WO2015091963A2 - Détermination de miarn plaquettaires dans l'infarctus aigu du myocarde - Google Patents

Détermination de miarn plaquettaires dans l'infarctus aigu du myocarde Download PDF

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WO2015091963A2
WO2015091963A2 PCT/EP2014/078766 EP2014078766W WO2015091963A2 WO 2015091963 A2 WO2015091963 A2 WO 2015091963A2 EP 2014078766 W EP2014078766 W EP 2014078766W WO 2015091963 A2 WO2015091963 A2 WO 2015091963A2
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platelet
whole blood
expression profile
blood sample
mirna
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WO2015091963A3 (fr
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Benjamin Meder
Markus Beier
Britta VOGEL
Hugo Katus
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Hummingbird Diagnostics GmbH
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Comprehensive Biomarker Center GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to methods, use and kits for the determination of platelet-miRNAs in Acute Myocardial Infarction (AMI).
  • AMI Acute Myocardial Infarction
  • miRNAs are a new class of biomarkers. They represent a group of small noncoding RNAs that regulate gene expression at the posttranslational level by degrading or blocking translation of messenger RNA (mRNA) targets. So far, miRNAs have been extensively studied in tissue material where it was found that miRNAs are expressed in a highly tissue-specific manner. Since recently it is known that miRNAs are not only present in tissues but also in body fluid samples, including blood. Nevertheless, the mechanism why miRNAs are found in blood, especially in the cellular blood fraction (blood cells of subfractions thereof) or in the extra-cellular fraction (serum/plasma), or their function in these blood fractions is not understood yet.
  • the inventors of the present invention assessed for the first time the expression of platelet- miRNAs in Acute Myocardial Infarction (AMI) in a platelet-comprising fraction derived from a whole blood sample. They surprisingly found that especially the platelet-miRNAs are significantly dysregulated in whole blood samples of subjects affected with AMI. Therefore, the inventors of the present invention followed different approaches to determine expression profiles of platelet-miRNA in platelet-comprising fractions of whole blood samples.
  • AMI Acute Myocardial Infarction
  • the inventors determined expression profiles of platelet-miRNAs in AMI directly from whole blood samples without prior depletion of removal of white blood cells and/or red blood cells, hence in front of a background of white blood cells and/or red blood cells (or without isolation of platelets). This is surprising since the RNA-content of the anucleated platelets is approximately 10.000 times lower when compared to the RNA-content of the nucleated white blood cells, with which the platelets are mixed in the whole blood sample collected in said whole blood collection tubes.
  • the expression of the platelet- miRNAs can be determined directly from total RNA isolated from the mixture of platelets, white blood cells and red blood cells present in the whole blood collected in whole blood collection tubes (preferably collected in Paxgene-like tubes, more preferably collected in PAXgeneTM Blood RNA tubes) without the need for prior depletion or removal of the white blood cells and/or the red blood cells.
  • the expression of the platelet- miRNAs can be determined in front of a background of white blood cells and/or red blood cells, without the need to isolate (or enrich) the platelets or the platelet fraction from the whole blood sample before.
  • platelet- miRNAs that are determined from total RNA isolated from whole blood samples without prior depletion and/or removal of white blood cells and/or red blood cells (or in front of a background of white blood cells and/or red blood cells; or without prior isolation of the platelet fraction), may be employed as biomarkers for non-invasive diagnosis of AMI, or for non-invasive diagnosis of a platelet-related (platelet-activated) component of AMI.
  • platelet-miRNAs that are determined from total RNA isolated from whole blood samples without prior depletion or removal of white blood cells and/or red blood cells (or in front of a background of white blood cells and/or red blood cells; or without prior isolation of the platelet fraction), may be employed as biomarkers for determining the platelet-activity in AMI and/or for monitoring the efficacy of anti-platelet therapy in AMI.
  • the inventors of the present invention found that the whole blood collection tubes, preferably Paxgene-like tubes, more preferably PAXgeneTM Blood RNA tubes, are suitable or useful for determination of expression profiles of platelet-miRNAs without prior depletion or removal of white blood cells and/or red blood cells (or in front of a background of white blood cells and/or red blood cells; or without prior isolation of the platelet fraction) and that such expression profiles may be used in a method for non-invasive diagnosis of AMI, or for non-invasive diagnosis of platelet-related (platelet- activated) components of AMI and for determining platelet-activity in AMI or for monitoring the efficacy of anti-platelet therapy in AMI.
  • the whole blood collection tubes preferably Paxgene-like tubes, more preferably PAXgeneTM Blood RNA tubes
  • the inventors determined expression profiles of platelet-miRNAs in AMI with prior depletion or removal of the white blood cells and/or the red blood cells, or with isolation of the platelet-fraction.
  • a platelet- comprising fraction e.g. platelet-rich-plasma, platelet concentrate
  • the expression profile of said set of at least one platelet-miRNA was determined.
  • the expression of the platelet-miRNAs can be determined without a background of white blood cells and/or red blood cells, allowing to determine platelets that are expressed at lower levels which would not possible to determine in front of a background of white blood cells and/or red blood cells.
  • This allows for a clear separation of (biomarker) information originating from the platelet- comprising fraction on the one side and from the white blood cells or the red blood cells on the other side.
  • This increased sensitivity for determining platelet-miRNAs may be of advantage to especially diagnostic applications, where the determination of certain platelet-miRNAs, especially those expressed at low levels, is required with high sensitivity, e.g. in the diagnosis of AMI.
  • Said expression profiles that were determined from total RNA isolated from whole blood samples with prior depletion and/or prior removal of white blood cells and/or red blood cells (or with prior isolation of the platelet-comprising fraction) may be employed as biomarkers for noninvasive diagnosis of AMI, for non-invasive diagnosis of platelet-related (platelet-activated) components of AMI, or for determining the platelet-activity in AMI or for monitoring the efficacy of anti-platelet therapy in AMI.
  • the invention provides for a method for diagnosing a platelet-related or platelet- activated component of Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • the invention provides for a method for diagnosing Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • the invention provides for a method for monitoring the progression of Acute Myocardial Infarction in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • the invention provides for a method for determining the platelet activity in a subject affected by Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • the invention provides for a method for monitoring the efficacy of an antiplatelet therapy in a subject affected by Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • the invention provides for kit for use in the method according to any of the first, second, third, fourth or fifth aspect of the invention, comprising: a) means for determining an expression profile of a set comprising at least one platelet-miR A
  • the expression profile and the reference expression profile are obtained from said set of at least one platelet-miRNAs selected from the group consisting SEQ ID NO: 1 to SEQ ID NO: 8 and wherein the expression profile and the reference expression profile are determined from a platelet-comprising fraction derived from a whole blood sample.
  • test compounds As used in this specification and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the content clearly dictates otherwise. For example, the term “a test compound” also includes “test compounds”.
  • micro RNA refers to single-stranded RNA molecules of at least 10 nucleotides and of not more than 35 nucleotides covalently linked together.
  • the miRNAs regulate gene expression and are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein (i.e. miRNAs are non-coding RNAs).
  • miRNAs are non-coding RNAs.
  • micro RNA* refers to miRNA molecules derived from the passenger strand upon processing. In the context of the present invention, the terms “miRNA” and “miRNA*” are interchangeable used.
  • the miRBase (www.mirbase.org) is a well established repository and searchable database of published miRNA sequences and annotation.
  • a human miRNA may also be suitable for detecting the respective miRNA orthologue(s) in another species, e.g. in another mammal, e.g. in an animal such as mouse or rat or vice versa.
  • the term "platelet” as used in the context of the present invention refers to the smallest type of blood cells, also known as "thrombocytes", which are released into the blood stream from bone marrow megakaryocytes. Platelets play a crucial role in haemostasis and thrombosis. Platelets do not contain a nucleus, therefore missing substantial parts of the microRNA machinery components required for transcription and nuclear processing.
  • Platelets comprise approximately 10.000 times less RNA when compared to nucleated cells (e.g. white blood cells). Platelets contain miRNAs (PMID 21415270, 22371016, 23323973), but it is not yet completely understood where these originate from and how these interact in translational control or repression of platelet mRNAs.
  • miRNAs PMID 21415270, 22371016, 23323973
  • platelet-miRNA(s) refers to a miRNA(s) that is (are) expressed in platelets (thrombocytes). This does not necessarily mean that these platelet-miRNAs are exclusively expressed in platelets and not in any other blood cells, in other cells, in other body fluids or in tissue. Currently, there are approximately 400 miRNA described to be expressed in platelets.
  • the platelet-miRNAs in AMI according to the present invention are selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 and are listed in Figure 1.
  • the expression level of said platelet- miRNAs selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 are in a range that allows for direct determination from a whole blood sample of a subject affected by AMI or suspected to be affected by AMI without the need for additional sample processing steps (e.g. removal or depletion of the red blood cells and/or white blood cell fractions, or isolation of the platelet fraction), which is of advantage to commercial applications, especially in the field of diagnostics.
  • the platelet-miRNAs selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 can be determined directly from a whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells.
  • the inventors of the present invention applied further criteria based on expression data for selection of said 8 platelet-miRNAs with SEQ ID NO: 1 to SEQ ID NO: 8 from the approximately 400 miRNAs that are described to be expressed in platelets.
  • the expression profile of said set of at least one platelet miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 may be determined after isolation of the platelet-comprising fraction, e.g by isolating the platelets from the whole blood sample or by depletion or removal of the white blood cells and/or the red blood cells from the whole blood sample.
  • the term "whole blood sample”, as used in the context of the present invention, refers to a blood sample originating from a subject containing all blood fractions, including both the cellular (red blood cells, white blood cells, platelets) and the extra-cellar blood fractions (serum, plasma).
  • the "whole blood sample” may be derived by removing blood from a subject by conventional blood collecting techniques, but may also be provided by using previously isolated and/or stored blood samples.
  • the whole blood sample from a subject e.g. human or animal
  • platelet-comprising fraction refers to a fraction of a whole blood sample that comprises platelets.
  • that platelet-comprising fraction may contain -besides platelets- also white blood cells and/or red blood cells or alternatively said platelet-comprising fraction comprises substantially (only) platelets.
  • An example for the first case is a mixture of platelets, white blood cells and red blood cells which is present in a whole blood sample.
  • An example for the second case is platelet-rich-plasma or platelet concentrate, which are derived from a whole blood sample from which the white blood cells and the red blood cells were removed.
  • a platelet-comprising fraction is a (useful) source from which an expression profile of a set comprising at least one platelet-miR A can be determined with or without prior depletion of removal of white blood cells and/or red blood cells (preferably not in front or in front of a background of white blood cells and/or red blood cells or prefereably with or without isolation of platelets).
  • whole blood collection tube relates to blood collection tube, that is used for collection of whole blood samples, preferebly a whole blood collection tube according to the present invention is a Paxgene-like tube, a PAXgeneTM Blood R A tube, a Tempus Blood R A tube, an EDTA-tube, a Na-citrate tube or a ACD-tube (Acid citrate dextrose).
  • a whole blood collection tube is a Paxgene-like tube, a PAXgeneTM Blood R A tube, a Tempus Blood R A tube, an EDTA-tube, a Na-citrate tube or a ACD-tube (Acid citrate dextrose).
  • the R A-fraction, -especially the miR A fraction- may be protected/guarded against degradation.
  • special whole blood collection tubes e.g.
  • RNAlaterTM Blood RNA tubes from Preanalytix, TempusTM Blood RNA tubes from Applied Biosystems
  • additives e.g. RNAlaterTM from Ambion, RNAsin ® from Promega, RNA Retain ® from Asuragen, catrimox-14 or tetradecyltrimethyl-ammonium oxalate or derivatives thereof
  • RNAlaterTM from Ambion
  • RNAsin ® from Promega
  • RNA Retain ® from Asuragen, catrimox-14 or tetradecyltrimethyl-ammonium oxalate or derivatives thereof
  • Paxgene-like tube as used in the context of the present invention relates to blood collection tubes, which are suited or used for collection of whole blood samples, which contain additives for stabilization of RNA and which are suited for expression analyses of the intracellular RNA and/or intracellular miRNAs and/or platelet-miRNAs.
  • additives for stabilization of RNA are employed, including, but are not limited to, RNAlaterTM, RNAsin ® , RNA Retain ® catrimox- 14 or tetradecyltrimethylammonium oxalate or derivatives thereof.
  • Paxgene-like tubes are “PAXgeneTM Blood RNA tubes", which are suited for collection of whole blood samples and which contain additives for cell lysis and stabilization of intracellular RNA and which are suited for expression analyses of RNAs and/or miRNAs and/or platelet-miRNAs.
  • additives for cell lysis and stabilization of intracellular RNA include, but are not limited to, catrimox- 1 4 or tetradecyltrimethylammonium oxalate or derivatives thereof.
  • PAXgeneTM Blood RNA tubes are currently marketed by PreAnalytix (www.preanalytix.com).
  • total RNA refers to the RNA isolated from a platelet-comprising fraction derived from the whole blood sample.
  • the total RNA comprising the miRNA-fraction or comprising a miRNA-enriched fraction, is obtained by lysis (e.g. Trizol) of the blood cells of the platelet-comprising fraction derived from the whole blood sample, followed by RNA isolation (extraction) e.g. by phenol/chloroform extraction and/or separation based techniques (e.g. glass fibre filter column, silica-membrane column).
  • kits for RNA isolation and purification include the miRNeasy Kits (Qiagen), PAXgeneTM Blood miRNA Kit (Qiagen), mirVana PARIS Kit (Life Technologies), PARIS Kit (Life Technologies), Tempus Spin RNA Isolation Kit (Life Technologies).
  • the total RNA according to the present invention contains the miRNA- fraction or contains a miRNA-enriched fraction.
  • intracellular RNA as used herein relates to the RNA present in the blood cells (platelets, white blood cells, red blood cells) of a whole blood sample, comprising the miRNA- fraction.
  • expression profile represents the a measure that correlates with the miRNA expression (level) in a sample.
  • each miRNA is represented by a numerical value. The higher the value of an individual miRNA, the higher is the expression level of said miRNA, or the lower the value of an individual miRNA, the lower is the expression level of said miRNA.
  • the expression profile may be generated by any convenient means, e.g. nucleic acid hybridization (e.g. to a microarray), nucleic acid amplification (PCR, RT-PCR, qRT-PCR, high-throughput RT-PCR), ELISA for quantitation, next generation sequencing (e.g.
  • the sample material measured by the aforementioned means is a platelet-comprising fraction derived from the whole blood sample (with or without prior depletion or removal of the white blood cells and/or the red blood cells or with or without isolation of the platelet fraction or not in front or in front of a background of white blood cells and/or red blood cells), comprising said platelet-miRNAs and may be total RNA, labeled total RNA, amplified total RNA, cDNA, labeled cDNA, amplified cDNA, miRNA, labeled miRNA, amplified miRNA or any derivatives that may be generated from the aforementioned RNA/DNA species.
  • the "expression profile”, as used herein, relates to a collection of expression (levels) of at least one miRNAs, preferably of least 1, 2, 3, 4, 5, 6, 7, 8 or more miRNAs.
  • determining an expression profile relates to the determination of the expression profile of set comprising at least one platelet-miRNA, hence from miRNA(s) expressed in platelets, preferably from miRNAs expressed in platelets selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8.
  • the determination of the expression profile is a measure that directly or indirectly correlates with the levels of platelet-miRNAs present in said platelet-comprising fraction derived from the whole blood sample.
  • nucleic acid hybridisation nucleic acid amplification, polymerase extension, mass spectroscopy, flow cytometry, sequencing
  • RNA- or miRNA-isolation RNA- or miRNA-enrichment
  • RNA- or miRNA- purification RNA- or miRNA-labeling
  • polymerase extension of RNA or miRNA ligation of RNA or miRNA, reverse-transcription of RNA or miRNA into cDNA, amplification of the cDNA, labelling of cDNA).
  • nucleic acid hybridization relates to a means for determining an expression profile.
  • the nucleic acid hybridization may be performed using a microarray/biochip or in situ hybridization.
  • the polynucleotides (probes) with complementarity to the corresponding platelet-miRNAs to be detected are e.g. attached to a solid phase to generate a microarray/biochip.
  • Said microarray/biochip is then incubated with a sample containing the platelet-miRNA(s) (or a species that is derived from said platelet- miRNA(s)), which may be labelled or unlabelled.
  • Quantification of the expression level of the miRNAs may then be carried out e.g. by direct read out of said label or by additional manipulations, e.g. by use of an enzymatic reaction.
  • the polynucleotides which are at least partially complementary to miRNAs having SEQ ID NO: 1 to SEQ ID NO: 8 are contacted with said sample containing said platelet-miRNA(s) (or a species that is derived from said platelet-miRNA(s), e.g. a cDNA- species) in solution to hybridize.
  • the hybridized duplexes are pulled down to the surface and successfully captured miRNAs are quantitatively determined (e.g. FlexmiR-assay, FlexmiR v2 detection assays from Luminex, Fireplex assayfrom Firefly Bioworks).
  • nucleic acid amplification relates to a means for determining an expression profile.
  • Nucleic acid amplification may be performed using real time polymerase chain reaction (RT-PCR) such as real time quantitative polymerase chain reaction (RT qPCR).
  • RT-PCR real time polymerase chain reaction
  • the standard real time polymerase chain reaction (RT-PCR) is preferred for the analysis of a single miRNA or a set comprising a low number of miRNAs (e.g. a set of at least 2 to 10 miRNAs), whereas high-throughput RT-PCR technologies (e.g.
  • OpenArray from Applied Biosystems, SmartPCR from Wafergen, Biomark System from Fluidigm are also able to measure large sets (e.g a set of 5, 10, 20, 30, 50, 80, 100, 200 or more) to all known miRNAs in a high parallel fashion.
  • RT-PCR real time polymerase chain reaction
  • steps may include the following steps: (i) extracting the total RNA from platelet-comprising fraction derived from the whole blood sample collected in a whole blood collection tube, preferably collected in a Paxgene-like tube, particularly preferably collected in a PAXgeneTM Blood RNA tube, obtained from a subject, (ii) obtaining cDNA samples by RNA reverse transcription (RT) reaction using universal or miRNA-specific primers; (iii) optionally pre-amplifying the cDNA of step (ii) via polymerase chain reaction (PCR), (iv) amplifying the optionally pre-amplified cDNA via polymerase chain reaction (PCR), thereby monitoring the amplification through a previously added fluorescent reporter dye (e.g.
  • Step (i) the isolation and/or extraction of RNA may be omitted in cases where the RT-PCR is conducted directly from the miRNA-containing sample.
  • Kits for determining a miRNA expression profile by real time polymerase chain reaction are e.g. from Life Technologies, Applied Biosystems, Ambion, Roche, Qiagen, Invitrogen, SABiosciences,Exiqon.
  • sequencing relates to a means for determining an expression profile, including conventional (Maxam-Gilbert, Sanger) sequencing technology, Pyrosequencing or next generation sequencing technology (e.g. ABI SOLID, Illumina Hiseq, Gnubio, Pacific Biosystems, 454) or any other sequencing technology, capable of determination of the expression profile of set comprising at least one platelet-miRNA.
  • reference refers to a reference to which the expression profile of a test sample of a subject affected by AMI or suspected to be affected by AMI is compared in the course of non- invasive diagnosis of AMI, or in the course of non-invasive diagnosis of a platelet-related (platelet-activated) component of AMI or in the course of determining the platelet-activity in AMI and/or in the course of monitoring the efficacy of anti-platelet therapy in AMI.
  • both the expression profile of the subject (affected by AMI or suspected to be affected by AMI) to be tested as well as the reference, are determined from the same platelet-miRNAs and the same sample type (collected and worked up in the same way), preferably they are determined from a platelet-comprising fraction derived from the whole blood sample collected in whole blood collection tubes, preferably collected in Paxgene-like tubes, more preferably collected in PAXgeneTM Blood RNA tubes.
  • the reference may be a reference expression profile obtained from determining one or more expression profiles of a set comprising at least one platelet-miRNA from a platelet-comprising fraction derived from the whole blood sample collected in whole blood collection tubes, preferably obtained from a platelet-comprising fraction derived from the whole blood sample collected in Paxgene-like tubes, more preferably collected in PAXgeneTM Blood RNA tubes, in one or more reference subjects.
  • the reference may be an algorithm, a mathematical function or a score that was developed from such aforementioned reference expression profiles.
  • AMI Acute Myocardial Infarction
  • ischemic injury occurs when the blood supply is insufficient to meet the tissue demand for metabolism.
  • AMIs are caused by rupture of coronary atherosclerotic plaques with superimposed coronary thrombosis.
  • Patients with MI usually present with signs and symptoms of crushing chest pressure, diaphoresis, malignant ventricular arrhythmias, heart failure (HF), or shock.
  • HF heart failure
  • AMI Alzheimer's disease .
  • ACS acute coronary syndromes
  • U unstable angina
  • NSTEMI non-ST-elevation myocardial infarction
  • ST-elevation myocardial infarction STEMI
  • diagnosis refers to the process of determining a possible disease (e.g. Acute Myocardial Infarction) or disorder or a certain component of a disease (e.g. an inflammatory component of a disease) and therefore is a process attempting to define the (clinical) condition of a subject.
  • a possible disease e.g. Acute Myocardial Infarction
  • a certain component of a disease e.g. an inflammatory component of a disease
  • the diagnosis comprises (i) determining the occurrence/presence of the disease ( or of a component of a disease), especially in an (very) early phase of the disease (ii) monitoring the course or progression of the disease, (iii) staging of the disease, (iv) measuring the response of a patient affected with the disease to therapeutic intervention, (v) monitoring the efficacy of a therapeutic intervention and/or (vi) segmentation of a subject suffering from the disease.
  • platelet-related component of a AMI refers to an aspect or feature of AMI that is platelet-related or related to the function/disfunction of platelets in AMI.
  • platelet-related components include, but are not limited to, haemo stasis-related, thrombosis-related, immunity-related, or inflammation-related aspects or features of the disease, more preferably the platelet-related component of AMI is an inflammatory component of AMI.
  • platelet-activated component of a AMI refers to an aspect or feature of AMI that is platelet-activated or activated due to function/dis function of platelets in AMI.
  • platelet-activated components include, but are not limited to, haemostasis-activated, thrombosis-activated, immunity-activated, or inflammation-activated aspects or features of AMI, more preferably the platelet-activated component of a disease is an inflammatory component of AMI.
  • An exemplarily approach to determine expression profiles in AMI ++-of a s e t comprising at least one platelet-miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 from a platelet-comprising fraction derived from the whole blood sample when starting from whole blood collected in a PAXgeneTM Blood R
  • a tube without prior depletion or removal of white blood cells and/or red blood cells is summarized below :
  • Step 1 Providing a whole blood sample of a subject : Whole blood is drawn from a subject (affected by AMI or suspected to be affected by AMI) into a PAXgeneTM Blood RNA tube, drawing of 2.5 ml of whole blood into a PAXgeneTM Blood RNA tube is sufficient for the downstream analyses. The tube should be carefully inverted to ensure that the reagents contained are thoroughly mixed with the blood. The tube maybe stored before analysis (e.g. at 4°C for up to 3 days, at -80°C for up to several months).
  • Step 2 Isolation of the total RNA from said whole blood sample :
  • the PAXgeneTM Blood RNA tube which was allowed to incubate at room temperature for at least 1 hour, is centrifuged to form a blood cell pellet (comprising white blood cells, red blood cells, platelets) at the bottom of the tube, which is collected, whereas the supernatant is discarded.
  • the total RNA (comprising the miRNA fraction) is isolated from the collected blood cell pellet (comprising white blood cells, red blood cells, platelets) using suitable kits (e.g. miRNeasy kit) and/or purification methods.
  • Step 3 Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated : From the total RNA isolated the expression profile of a set of at least one platelet-miRNA selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 is measured using experimental techniques. These techniques include but are not limited to nucleic acid hybridisation based approaches, nucleic acid amplification methods (PCR, RT-PCR, qPCR), sequencing, next generation sequencing, flow cytometry and/or mass spectroscopy. In a preferred embodiment, it may be required in Step 3 that the total RNA or that individual platelet-miRNAs are reverse-transcribed into cDNA and optionally be amplified before the expression profile is determined.
  • Step 4 Comparing said expression profile to a reference : The expression profile obtained in Step 3) is compared to a reference.
  • the reference may be e.g. a reference expression profile, obtained from determining the expression profile of said at least one platelet- miRNA from a whole blood sample collected in the same type of whole blood collection tube as in Step 1 (here: collected in a PAXgeneTM Blood RNA) in one or more reference subjects or the reference may be an algorithm, a mathematical function or a score that was developed from such a reference expression profile.
  • Step 5 Identifying if said subject is affected by an AMI -related condition : The comparison to the reference then allows to identify if said subject is affected or not affected by AMI (and/or the platelet- related (or platelet-activated) component of AMI and/or the platelet-related disease, such as AMI) or alternatively if certain thresholds for monitoring the progression of a platelet-related (platelet-activated) disease, such as AMI, and/or if thresholds for determining the platelet activity in AMI and/or if thresholds for monitoring the efficacy of an anti-platelet therapy in AMI are reached.
  • Step 6 Optionally administering said affected subject to therapy:
  • said subject that was identified to be affected by AMI (and/or was identified to be affected by the platelet- related (or platelet-activated) component of AMI) is administered to therapy, e.g. by treating the subject with drugs suited for therapy of AMI (and/or drugs suited for said the platelet- related (or platelet-activated) component of AMI).
  • administering said affected subject to therapy may include primary percutaneous coronary intervention (PCI), administration of fibrinolytic drugs, antithrombotic therapy without reperfusion therapy, coronary bypass surgery, administration of antiplatelet agents, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, further management with angiography and coronary revascularization.
  • PCI percutaneous coronary intervention
  • ACE angiotensin-converting enzyme
  • An exemplarily approach to determine expression profiles in AMI of a set comprising at least one platelet-miR A selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 platelet-comprising fraction derived from the whole blood sample when starting from whole blood collected in an EDTA-tube, with prior depletion or removal of white blood cells and/or red blood cells (or with prior isolation of the platelet fraction or not in front of a background of white blood cells and/or red blood cells) is summarized below :
  • Step 1 Providing a whole blood sample of a subject : Whole blood is drawn from a subject (affected by AMI or suspected to be affected by AMI) into a EDTA-tube (Sarstedt, S- Monovette EDTA-K 2 , 7.5 ml); drawing of 7.5 ml of whole blood into EDTA-tube tube is sufficient for the downstream analyses.
  • the tube should be carefully inverted to ensure that the reagents contained are thoroughly mixed with the blood.
  • the tube should not kept at room temperature for longer than 4 hours before centrifugation.
  • the collected whole blood sample is centrifuged with soft spin (170g, 15 min) to separate the blood cell fractions. After centrifugation the whole blood sample is separated into Platelet- Rich-Plasma (PRP, top-fraction), buffy coat (white blood cells; middle- fraction, interphase) and red blood cells (bottom fraction). From this the platelet-comprising fraction, namely the Platelet-Rich-Plasma is collected.
  • PRP Platelet- Rich-Plasma
  • buffy coat white blood cells; middle- fraction, interphase
  • red blood cells bottom fraction
  • platelet concentrate as a further platelet-comprising fraction, may be prepared by subjecting the PRP to a second hard spin centrifugation (5000g, 3min), where platelets are pelleted out of the plasma to yield platelet-concentrate and platelet-poor-plasma (PPP).
  • PRP platelet-concentrate and platelet-poor-plasma
  • Step 3 Isolation of the total RNA from said platelet-comprising fraction derived from the whole blood sample : After cell lysis the total RNA is isolated from the platelet- comprising fraction obtained in Step 2, namely from Platelet-Rich-Plasma or from platelet concentrate (each of them comprising said platelet-miRNAs) using suitable kits (e.g. miRNeasy kit) and/or purification methods.
  • suitable kits e.g. miRNeasy kit
  • Step 4 Determining an expression profile of a set comprising at least one platelet-miRNA from the total RNA isolated : From the total RNA isolated the expression profile of a set of at least one platelet-miRNA, selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8 is measured using experimental techniques. These techniques include but are not limited to nucleic acid hybridisation based approaches, nucleic acid amplification methods (PCR, RT-PCR, qPCR), sequencing, next generation sequencing, flow cytometry and/or mass spectroscopy. In a preferred embodiment, it may be required in Step 4 that the total RNA or that individual platelet-miRNAs are reverse-transcribed into cDNA and optionally be amplified before the expression profile is determined.
  • Step 5 Comparing said expression profile to a reference : The expression profile obtained in Step 3) is compared to a reference.
  • the reference may be e.g. a reference expression profile, obtained from determining the expression profile of said at least one platelet- miRNA from platelet-comprsing fraction derived from the whole blood sample collected in the same type of whole blood collection tube as in Step 1 (here: collected in a EDTA-tube; Sarstedt S-Monovette) in one or more reference subjects or the reference may be an algorithm, a mathematical function or a score that was developed from such a reference expression profile.
  • Step 6 Identifying if said subject is affected by AMI related condition : The comparison to the reference then allows to identify if said subject is affected or not affected by AMI (and/or the platelet- related (or platelet-activated) component of AMI or alternatively if certain thresholds for monitoring the progression of AMI, and/or if thresholds for determining the platelet activity in AMI and/or if thresholds for monitoring the efficacy of an anti-platelet therapy in AMI are reached.
  • Step 7 Optionally administering said affected subject to therapy:
  • said subject that was identified to be affected by AMI and/or a platelet- related (or platelet-activated) component of AMI, is administered to therapy, e.g. by treating the subject with drugs suited for therapy of AMI (and/or drugs suited for said the platelet- related (or platelet-activated) component of AMI).
  • administering said affected subject to therapy may include primary percutaneous coronary intervention (PCI), administration of fibrinolytic drugs, antithrombotic therapy without reperfusion therapy, coronary bypass surgery, administration of antiplatelet agents, beta- blockers, angiotensin- converting enzyme (ACE) inhibitors, further management with angiography and coronary revascularization.
  • PCI percutaneous coronary intervention
  • ACE angiotensin- converting enzyme
  • the present invention relates to method for diagnosing a platelet-related (or platelet-activated) component of Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8.
  • the platelet-comprising fraction derived from the whole blood sample is obtained with or without prior depletion or removal of the white blood cells and/or the red blood cells. It is further preferred that the platelet-comprising fraction derived from the whole blood sample is obtained with or without prior isolation of the platelets from the whole blood. It is further preferred that the expression profile of set comprising at least one platelet-miR A is determined in front or not in front of a background of white blood cells and/or red blood cells.
  • the platelet-related or platelet-activated component of Acute Myocardial Infarction to be determined is an inflammatory component of AMI, more prefereably it is an inflammatory component of AMI.
  • the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a EDTA-, Na-citrate-, ACD- or a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood R A tube.
  • the expression profile is determined directly from a whole blood sample.
  • the mixture of white blood cells, red blood cells and platelets represents the platelet-comprising fraction derived from said whole blood sample.
  • the expression profile is directly determined from the total RNA isolated from the whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells or the expression profile is determined directly from the total RNA isolated from the whole blood sample in front of a background of white blood cells and red blood cells or the expression profile is determined directly from the total RNA isolated from the whole blood sample without prior isolation (or extraction) of the platelet fraction.
  • the method of the first aspect of the present invention for diagnosing a platelet-related (or platelet-activated) component of Acute Myocardial Infarction comprises the following steps :
  • the expression profile is determined not directly from a whole blood sample, but from a platelet-comprising fraction of said whole blood sample, that is derived from said whole blood sample.
  • a platelet-comprising fraction of said whole blood sample that is derived from said whole blood sample.
  • the platelet-comprising fraction is prepared by isolation of the platelet fraction or by depletion or removal of the white blood cells and/or the red blood cells from said whole blood sample, hence the expression profile is determined not front of a background of white blood cells and red blood cells.
  • the platelet-comprising fraction may be isolated from said whole blood sample by convenient means (e.g.
  • the method of the first aspect of the present invention for diagnosing a platelet- related or platelet-activated component of Acute Myocardial Infarction comprises the following steps :
  • Providing a whole blood sample of a subject b. Depletion or removal of the white blood cells and/or the red blood cells from the whole blood sample
  • the invention in a second aspect, relates to a method for diagnosing Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a platelet- comprising fraction derived from said whole blood sample.
  • the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8.
  • the platelet-comprising fraction derived from the whole blood sample is obtained with or without prior depletion or removal of the white blood cells and/or the red blood cells. It is further preferred that the platelet-comprising fraction derived from the whole blood sample is obtained with or without prior isolation of the platelets from the whole blood. It is further preferred that the expression profile of set comprising at least one platelet-miRNA is determined in front or not in front of a background of white blood cells and/or red blood cells.
  • Acute Myocardial Infarction may be diagnosed from a platelet- related component of AMI , such as an platelet-related inflammatory component of AMI, such as an platelet-related an inflammatory component of AMI which may be determined from an expression profile of a set comprising at least one platelet-miRNA selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 8. It is preferred that the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a EDTA-, Na-citrate-, ACD- or a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood R A tube.
  • the expression profile is determined directly from a whole blood sample.
  • the mixture of white blood cells, red blood cells and platelets represents the platelet-comprising fraction derived from said whole blood sample.
  • the expression profile is directly determined from the total RNA isolated from the whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells or the expression profile is determined directly from the total RNA isolated from the whole blood sample in front of a background of white blood cells and red blood cells or the expression profile is determined directly from the total RNA isolated from the whole blood sample without prior isolation (or extraction) of the platelet fraction.
  • the method of the second aspect of the present invention for diagnosing Acute Myocardial Infarction comprises the following steps :
  • the expression profile is determined not directly from a whole blood sample, but from a platelet-comprising fraction of said whole blood sample, that is derived from said whole blood sample.
  • a platelet-comprising fraction of said whole blood sample that is derived from said whole blood sample.
  • the platelet-comprising fraction is prepared by isolation of the platelet fraction or by depletion or removal of the white blood cells and/or the red blood cells from said whole blood sample, hence the expression profile is determined not front of a background of white blood cells and red blood cells.
  • the platelet-comprising fraction may be isolated from said whole blood sample by convenient means (e.g.
  • the method of the second aspect of the present invention for diagnosing Acute Myocardial Infarction comprises the following steps :
  • Providing a whole blood sample of a subject b. Depletion or removal of the white blood cells and/or the red blood cells from the whole blood sample c. Isolation of the total RNA from sample of step b.
  • the invention in a third aspect, relates to a method for monitoring the progression of Acute Myocardial Infarction in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample. It is preferred that in said method the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8.
  • the platelet-comprising fraction derived from the whole blood sample is obtained with or without prior depletion or removal of the white blood cells and/or the red blood cells. It is further preferred that the platelet-comprising fraction derived from the whole blood sample is obtained with or without prior isolation of the platelets from the whole blood. It is further preferred that the expression profile of set comprising at least one platelet-miRNA is determined in front or not in front of a background of white blood cells and/or red blood cells.
  • the progression of Acute Myocardial Infarction may be monitored by monitoring the progression of a platelet-related componenet of AMI, such as a platelet-related inflammatory component of AMI, such as a platelet-related an inflammatory component of AMI which may be monitored from an expression profile of a set comprising at least one platelet- miRNA selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 8.
  • the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a EDTA-, Na-citrate-, ACD- or a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood RNA tube.
  • the expression profile is determined directly from a whole blood sample.
  • the mixture of white blood cells, red blood cells and platelets represents the platelet-comprising fraction derived from said whole blood sample.
  • the expression profile is directly determined from the total RNA isolated from the whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells or the expression profile is determined directly from the total RNA isolated from the whole blood sample in front of a background of white blood cells and red blood cells or the expression profile is determined directly from the total RNA isolated from the whole blood sample without prior isolation (or extraction) of the platelet fraction.
  • the method of the third aspect of the present invention for monitoring Acute Myocardial Infarction comprises the following steps :
  • the expression profile is determined not directly from a whole blood sample, but from a platelet-comprising fraction of said whole blood sample, that is derived from said whole blood sample.
  • a platelet-comprising fraction of said whole blood sample that is derived from said whole blood sample.
  • the platelet-comprising fraction is prepared by isolation of the platelet fraction or by depletion or removal of the white blood cells and/or the red blood cells from said whole blood sample, hence the expression profile is determined not front of a background of white blood cells and red blood cells.
  • the platelet-comprising fraction may be isolated from said whole blood sample by convenient means (e.g.
  • the method of the third aspect of the present invention for monitoring the progression of Acute Myocardial Infarction comprises the following steps :
  • the invention in a fourth aspect, relates to a method for determining the platelet activity in a subject affected by Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8.
  • the platelet-comprising fraction derived from the whole blood sample is obtained with or without prior depletion or removal of the white blood cells and/or the red blood cells. It is further preferred that the platelet-comprising fraction derived from the whole blood sample is obtained with or without prior isolation of the platelets from the whole blood. It is further preferred that the expression profile of set comprising at least one platelet-miRNA is determined in front or not in front of a background of white blood cells and/or red blood cells.
  • the platelet activity of a subject affected (or suspected to be affected) by Acute Myocardial Infarction may be determined by determining an expression profile of a set comprising at least one platelet-miRNA selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 8. It is preferred that a pathological platelet activity, hence a platelet activity that is below or above a (pathological) platelet activity threshold may be determined by determining an expression profile of a set comprising at least one platelet-miRNA selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 8 from a platelet-comprising fraction derived from said whole blood sample.
  • a subject with a pathological platelet activity may be identified from comparison of said expression profile of a set comprising at least one platelet-miRNA selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 8 to a reference.
  • the reference may be a reference expression profile derived from reference subjects with known platelet activity, preferably from subjects with known platelet activity below or above a pathological threshold.
  • the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a EDTA-, Na-citrate-, ACD- or a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood RNA tube.
  • the expression profile is determined directly from a whole blood sample.
  • the mixture of white blood cells, red blood cells and platelets represents the platelet-comprising fraction derived from said whole blood sample.
  • the expression profile is directly determined from the total RNA isolated from the whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells or the expression profile is determined directly from the total RNA isolated from the whole blood sample in front of a background of white blood cells and red blood cells or the expression profile is determined directly from the total RNA isolated from the whole blood sample without prior isolation (or extraction) of the platelet fraction.
  • the method of the fourth aspect of the present invention for determining the platelet activity in a subject affected with (or suspected to be affected with) Acute Myocardial Infarction comprises the following steps :
  • the expression profile is determined not directly from a whole blood sample, but from a platelet-comprising fraction of said whole blood sample, that is derived from said whole blood sample.
  • a platelet-comprising fraction of said whole blood sample that is derived from said whole blood sample.
  • the platelet-comprising fraction is prepared by isolation of the platelet fraction or by depletion or removal of the white blood cells and/or the red blood cells from said whole blood sample, hence the expression profile is determined not front of a background of white blood cells and red blood cells.
  • the platelet-comprising fraction may be isolated from said whole blood sample by convenient means (e.g.
  • the method of the fourth aspect of the present invention for determining the platelet activity in a subject affected with (or suspected to be affected with) Acute Myocardial Infarction comprises the following steps :
  • Providing a whole blood sample of a subject b. Depletion or removal of the white blood cells and/or the red blood cells from the whole blood sample
  • the invention relates to a method for monitoring the efficacy of an anti-platelet therapy in a subject affected by Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • the set comprising at least one platelet-miRNA is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8.
  • the platelet-comprising fraction derived from the whole blood sample is obtained with or without prior depletion or removal of the white blood cells and/or the red blood cells. It is further preferred that the platelet-comprising fraction derived from the whole blood sample is obtained with or without prior isolation of the platelets from the whole blood. It is further preferred that the expression profile of set comprising at least one platelet-miRNA is determined in front or not in front of a background of white blood cells and/or red blood cells.
  • the efficacy of an anti-platelet therapy of a subject affected (or suspected to be affected ) with Acute Myocardial Infarction may be monitored by determining an expression profile of a set comprising at least one platelet-miRNA selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 8. It is preferred that a pathological efficacy of an anti-platelet therapy, hence an efficacy of an anti-platelet therapy that is below or above a (pathological) efficacy threshold may be monitored by determining an expression profile of a set comprising at least one platelet-miRNA selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 8 from a platelet-comprising fraction derived from said whole blood sample.
  • a subject with a pathological efficacy of an anti-platelet therapy may be identified from comparison of said expression profile of a set comprising at least one platelet-miRNA selected from the group comprising SEQ ID NO: 1 to SEQ ID NO: 8 to a reference.
  • the reference may be a reference expression profile derived from reference subjects with known efficacy of an anti-platelet therapy, preferably from subjects with known efficacy of an anti-platelet therapy below or above a pathological threshold.
  • the whole blood sample is collected in a whole blood collection tube, preferably it is collected in a EDTA-, Na-citrate-, ACD- or a Paxgene-like tube, particularly preferred it is collected in a PAXgeneTM Blood RNA tube.
  • the expression profile is determined directly from a whole blood sample.
  • the mixture of white blood cells, red blood cells and platelets represents the platelet-comprising fraction derived from said whole blood sample.
  • the expression profile is directly determined from the total RNA isolated from the whole blood sample without prior depletion or removal of the white blood cells and/or the red blood cells or the expression profile is determined directly from the total RNA isolated from the whole blood sample in front of a background of white blood cells and red blood cells or the expression profile is determined directly from the total RNA isolated from the whole blood sample without prior isolation (or extraction) of the platelet fraction.
  • the method of the fifth aspect of the present invention for monitoring the efficacy of an anti-platelet therapy in a subject affected with (or suspected to be affected with) Acute Myocardial Infarction comprises the following steps :
  • the expression profile is determined not directly from a whole blood sample, but from a platelet-comprising fraction of said whole blood sample, that is derived from said whole blood sample.
  • a platelet-comprising fraction of said whole blood sample that is derived from said whole blood sample.
  • the platelet-comprising fraction is prepared by isolation of the platelet fraction or by depletion or removal of the white blood cells and/or the red blood cells from said whole blood sample, hence the expression profile is determined not front of a background of white blood cells and red blood cells.
  • the platelet-comprising fraction may be isolated from said whole blood sample by convenient means (e.g.
  • the method of the fifth aspect of the present invention for monitoring the efficacy of an anti-platelet therapy in a subject affected with (or suspected to be affected with) Acute Myocardial Infarction comprises the following steps :
  • Providing a whole blood sample of a subject b. Depletion or removal of the white blood cells and/or the red blood cells from the whole blood sample
  • the determining of an expression profile of a set comprising at least one platelet-miR A (selected from SEQ ID NO: 1 to 8) from the total RNA isolated according to the first aspect (diagnosing a platelet-related component of AMI), second aspect (diagnosing AMI), third aspect (monitoring the progression of AMI), fourth aspect (determining the platelet activity in AMI) or fifth aspect (monitoring the efficacy of an anti-platelet-therapy in AMI) of the invention comprises the steps :
  • step (b) optionally amplifying the cDNA of step (a)
  • miRNA-specific or universal reverse transcription DNA-primers are used for reverse transcription in step (a).
  • miRNA-specific reverse transcription primers are listed in column B of Figure 3
  • examples of universal reverse transcription primers are 01igo-d(T)-Primers or RT primers listed in column C of Figure 6.
  • miRNA-specific forward primer and universal reverse primer or alternatively universal forward and universal reverse primer are used for optionally amplifying the cDNA in step (b).
  • Examples of miRNA-specific forward primer and universal reverse primer are listed in column C and D of Figure 3, examples of universal forward and universal reverse primer are listed in column D and E of Figure 6.
  • miRNA-specific forward primer and universal reverse primer or alternatively miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c).
  • the quantifying in step (c) is performed by real-time PCR, nucleic acid hybridization (e.g. microarray) or sequencing (e.g. next generation sequencing) techniques.
  • the quantifying in step (c) by real-time PCR is utilizing dual-labeled hydrolysis probes that make use of the 5 ' -3 ' exonuclease activity of polymerase (e.g. Taqman-probes) or DNA-intercalating dyes (e.g. SYBRgreen).
  • Examples of miRNA-specific forward primer and universal reverse primer are listed in column C and D of Figure 3, examples of miRNA-specific forward and partially universal reverse primer are listed in column B and C of Figure 4; examples of dual-labeled hydrolysis probes are listed in column E of Figure 3.
  • the determining of an expression profile of a set comprising at least one platelet-miR A (selected from SEQ ID NO: 1 to 8) from the total RNA isolated according to the first aspect (diagnosing a platelet-related component of AMI), second aspect (diagnosing AMI), third aspect (monitoring the progression of AMI), fourth aspect (determining the platelet activity in AMI) or fifth aspect (monitoring the efficacy of an anti-platelet-therapy in AMI) of the invention comprises the steps :
  • RNA-DNA hybrids (a) adding a DNA- fragment to the 3 '-end of the miRNAs comprised in the total RNA isolated (from a whole blood or from a whole blood sample with or without prior depletion or removal of the white blood cells and/or the red blood cells), thereby forming non-naturally occurring RNA-DNA hybrids
  • DNA- fragments of 1 to 150 nucleotides in length are added in step (a) to the 3 '-end of the miRNAs by ligation or by polymerase-based elongation.
  • Examples of said DNA-fragments are listed in Figure 5 or column A of Figure 6.
  • step (miRNA-specific or) universal reverse transcription DNA-primers are used for reverse transcription in step (b). Further, it is preferred that miRNA-specific forward primer and universal reverse primer or alternatively miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c). Examples of miRNA-specific reverse transcription primers are listed in column B of Figure 3, examples of universal reverse transcription primers are oligo-d(T)-Primers or RT primers listed in column C of Figure 6. It is preferred that the quantifying in step (c) is performed by real-time PCR, nucleic acid hybridization or sequencing (e.g. next generation sequencing) techniques.
  • the quantifying in step (c) by real-time PCR is utilizing dual-labeled hydrolysis probes that make use of the 5 ' -3 ' exonuclease activity of polymerase (e.g. Taqman-probes) or DNA-intercalating dyes (e.g. SYBRgreen).
  • polymerase e.g. Taqman-probes
  • DNA-intercalating dyes e.g. SYBRgreen
  • the determining of an expression profile of a set comprising at least one platelet-miRNA (selected from SEQ ID NO: 1 to 8) from the total RNA isolated according to the first aspect (diagnosing a platelet-related component of AMI), second aspect (diagnosing AMI), third aspect (monitoring the progression of AMI), fourth aspect (determining the platelet activity in AMI) or fifth aspect (monitoring the efficacy of an anti-platelet-therapy in AMI) of the invention comprises the steps :
  • RNA-RNA hybrids (a) adding a RNA- fragment to the 3 '-end of the miRNAs comprised in the total RNA isolated (from a whole blood or from a whole blood sample with or without prior depletion or removal of the white blood cells and/or the red blood cells), thereby forming non-naturally occurring RNA-RNA hybrids
  • RNA- fragments of 1 to 150 nucleotides in length are added in step (a) to the 3 '-end of the miRNAs preferably by poly(A)-tailing reaction.
  • the reverse- transcription of step (b) is preferably with universal RT -primers, e.g. oligo-d(T)-primers.
  • the quantifying in step (c) is preferably utilizing miRNA-specific forward and partially universal reverse primer, e.g. miRNA-specific forward and partially universal reverse primer as listed in column B and C of Figure 4.
  • kits for use in the method according to any of the first, second, third, fourth or fifth aspect of the present invention including all of its embodiments.
  • Said kit for use in the method according to any of the first, second, third, fourth or fifth aspect of the present invention comprises : a) means for determining an expression profile of a set comprising at least one platelet-miRNA
  • the expression profile and the reference expression profile are obtained from said at least one platelet-miRNAs selected from the group consisting SEQ ID NO: 1 to SEQ ID NO: 8 and wherein the expression profile and the reference expression profile are determined from a platelet-comprising fraction derived from a whole blood sample (from a subject affected or suspected to be affected by AMI).
  • the reference may be contained in the data carrier of the kit.
  • the kit may contain a reference sample and/or a reference standard that is included in the kit and which is employed when performing the kit, e.g. in the determining of the expression profile.
  • the kit optionally comprises a data carrier.
  • the data carrier is an electronic or a nonelectronic data carrier, more preferably it is an electronic data carrier, such as a storage medium.
  • the data carrier comprised in the kit comprises a guide for use of the kit in the method according to any of the first, second, third, fourth or fifth aspect of the present invention.
  • This guide may include instructions for the doctor and/or the diagnostic laboratory that are involved in the method according to any of the first, second, third, fourth or fifth aspect of the present invention.
  • the guide may include a reference according to the present invention.
  • the data carrier further comprises tools for analysis and evaluation of the determined expression profile(s).
  • tools may be any tools to assist the doctor and/or the diagnostic laboratory in the method according to any of the first, second, third, fourth or fifth, aspect of the present invention.
  • these tools are software-tools that assist in analysis of the determined expression profile(s) and/or assist in the subsequently diagnosis.
  • the tools for analysis and evaluation may include a reference according to the present invention.
  • the kit optionally comprises optionally means for deriving a platelet-comprising fraction from a whole blood sample.
  • Said means are for preparing said platelet-comprising fraction from said whole blood sample, wherein said means may include means for removal or depletion of white blood cells and/or red blood cells from said whole blood sample (e.g. by centrifugation, by size selection techniques, by differential cell lysis , e.g. by differential red blood cell lysis), means for isolation of platelets from said whole blood sample (e.g. by centrifugation, by size selection techniques).
  • the kit optionally comprises a whole bood collection tube, which is preferably selected from group consisting of EDTA- , Na-citrate-, ACD-, PAXgeneTM Blood R A-, Tempus Blood R A- tubes and which optionally may contain an additive for stabilizing the R A-and/or the miRNA- fraction.
  • a whole bood collection tube which is preferably selected from group consisting of EDTA- , Na-citrate-, ACD-, PAXgeneTM Blood R A-, Tempus Blood R A- tubes and which optionally may contain an additive for stabilizing the R A-and/or the miRNA- fraction.
  • the present invention is composed of the following items :
  • Method for diagnosing a platelet-related (or platelet-activated) component of Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miR A from a whole blood sample of a subject (affected or suspected to be affected by Acute Myocardial Infarction), wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • Method for diagnosing Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject (affected or suspected to be affected by Acute Myocardial Infarction), wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • Method for monitoring the progression of Acute Myocardial Infarction in a subject by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject (affected or suspected to be affected by Acute Myocardial Infarction), wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • Method for determining the platelet activity in a subject affected or suspected to be affected by Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • Method for monitoring the efficacy of an anti-platelet therapy in a subject affected or suspected to be affected by Acute Myocardial Infarction by determining an expression profile of a set comprising at least one platelet-miRNA from a whole blood sample of a subject, wherein the expression profile is determined from a platelet-comprising fraction derived from said whole blood sample.
  • the platelet-comprising fraction is derived from a whole blood sample without prior removal of the white blood cells and/or the red blood cells.
  • the platelet-comprising fraction comprises white blood cells, red blood cells and platelets and/or wherein the expression profile of said set comprising at least one platelet-miRNA is determined in front of a background of white blood cells and red blood cells.
  • the whole blood sample is collected in a whole blood collection tube, optionally containing an additive for RNA stabilization, preferably optionally containing an additive for stabilization of intracellular RNA, more preferably the whole blood collection tube is a PAXgeneTM Blood RNA tube
  • RNA-stabilization is selected from the group consisting of catrimox-14, tetradecyltrimethylammonium oxalate, RNA- later, RNAsin or RNAretain.
  • the expression profile and the reference expression profile are determined from said at least one platelet-miRNA selected from the group consisting SEQ ID NO: 1 to SEQ ID NO: 8 and wherein the expression profile and the reference expression profile are determined from a platelet-comprising fraction derived from a whole blood sample.
  • the data carrier comprises instructions for performing the method according to any of the items 1 to 14 and/or comprises said reference.
  • the determining of an expression profile of a set comprising at least one platelet-miR A comprises the steps:
  • step (b) optionally amplifying the cDNA of step (a)
  • step (c) quantifying the optionally amplified cDNA, thereby determining the expression profile of said miRNAs 20.
  • step (c) The method according to any of the items 19 to 20, wherein miRNA-specific forward primer and universal reverse primer or miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c).
  • step (b) The method according to any of the items 19 to 21, wherein miRNA-specific forward primer and universal reverse primer or miRNA-specific forward and partially universal reverse primer are used for optionally amplifying the cDNA in step (b).
  • step (c) The method according to any of the items 19 to 22 wherein that the quantifying in step (c) is performed by real-time PCR, nucleic acid hybridization or sequencing techniques.
  • RNA-DNA hybrids (a) adding a DNA- fragment to the 3 '-end of the miRNAs comprised in the total RNA isolated (from a whole blood sample or from whole blood sample from which white blood cells and/or red blood cells have been depleted or removed), thereby forming non-naturally occurring RNA-DNA hybrids
  • step (c) The method according to any of the items 24 to 26, wherein miRNA-specific forward primer and universal reverse primer or miRNA-specific forward and partially universal reverse primer are used for quantifying the optionally amplified cDNA in step (c).
  • step (c) is performed by real-time PCR, nucleic acid hybridization or sequencing techniques.
  • the administering of said affected subject to therapy comprises primary percutaneous coronary intervention (PCI), administration of fibrinolytic drugs, antithrombotic therapy without reperfusion therapy, coronary bypass surgery, administration of antiplatelet agents, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, further management with angiography and coronary revascularization.
  • PCI percutaneous coronary intervention
  • fibrinolytic drugs antithrombotic therapy without reperfusion therapy
  • coronary bypass surgery administration of antiplatelet agents, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors
  • ACE angiotensin-converting enzyme
  • Figure 1 Overview of the platelet-miRNAs (with SEQ ID NO: 1 to SEQ ID NO: 8) well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated component of AMI, for diagnosing AMI, for monitoring the progression of AMI, for determining the platelet- activity in AMI and/or for monitoring the efficacy of anti-platelet therapy in AMI and for determining an expression profile according to the present invention.
  • SEQ ID NO: sequence identification number
  • miRNA identifier of the miRNA according to miRBase (www.mirbase.org)
  • “Sequence” (5'-3')-sequence of the miRNA.
  • FIG. 1 ACUTE MYOCARDIAL INFARCTION (AMI): Platelet-miRNAs well suited in the method, use or kit for diagnosing a platelet-related or platelet-activated component of AMI, for diagnosing AMI, for monitoring the progression of AMI, for determining the platelet-activity in AMI and/or for monitoring the efficacy of anti-platelet therapy in AMI and for determining an expression profile according to the present invention.
  • AMI ACUTE MYOCARDIAL INFARCTION
  • FIG. 3 miRNA-specific DNA-primers (column B) used for reverse transcription (RT) of at least one platelet-miRNA with SEQ ID NO: 1 to 8 to non-naturally occurring cDNA; miRNA-specific forward and universal reverse primers (column C, D) for quantification and optionally amplification of at least one platelet-miRNA with SEQ ID NO: 1 to 8 employed for determining of an expression profile of a set comprising at least one platelet-miRNA representative for AMI; dual-labeled hydrolysis probes (Taqman-probes, column E) utilized for quantifying of at least one platelet-miRNA with SEQ ID NO: 1 to 8 by real-time PCR .
  • column B miRNA-specific DNA-primers
  • RT reverse transcription
  • C, D miRNA-specific forward and universal reverse primers
  • Taqman-probes, column E dual-labeled hydrolysis probes
  • Figure 4 miRNA-specific forward primer (column B) and partially universal reverse primer (column C) for quantification and optionally amplification of cDNA-transcripts of at least one platelet-miRNA with SEQ ID NO: 1 to 8 employed for determining of an expression profile of a set comprising at least one platelet-miRNA representative for AMI.
  • Figure 5 DNA-fragments added to the 3'-end of the miRNAs with SEQ ID NO : 1 to 8 employed for determining of an expression profile of a set comprising at least one platelet- miRNA representative for AMI, thereby forming non-naturally occurring RNA-DNA hybrids.
  • EXAMPLE 1 Preparation of total RNA for determination of platelet-miRNAs from whole blood samples without prior depletion or removal of the white blood cells and/or the red blood cells
  • Blood of AMI-patients (STEMI) and healthy controls was drawn by venipuncture in PAXgeneTM Blood RNA tubes (PreAnalytiX GmbH, Hombrechtikon, Switzerland). The tubes were gently inverted 10-15 times. For each blood donor, 2.5 ml of peripheral whole blood was collected. Herein, the blood cell pellet (the intra-cellular blood fraction comprising red blood cells, white blood cells and platelets) was collected at the bottom of the tube by centriiugation. For further processing the blood cell pellet was used, while the supernatant (including the extra-cellular blood fraction) was discarded.
  • PAXgeneTM Blood RNA tubes PreAnalytiX GmbH, Hombrechtikon, Switzerland
  • RNA including the small RNA (miRN A- fraction) was isolated from the pelleted blood cells using the miRNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and the resulting RNA were stored at -80°C before use in expression profiling experiments.
  • EXAMPLE 2 Microarray-based determination of expression profiles The RNA-samples of AMI (patients with acute ST elevation myocardial infarction) and healthy controls were analyzed employing microarray hybridization on the Geniom Realtime Analyzer (febit biomed GmbH, Heidelberg, Germany) using the Geniom Biochip miRNA homo sapiens. Each micro fluidic microarray contains complementary dna-probes of 866 miRNAs and miRNA* (each represented by 7 replicates) as annotated in the Sanger miRBase 12.0. Sample labeling with biotin has been carried out by enzymatic on-chip labeling of miRNAs employing the MPEA-assay (Vorwerk et.al.
  • a parametric t-test (unpaired, two-tailed) was carried out for each miRNA separately, to detect miRNAs that show a different behavior in different groups of blood donors.
  • receiver operating characteristics and calculated the "Area under the Curve"-value ( AUC).
  • the AMI quantile normalized dataset (Healthy Control vs. Acute Myocardial Infarction subjects) was subjected to strict filtering rules in order identify the platelet-miRNAs with SEQ IDNO: 1 to SEQ ID NO: 8, that are employed in the determination of expression profiles in a platelet- comprising fraction derived from the whole blood sample starting from whole blood collected in a PAXgeneTM Blood RNA tube without prior depletion or removal of white blood cells and/or red blood cells (or without prior isolation of the platelet fraction or in front of a background of white blood cells and/or red blood cells), which may e.g.
  • biomarkers for noninvasive diagnosis of AMI for non-invasive diagnosis of a platelet-related (platelet-activated) component of AMI, or . may be employed as biomarkers for determining the platelet-activity in AMI and/or for monitoring the efficacy of anti-platelet therapy in AMI.
  • the remaining miRNA-biomarker candidates were aligned to a list of selected platelet- miRNAs described to be highly expressed in platelets (PMID 21415270, 22371016, 23323973), wherein the list of highly expressed miRNAs contained the 30% highest expressed platelet- miRNAs.
  • AMI acute ST elevation myocardial infarction
  • EXAMPLE 6 Preparation of platelet-comprising fraction of a whole blood sample with prior depletion or removal of white blood cells and/or red blood cells
  • Platelet-comprising fractions include, but are not limited to, platelet-rich-plasma (PRP), Leukocyte-depleted platelet-rich-plasma, platelet-concentrate or Leukocyte-depleted platelet concentrate.
  • PRP platelet-rich-plasma
  • Leukocyte-depleted platelet-rich-plasma Leukocyte-depleted platelet-rich-plasma
  • platelet-concentrate Leukocyte-depleted platelet concentrate.
  • venous blood is conveniently drawn into EDTA-tubes (7.5 ml S-Monovette, Sarstedt / 10ml, Vaccutainer, BD Heidelberg, Germany), Na- citrate tubes (380% ;4.5 ml Vaccutainer, BD Heidelberg, Germany) or ACD-tubes (ACD type A, 8.5 ml, ACD type B, 6.5 ml Vaccutainer, BD Heidelberg, Germany).
  • EDTA-tubes 7.5 ml S-Monovette, Sarstedt / 10ml, Vaccutainer, BD Heidelberg, Germany
  • Na- citrate tubes 380% ;4.5 ml Vaccutainer, BD Heidelberg, Germany
  • ACD-tubes ACD type A, 8.5 ml, ACD type B, 6.5 ml Vaccutainer, BD Heidelberg, Germany.
  • Freshly collected whole blood is centrifuged with soft spin (170g, 15 min) to make Platelet-Rich- Plasma (PRP), buffy coat (white blood cells) and red blood cells, from which the PRP is collected.
  • PRP Platelet-Rich- Plasma
  • buffy coat white blood cells
  • red blood cells from which the PRP is collected.
  • Leukocyte-depleted PRP is obtained by either filtering the collected PRP through leukocyte depletion filters (Pall corporation, Port Washington, NY, ISA) or by negative selection employing magnetic cell sorting using human CD45+ magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany).
  • Platelet-concentrate is obtained from PRP by a second hard spin centrifugation (5000g, 3min), where platelets are pelleted out of the plasma to yield platelet-concentrate and platelet-poor- plasma (PPP).
  • the collected whole blood is centrifuged with hard spin (5000g, 7 min) to make Platelet-Poor- Plasma (PPP), buffy coat (including white blood cells & platelets) and red blood cells, from which the buffy coat comprising the platelets is collected.
  • PPP Platelet-Poor- Plasma
  • buffy coat including white blood cells & platelets
  • red blood cells from which the buffy coat comprising the platelets is collected.
  • the buffy coat is further centrifuged (2000g, 3min).
  • Leukocyte-depleted platelet concentrate is obtained by either filtering through leukocyte depletion filters (Pall corporation, Port Washington, NY, ISA) or by negative selection employing magnetic cell sorting using human CD45+ magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany).

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Abstract

La présente invention concerne des méthodes, des trousses et des utilisations pour déterminer des miRn plaquettaires dans un échantillon de sang entier d'un sujet atteint ou suspecté d'être atteint par l'infarctus aigu du myocarde.
PCT/EP2014/078766 2013-12-19 2014-12-19 Détermination de miarn plaquettaires dans l'infarctus aigu du myocarde Ceased WO2015091963A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105713972A (zh) * 2016-03-16 2016-06-29 上海中医药大学 miRNA在制备药物性心脏病生物标志物中的用途
US9944734B2 (en) 2013-12-20 2018-04-17 Saudi Basic Industries Corporation Catalyst system for polymerization of an olefin
EP4326894A4 (fr) * 2021-04-24 2025-06-04 University of Notre Dame du Lac Procédé et dispositif de détection d'infarctus du myocarde et de lésion de reperfusion

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DK3150721T3 (da) * 2009-12-24 2019-07-01 Micromedmark Biotech Co Ltd Pankreascancermarkører og detekteringsfremgangsmåder
TW201239097A (en) * 2010-12-17 2012-10-01 Sanofi Sa MiRNAs in joint disease
WO2013150105A1 (fr) * 2012-04-04 2013-10-10 Febit Holding Gmbh Ensembles complexes de miarn en tant que biomarqueurs non invasifs pour le diagnostic précoce de l'infarctus du myocarde aigu

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9944734B2 (en) 2013-12-20 2018-04-17 Saudi Basic Industries Corporation Catalyst system for polymerization of an olefin
CN105713972A (zh) * 2016-03-16 2016-06-29 上海中医药大学 miRNA在制备药物性心脏病生物标志物中的用途
EP4326894A4 (fr) * 2021-04-24 2025-06-04 University of Notre Dame du Lac Procédé et dispositif de détection d'infarctus du myocarde et de lésion de reperfusion

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