

4 CEN/TC 140 Date: TC 140 WI CEN/TC 140 Secretariat: DIN Molecular in-vitro diagnostic examinations Specifications for preexamination processes for blood Circulating cell free DNA Einführendes Element Haupt-Element Ergänzendes Element Élément introductif Élément central Élément complémentaire ICS: Descriptors: Document type: Technical Specification Document subtype: Document stage: Working Document Document language: E C:\Users\krl\Desktop\140\NWIPs 2013\CENTC_140_NWIP_blood - circulating cell free DNA.doc STD Version 2.5a

5 Contents Foreword...3 Introduction Scope Normative references Terms and definitions General Considerations Primary blood collection manual Outside the laboratory General Selection of collection containers for blood samples for ccfdna isolation Primary sample collection from the patient Storage and Transport requirements for blood sample Inside the laboratory Sample reception Storage requirements Plasma preparation Storage requirements for plasma sample Isolation of the ccfdna Storage of isolated ccfdna Quality assessment of isolated ccfdna...8 Bibliography...9 Page 2

6 Foreword This document (TC 140 WI ) has been prepared by Technical Committee CEN/TC 140 in vitro diagnostic medical devices, the secretariat of which is held by DIN. This document is a working document. 3

7 Introduction Molecular in-vitro diagnostics has enabled a significant progress in medicine. Further progress is expected by new technologies analyzing signatures of nucleic acids, proteins, and metabolites in human tissues and body fluids. However, the profiles of these molecules can change drastically during collection, preservation, transport, and storage thus making a reliable diagnostic or pharmaceutical research unreliable or even impossible because the subsequent analytical assay will not determine the situation in the patient but an artificial profile generated during sample processing. Therefore, a standardization of the entire process from sample collection to analyte measurement is needed. Studies have been undertaken working on the missing standardization and improvement of pre-analytical procedures for in-vitro diagnostics, to determine the important influencing factors. This Technical Specification draws upon such work to codify and standardize the steps for circulating cell free DNA (ccfdna) analysis from plasma prepared from human blood in what is referred to as the pre-analytical phase. 4

8 1 Scope This Technical Specification recommends the handling, documentation and processing of blood specimens intended for circulating cell free DNA (ccfdna) analysis during the pre-analytical phase before a molecular assay is performed. This Technical Specification is applicable to molecular in-vitro diagnostic examinations (e.g. in-vitro diagnostic laboratories, laboratory customers, in-vitro diagnostics developers and manufacturers, institutions and commercial organizations performing biomedical research, biobanks, and regulation authorities). Blood ccfdna can change significantly after collection (e.g. ccfdna fragmentation, ccfdna quantity, release of genomic DNA from white blood cells). Special measures have to be taken to secure good quality blood samples for ccfdna analysis. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 15189:2012, Medical laboratories Requirements for quality and competence (ISO 15189:2012). 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 analytical phase 3.2 ccfdna circulating cell free DNA is specific extracellular fragmented human DNA present in blood, serum and plasma 3.3 pre-analytical phase processes that start, in chronological order, from the clinician s request and include the examination request, preparation and identification of the patient, collection of the primary sample(s), temporary storage, transportation to and in the analytical laboratory, aliquoting, retrieval, isolation of analytes, and ending when the analytical examination begins. Note to entry: The pre-analytical phase may include preparative processes that may influence the outcome of the intended examination. 3.4 primary sample specimen discrete portion of a body fluid, breath, hair or tissue taken for examination, study or analysis of one or more quantities or properties assumed to apply for the whole. [SOURCE: EN ISO 15189:2012, definition 3.16] 4 General Considerations Generally for the primary sample collection and handling also see EN ISO 15189, Specifically, for samples intended to be analysed for ccfdna the following steps have to be performed. 5

9 1. Primary sample collection from the patient; the documentation should include, but is not limited to: a) physical health condition of the sample donor (healthy, disease type etc.); b) information about routine medical treatment and special treatment prior to blood collection (e.g. anaesthetics treatment information); c) time of blood collection. 2. Selection of collection containers and packages (e.g. cooling box, box for storing and transportation); 3. Selection of stabilization procedures (procedures or reagents suitable for stabilization of the ccfdna profile in the specimen); 4. Recording of temperature at collection, of sample stabilization procedure used and of the time intervals from collection until stabilization, storage, and transportation; 5. Recording of any additions or modifications to the primary sample; 6. Types and quantity and description of samples. 5 Primary blood collection manual 5.1 Outside the laboratory General The documentation should include, but is not limited to: a) physical health conditions of the blood donor (disease type etc.) and disease treatments (drugs etc.). b) the time of blood sample collection. c) instructions for the preparation of the patient for the blood draw procedure. NOTE 1 NOTE 2 There is no known specific effect of this step on the ccfdna. Routine procedures can therefore be used. See also EN ISO 15189:2012 (Clause) Selection of collection containers for blood samples for ccfdna isolation The ccfdna profile can be influenced by inadequate blood collection procedures and inappropriate storage/shipping conditions, plasma separation as well as ccfdna extraction procedures. Blood should be collected in appropriate collection devices. EDTA blood collection tubes should be preferred to other collection tubes as EDTA shows a stabilizing effect during the time between blood draw and processing (see [1], [2]). NOTE New blood collection tubes containing ccfdna profile stabilization reagents are currently under development or are available as for research use only products. (Uwe, Martin - references) Primary sample collection from the patient 1. Record the identity of the person collecting the sample and the time of blood collection according to EN ISO 15189, , f). 2. For blood collection tube labelling (sample identification), use a routine procedure or a procedure with additional information (e.g. 2D-barcode). 6

10 3. Standard venipuncture technique can be used. NOTE Also, steps for preventing possible backflow may be required (follow manufacturers instructions). 4. Blood collection tubes shall be filled and treated (mixing) in accordance to the manufacturers instructions. Follow blood collection tube manufacturers' instructions for mixing or inverting the tube immediately after blood collection. Otherwise, reagents in the tube will not be homogenously mixed with the blood causing compromised samples, which can lead to wrong or questionable analytical test results. 5. Routine procedures including needle disposal container for used needle or needle/holder combination can be used for safe disposal of materials used in the sample collection Storage and Transport requirements for blood sample The instruction of the assay provider on blood filled tubes' transport, storage conditions and duration shall be followed. If there are no such instructions and blood collection tubes containing ccfdna stabilizers are used, the instructions of the blood tube manufacturer shall be followed. NOTE The blood storage duration includes the time for storage at the point of blood collection, transportation to the testing laboratory and further storage at the testing laboratory or other institutions until the plasma separation. After blood collection genomic DNA is continuously released from white blood cells. Therefore, the entire process from blood collection until the plasma separation shall be performed as soon as possible or at least within 6 h [reference, Mario, Martin, Uwe, Ralf], if the blood sample is stored at room temperature or within 24 h, if the blood sample is stored at 4 C [1]. In the case of the availability of blood collection tubes containing ccfdna stabilizers, follow the manufactures instructions for storage/transport conditions. NOTE When using blood collection tubes without a ccfdna stabilizer (also those containing EDTA as anticoagulant), the quality of the ccfdna after blood draw during the entire pre-analytical workflows should be investigated. 5.2 Inside the laboratory Sample reception Record the blood sample arrival time and conditions (e.g. labelling, transport conditions, blood volume, leaking) of the received samples. Report nonconformities of labelling, transport conditions and obvious blood volume differences to specifications described for the blood collection tube and any variations from assay requirements. If there is nonconformity in transport conditions, overall storage and transport time or blood volume, a new sample should be ordered as this will risk the validity of the analytical test result Storage requirements Record the temperature and time interval between sample receipt and sample processing for ccfdna extraction. Total storage temperature and time interval conditions should not exceed specifications identified in NOTE Total storage time includes storage time at the point of blood collection, transport time and storage time of blood and plasma inside the laboratory or any other institution. The total storage time may not exceed specification given by the analytical test manufacturer Plasma preparation EDTA blood sample should be centrifuged at 1 600g at 4 C for 10 min and plasma should be carefully recovered without disturbing the buffy coat layer in order to remove additional cellular debris and contamination from genomic DNA and RNA derived from damaged blood cells. A second centrifugation 7

11 should be performed at g to g speed at 4 C for 10 min and carefully transfer the supernatant in a new tube without disturbing the pellet [3]. When using blood collection tubes with ccfdna stabilizers or alternatives validated to the same requirements, the manufacturers' instructions to obtain plasma separation shall be followed Storage requirements for plasma sample Plasma should be stored at 4 C for up to 6 h; for long-term storage the specimens should be frozen and stored at approximately -20 C or -80 C (Refs available, Mario). Frozen plasma samples shall not be thawed more than once, if cryoprecipitates are visible they should be pelleted by centrifugation at maximum speed for 5 min at 4 C Isolation of the ccfdna In the case of the availability of blood collection tubes containing ccfdna stabilizers, follow the manufactures instructions for ccfdna isolation. When using blood collection tubes containing EDTA as anticoagulant, specific ccfdna isolation kits or alternatives validated to the same requirements shall be used. The kit manufacturers instructions or the instructions for the validated alternative for isolating the ccfdna shall be followed. NOTE Dedicated procedures can be included in the manufacturers instructions for processing frozen blood samples. When using blood collection tubes not containing EDTA as anticoagulant, the testing laboratory shall validate the process from the blood collection procedure, to plasma separation process and ccfdna isolation, to the final analytical test result according to its internal quality management system (see EN ISO 15189). NOTE The ccfdna obtained by different ccfdna isolation procedures can have different recovery amount (Refs., Mario). It is recommended to check, if the obtained ccfdna quantity is suitable/adequate for requested analytical assay Storage of isolated ccfdna For storing and archiving the isolated ccfdna follow the ccfdna isolation kit providers specific instructions. If there is no information available from the ccfdna isolation kit provider or if testing laboratories own validated ccfdna isolation procedures are used, the isolated ccfdna should be assayed immediately. In the case that the isolated ccfdna cannot be assayed immediately, the testing laboratory shall have validated procedures in place how to store the isolated ccfdna Quality assessment of isolated ccfdna When a testing laboratory is using its own validated ccfdna isolation procedure or even if it is using a commercial kit, it is advisable to check for ccfdna quality by generally accepted physical, chemical and biochemical procedures (e.g. wavelength scan, A 260/A 280 ratio, cellular RNA integrity by electrophoresis, laboratory chip analysis, interfering substances testing). Quality assessment: How (quality and/or quantity? By UV? By qpcr? Fragmentation: yes/not and eventually how? In all the samples? (Uwe, Martin) 8

12 Bibliography [1] M. Jung, Clinical Chemistry, 49, 2003, p [2] N. Y. Lam, Clinical Chemistry, 50, 2004, p [3] P. Pinzani, Methods, 50, 2010, p