Each series will contain separately published parts under the generic specimen type according to specific methods.

Transcription

1 CEN/TC 140 N 919 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 metabolomics analyses in urine, plasma and serum' 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 invitro 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 metabolomics analyses in urine, plasma and serum 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 urine, plasma and serum generated from whole blood intended for metabolomics analysis during the pre-analytical phase before metabolomic profiling is performed. This Technical Specification is applicable to molecular 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: 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): The concentration of some metabolites can undergo significant changes after collection, as an answer to different operating procedures. Special measures have to be taken to secure good quality of urine, serum and plasma samples for the metabolomics analysis of those biofluids. Version 1 September 2004

3 Form N A2.4 Urgency high medium low 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 metabolomics in urine, serum and plasma 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_metabolomics_urine_serum_plasma.doc STD Version 2.5a

5 Contents Foreword...3 Introduction Scope Normative references Terms and definitions General Considerations Urine collection manual Outside the laboratory General Selection of collection containers Urine collection from the patient Transport requirements Inside the laboratory Sample reception Urine sample processing Storage requirements Urine thawing and use Blood collection manual General Selection of collection containers Blood collection from the patient Transport to laboratory Inside the laboratory Sample reception Sample stabilization/processing Transport to biobank or laboratory for metabolomics analysis Storage requirements Serum and plasma thawing and use...9 Bibliography Page 2

6 Foreword This document (TC 140 WI ) has been prepared by Technical Committee CEN/TC 140 In vitro diagnostic and 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 to determine the important influencing factors. This Technical Specification draws upon such work to codify and standardize the steps for urine, serum and plasma metabolomics analysis in what is referred to as the pre-analytical phase. Metabolomics, the global profiling of metabolites (namely molecules with a molecular weight MW < Da) in biological samples, is the quantitative measurement of the dynamic multi-parametric metabolic response of living systems to pathophysiological stimuli or genetic modification. Metabolomics studies help identifying metabolic profiles that are characteristic for given pathological conditions, for disease prognosis, for the evaluation of the individual response to medical intervention and pharmaceutical treatments. Metabolites are physically and chemically different, and include salts, sugars, acids, bases, lipids, hormonal steroids, and other compounds. This diversity of metabolites and their concentration dynamic range in biological samples complicates the separation and detection methods and makes it impossible to identify all the metabolites in a single experiment. However new high-throughput technologies such as NMR (nuclear magnetic resonance) and mass spectrometry hold great potential due to their ability to look at large parts of the whole metabolome, although with different sensitivity. These two main analytical platforms are well standardized. Equally well established are the statistical approaches needed to extract information from the huge amount of data resulting from metabolomics analysis. Metabolic profiles can be regarded as the ultimate response of biological systems to genetic or environmental changes since they are the end products of cellular regulatory processes. Their value in biomedical research, biomarker discovery at the pharma level and for possible future clinical applications implies that what is measure by MS (mass spectrometry) and NMR represents as much as possible the original metabolome of the sample. On the other hand the metabolome is very sensitive to a number of variations that may due to residual enzymatic activity in the samples, chemical oxidative reactions and, in tissue and cells, apoptotic processes during sample collection, handling and storage. No best handling procedures are available for the pre-analytical steps and the importance of this aspect is often ignored in metabolomics studies. This Technical Specification series provides guidelines arising from systematic studies conducted on the most commonly employed biofluids: urine, serum, plasma. 4

8 Scope This Technical Specification recommends the handling, documentation and processing of urine, plasma and serum generated from whole blood intended for metabolomics analysis during the pre-analytical phase before metabolomic profiling is performed. This Technical Specification is applicable to molecular 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). The concentration of some metabolites can undergo significant changes after collection, as an answer to different operating procedures. Special measures have to be taken to secure good quality of urine, serum and plasma samples for the metabolomics analysis of this biofluids. 1 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). 2 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 analytical phase 3.2 blood cellular RNA RNA species present in blood cells 3.3 blood cellular RNA profile quantitative presens of all cellular RNA species in human bodies blood 3.4 blood cellular RNA profile stabilizers compounds solutions, mixtures or other technologies preventing changes of the cellular RNA profile in a blood sample 3.5 metabolites the global profiling of metabolites (namely molecules with a molecular weight MW < Da 3.6 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, aliquotting, 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. 3.7 primary sample specimen 5

9 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] 3 General Considerations Generally for the primary sample collection and handling also see EN ISO 15189, Specifically for samples intended to be analysed by metabolomics the following steps have to be performed. 1. Primary sample collection from the patient; the documentation should include, but is not limited to: a) physical health condition and relevant lifestyle factors of the sample donor (healthy, disease type etc.); b) information about routine medical treatment and special treatment prior to sample collection (e.g. anesthetics treatment information); c) time of sample collection. 2. selection of collection containers and packages (e.g. cooling box, box for storing and transportation); 3. selection of stabilization procedures; 4. recording of temperature at collection, sample stabilization procedure used and time intervals from collection until stabilization, storage, and transportation; 5. recording of any additions or modifications to the sample; 6. types and quantity and description of samples. 4 Urine collection manual 4.1 Outside the laboratory General The documentation should include, but is not limited to: a) physical health conditions of the urine donor (disease type etc.), disease treatments (drugs etc.) and diet; b) the time of urine collection Selection of collection containers No specific collection containers are required for urine collection. The minimum recommended volume is 5 ml. Stabilizers are usually not required. NOTE In case stabilizers are added, the impact on the analytical metabolomics profile should be analysed Urine collection from the patient 1. Record the identity of the person collecting the sample and the time of urine collection according to EN ISO 15189, , f). 6

10 2. For urine collection tube labelling (sample identification) use a routine procedure or a procedure with additional information (e.g. 2D-barcode). 3. The donor shall collect the first urine of the morning under fasting conditions. Specify if collected at different times. 4. The urine sample should be kept refrigerated at 4 C for a maximum of 2 h and shall not be frozen prior processing (to avoid cell breaking upon ice crystal formation) Transport requirements During transport the sample should be kept cool (temperature range 0 C to 4 C). Appropriate measures shall be taken to secure temperature specifications and to reduce time for the delivery, which should ideally be completed within 2 h from collection. 4.2 Inside the laboratory Sample reception Record the urine sample reception time and conditions (e.g. labelling, transport conditions, volume, leaking) of the received samples. Report nonconformities of labelling, transport conditions and obvious urine volume differences to specifications described for the urine collection tube and any variations from assay requirements. If there is nonconformity in transport conditions, overall storage and transport time or urine volume, a new sample should be ordered as this will risk the validity of the analytical test result Urine sample processing Mild centrifugation (1 000 RCF to RCF for 5 min at 4 C) followed by filtration with 0,20 µm cut-off, to remove particulate matter and cells. NOTE Mild centrifugation is important to avoid cell fragmentation that would contaminate the primary sample. Addition of additives, like enzyme inhibitors, should be avoided because the required concentrations will introduce signals in the NMR spectra covering the resonances of the metabolites and because may induce changes in ph, ionic strength, altering the original profiles Storage requirements Record the temperature and time interval between sample receipt, sample processing and freezing. Samples should be aliquoted to minimal volumes in cryo-vials for low temperature storage enabling the metabolic profile analytical test. The best procedure would require storage below the critical ice crystal temperature (-130 C) to avoid ice crystal formation which may cause cell breaking. NOTE Given the cost associated to storage in liquid nitrogen, long-term storage at approximately -80 C can be acceptable provided the processing step in has been carefully conducted Urine thawing and use NMR profile analytical test is performed at room temperature. The thawed sample should be introduced to the analytical test procedure within 5 min to10 min after thawing. 7

11 5 Blood collection manual General The documentation should include, but is not limited to: a) physical health conditions of the blood donor (disease type etc.), disease treatments (drugs etc.). b) the time of blood collection. c) instructions for the preparation of the patient for the blood draw procedure. NOTE 1 used. NOTE 2 There is no known specific effect of this step on the cellular RNA levels. Routine procedures can therefore be See also EN ISO 15189:2012 (Clause) Selection of collection containers Draw at least 5 ml of peripheral blood using 5 ml conventional blood collection tubes with appropriate anticoagulants. Depending on the metabolomics profile downstream analysis this shall be a serum or plasma enabling tube. For plasma, either EDTA or citrate can be used as anticoagulants. EDTA is preferred because, at variance with citrate, it is not present in the original metabolome and therefore less interfering with possible NMR analysis, although it signals may overlap and cover some of those of the original metabolites. The anticoagulant shall be recorded Blood collection from the patient 1. Record the identity of the person collecting the sample and the time of 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). 3. If processing of the primary blood sample is not possible within 30 min from collection, it shall be temporarily stored at 4 C Transport to laboratory The primary blood sample shall be transported at 4 C, unless transport to the laboratory is completed within 30 min after blood collection. Appropriate measures shall be taken to secure temperature specifications and to reduce time for the delivery, see also 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 urine collection tube and any variations from assay requirements. If there is nonconformity in transport conditions, overall storage and transport time or volume, a new sample should be ordered as this will risk the validity of the analytical test result. 8

12 5.2.2 Sample stabilization/processing Sample processing shall occur within 2 h from collection (including transport). Sample should be maintained at 4 C. Serum and plasma should be prepared according to standard procedures and frozen immediately after processing Transport to biobank or laboratory for metabolomics analysis Specimens shall be transported at -20 C. Upon receipt, record the serum or plasma arrival time and conditions (e.g. labelling, transport conditions, sample volume, leaking) of the received samples. Report nonconformities of labelling, transport conditions and obvious sample volume differences to specifications described for the collection tubes and any variations from assay requirements. If there is nonconformity in transport conditions, processing, overall storage and transport time or 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 specimens receipt and specimen storage. Specimens should be aliquoted to minimal volumes in cryo-vials for low temperature storage enabling the metabolic profile analytical test. Serum and plasma shall be cryopreserved in freezers below -70 C, Storage below -70 C shall be initiated within 24 h from blood collection Serum and plasma thawing and use NMR profile analytical test is performed at room temperature. The thawed specimen should be introduced to the analytical test procedure within 5 min to10 min after thawing. 9

13 Bibliography Bernini P. et al., 2011, Standard operating procedures for pre-analytical handling of blood and urine for metabolomics studies and biobanks. Journal of Biomolecular NMR, Vol. 48, p: