EP3607094A1 - Procédé de détection et dispositif - Google Patents

Procédé de détection et dispositif

Info

Publication number
EP3607094A1
EP3607094A1 EP18719437.8A EP18719437A EP3607094A1 EP 3607094 A1 EP3607094 A1 EP 3607094A1 EP 18719437 A EP18719437 A EP 18719437A EP 3607094 A1 EP3607094 A1 EP 3607094A1
Authority
EP
European Patent Office
Prior art keywords
pcr
objects
detection
whole blood
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18719437.8A
Other languages
German (de)
English (en)
Inventor
Rolf Günther
Tobias PÖHLMANN
Heinrich Maria SCHULTE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP3607094A1 publication Critical patent/EP3607094A1/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • 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
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the invention relates to a method and a device for the enrichment of cells, cell fragments and molecules from whole blood for a specific detection of at least one population of cells, cell fragments or molecules contained therein.
  • Cells within the meaning of the invention are biological cells, eukaryotic and prokaryotic cells, nucleated or coreless, as well as unicellular organisms, and multicellular organisms.
  • Cell fragments within the meaning of the invention are parts of cells, including cell wall components, organelles, membrane constituents, platelets and micelles, as well as viruses.
  • Molecules in the sense of the invention include macromolecules including proteins, peptides, and oligonucleotides such as DNA and RNA, or lipids, carbohydrates and small molecules.
  • Target objects Cells or viruses to be detected are hereinafter referred to as target objects.
  • Pathogens such as bacteria, fungi, protozoa and viruses can also be targets.
  • target objects can also be fetal cells in the maternal blood or circulating tumor cells.
  • the purpose of the detection may be to detect the DNA, RNA or antigens of the target objects, fragments of the cells, or the whole cells. To simplify matters, these proofs are also referred to below as proof of target objects.
  • matrix cells include the various types of immune cells (lymphocytes) as well as erythrocytes and cell fragments, such as platelets.
  • Methods by which target objects, their molecules and in particular their DNA are detected from whole blood include, for example, molecular biology techniques such as PCR (polymerase chain reaction) or arrays, recently also sequencing, NMR or mass spectroscopy.
  • PCR polymerase chain reaction
  • NMR mass spectroscopy
  • the "Cell Collector” from Gilupi, Potsdam is a device that enriches circulating tumor cells on the surface of a metal rod in vivo.Magna Lyser beads from Roche Diagnostics, Mannheim, are ceramic magnetobeads for the mechanical disruption of cells and subsequent ones Similarly, the beads of the MACS series from Miltenyi, Bergisch-Gladbach are used for the enrichment of DNA and RNA from cells, thus increasing the sensitivity and specificity of the detection, but these processes cost a lot of time and are usually associated with large amounts In addition, these methods are usually based on individual molecular target structures which are characteristic of the target objects, meaning that target objects or their molecules are specifically enriched depending on the capture structures used, for example antibodies Enrich one However, certain pathogen species may not be enriched for other coexisting pathogens.
  • non-molecular methods such as blood culture are therefore often used in the prior art, in which the pathogens are cultured for several hours or even days before detection in order to multiply them and thus the sensitivity of proof.
  • This cultivation can also be preceded by a molecular detection.
  • these culturing procedures are time consuming, labor-intensive, and problematic in that not all pathogens in the blood culture can be equally cultured or propagated and detected (Striebel, 2014, Thalhammer et al, 2016). This results in a limited sensitivity or specificity.
  • a disadvantage of all methods in the prior art is that they are much too slow in practical application.
  • rapid analysis is crucial for successful therapy (Afshari et al., 2012).
  • the high technical and logistical effort, and the complexity of the methods described require that the proof can be reliably performed only by specially qualified employees with laboratory experience and thus not or not sure at the point-of-care. Instead, the samples are taken to central laboratories and analyzed there, whereby the transit times from the patient to the addressee of the proof, in particular of the attending physician, are usually too long, especially outside normal working hours (Afshari et al., 2012).
  • the object of the invention is to detect a broad spectrum of target objects, including their molecules, simply, quickly and yet sensitively and specifically from whole blood.
  • Matrix objects of whole blood can be enriched prior to sample preparation. This is surprising, above all, because it is argued in the prior art that the sensitivity and specificity primarily depends on the complete preparation of the target objects (Banada et al., 2012). For example, in the case of pathogen infections in whole blood by the enrichment of matrix objects, in particular immune cells or individual immune cell populations, their processing, DNA isolation and subsequent PCR even a few pathogens (3-1000 pathogens / ml whole blood) by PCR, be detected specifically and sensitively. A specific connection of the matrix objects to target objects such as pathogens or specific phagocytosis processes may be related to this.
  • the enrichment of the matrix objects can be carried out with the aid of particles which bind specifically or unspecifically to specific matrix objects.
  • the binding of the particles to the matrix objects can be mediated by the coating or coupling of the particles with capture molecules, in particular antibodies or other proteins, peptides or aptamers.
  • the particles may have other properties, such as magnetic, acoustic or dielectric properties, that allow them to be directly enriched or isolated.
  • Size of particles are in a specific range, so that they can be enriched with a particle filter.
  • the enrichment can, however, also be carried out chromatographically, albeit with a greater outlay on equipment of the device.
  • the matrix objects flow past the immobilized beads, in particular beads with catcher molecules, with which the surface of a flow device is coated.
  • the described beads can also be used as packing material of a chromatographic column.
  • An additional centrifugation step can improve the enrichment, but increases the complexity of the process. Therefore, it may be advantageous to carry out the enrichment without centrifugation step.
  • the enrichment can also be done with a flow cytometer.
  • Matrix objects that can be enriched according to the method of the invention include leukocytes, in particular monocytes, macrophages, 20 B cells, T cells, NK cells, eosinophilic, basophilic and neutrophilic granulocytes, dendritic cells, but also erythrocytes.
  • Matrix objects may also be natural cell fragments, including platelets, or debris produced by mechanical or chemical action or biological processes such as apoptosis. It may be preferable to enrich more than one population of matrix objects in the method according to the invention, in particular by using particles or beads which are specific for a plurality of matrix objects or of different particles which are specific for each kind of matrix objects. However, accumulation of the entire lymphocyte spectrum is apparently unfavorable (Banada et al., 2012).
  • the enrichment of the immune cells of the exemplary embodiment is advantageous, for example, via antibodies which are bound to a carrier system (in particular particles, in particular beads, or solid matrices).
  • a carrier system in particular particles, in particular beads, or solid matrices.
  • the enrichment can be carried out in fluidic or microfluidic systems, in which - -
  • the immune cells or the particles are enriched by forces or energy input, in particular by acoustic or dielectrophoretic effects in the liquid flow, or in the flow cytometer.
  • nucleic acids in particular DNA and RNA of the target objects and in particular the pathogens.
  • Various prior art methods are available, including trizolysis, other chemical methods, and mechanical digestion. Subsequently, a precipitation of the DNA or RNA with ethanol, as well as a purification over columns are often advantageous.
  • PCR For the detection of DNA or RNA, PCR, isothermal amplification methods, sequencing or array-based methods are suitable. With the PCR one can distinguish multiplex and single PCR, of which the latter is particularly advantageous, because it allows a particularly simple procedure. Thus, the single PCR is particularly suitable to perform the procedure at the point of care and to detect specific pathogens and resistance indicators or genes.
  • the invention also includes a device for isolation, optionally supplemented by the detection of target objects.
  • a cartridge system comprises a device for sample input, storage locations for various wash buffers, storage locations for a bead antibody system for binding specific matrix objects, in particular immune cells, as well as a possibility for lysing isolated matrix objects, and optionally a device for removing the RNA or DNA.
  • the antibody bead system is added to the whole blood, washed with washing buffer, isolated via a screening system or magnetically the antibody-bead system with bound matrix objects, with cell - -
  • Lysis buffer added and removed the precipitated DNA.
  • a quick and easy enrichment of the matrix objects, as well as the lysis and DNA isolation in a nearly closed container is carried out, thus reducing the risk of contamination of the sample with foreign DNA.
  • the RNA or DNA remains in the cartridge system and is not removed. Instead, the PCR is performed inside the cartridge through a window in the cartridge or with integrated heating and detection elements. It then contains another location for the PCR reagents.
  • the isolated DNA is applied directly to a PCR analysis plate which contains standardized all chemicals for the analysis of different pathogens.
  • This plate can be designed as a multiplex PCR or as a parallel running single PCR.
  • the application of the isolated DNA from the cartridge into the PCR plate can be done manually and in particular with a pipette or directly from the cartridge via a special dosing system.
  • the DNA lysis in the cartridge and the subsequent PCR only one way of analysis; Further embodiments include the analysis of the cells via spectroscopic, enzymatic, colorimetric, histological, chemical-analytical (in particular MALDI-TOF-MS), electrochemical and other physical methods (in particular NMR).
  • inventive method, the inventive device and the application kit according to the invention can be used for the detection of a variety of - -
  • SIRS Systemic Inflammatory Response Syndrome
  • pneumonia bacteria are detectable in urine after a few days. Especially in the increasingly occurring nocosomal and conditioned by air conditioning pneumonia blood cultures are recommended in the prior art.
  • the method according to the invention also offers the advantage of faster detection in these infections.
  • Zoonoses are infections with a wide range of manifestations, some of which are also blood cultures for diagnosis, sometimes over 4 - 6 weeks such as suspected brucellosis.
  • the method according to the invention also offers the advantage of faster detection in these infections.
  • the method according to the invention also has application potential in viral diseases, in particular in HIV - -
  • CAR-T therapies Chimeric antigen receptor T cell therapies
  • inventive method can also be used.
  • the method according to the invention comprises the following steps: First, one or more blood samples are taken from the patient whose coagulation in the EDTA / citrate buffer is avoided. To this sample, beads, such as anti-CD 14 beads of pluriSelect are added, which bind the matrix objects, for example, all CD14-bearing cells such as monocytes and macrophages. The beads are then separated through a sieve, for example with the Cell Strainer from pluriSelect. The sample with the matrix objects enriched in this way is subjected to DNA lysis in a subsequent step (for example trizol precipitation). Subsequently, the sample is distributed to several sample vessels, for example wells of a qPCR-capable microtiter plate.
  • beads such as anti-CD 14 beads of pluriSelect are added, which bind the matrix objects, for example, all CD14-bearing cells such as monocytes and macrophages.
  • the beads are then separated through a sieve, for example with the Cell Strainer from pluri
  • the invention also encompasses an application kit for the isolation and analysis of cells, cell fragments and molecules, consisting at least of
  • At least one cartridge for the isolation of matrix objects from whole blood which may optionally be extended by the possibility of DNA isolation - -
  • a sample carrier for the detection in particular a PCR sample carrier and in particular a PCR sample plate containing all reagents for the detection and in particular for carrying out a PCR and the detection of target objects, for example pathogens and optionally their resistances - dilution and reaction buffer
  • Fig. 2 Representation of the proposed workflow according to the invention for the enrichment of matrix objects and the detection of pathogens
  • Fig. 1 the workflow of the invention is shown. From a sample, matrix objects are specifically isolated that are associated with target structures. After processing the samples, the actual detection procedure of the target structures takes place.
  • Fig. 2 a specific inventive workflow for the detection of pathogens in the blood is shown.
  • EDTA whole blood is introduced into the cartridge system according to the invention, in which the sample is incubated with CD14 beads, washed with buffer and a sieve system, the beads together with trapped monocytes / macrophages (matrix objects) and the pathogens contained therein / (target ) isolated. This is followed by the cell lysis and isolation / purification of the DNA before the DNA sample is transferred by means of pipettes from the cartridge into the PCR well plates according to the invention. All of these plates are already in lyophilized form for PCR - -
  • the polymerase is added to the well plate together with the DNA sample. This is followed by the PCR and thus the specific detection of the pathogens.
  • Streibel 2014 H.-W. Striebel: "Intensive Care Medicine: Safety in Clinical Practice", Schattauer Verlag, 6 Oct 2014, p. 869
  • Gattringer A. Grisold, R. Krause, Ch. Loewe, L. Müller, HJ Nesser, A. Wechsler, 10 Fördos, G. Weiss, Ch. Wenisch, S. Winkler, A. Zuckermann, R. Zweiker, K. Huber: "Infectious Endocarditis” Austrian Medical Journal, Supplementum, 8/2016, p. 2

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'invention concerne un procédé et un dispositif d'augmentation de la concentration en cellules, en fragments cellulaires et en molécules de sang total pour une détection spécifique d'au moins une population de cellules, de fragments cellulaires ou de molécules contenue dans le sang total. Le but de l'invention est de détecter un large spectre d'objets cibles, y compris leurs molécules, de manière simple, rapide et néanmoins sensible et spécifique dans du sang total. Selon l'invention, c'est la concentration en objets matriciels et non en objets cibles qui est augmentée spécifiquement, toutefois, les objets cibles sont analysés spécifiquement et une détection spécifique et sensible est ainsi obtenue.
EP18719437.8A 2017-04-05 2018-04-04 Procédé de détection et dispositif Pending EP3607094A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102017003312.9A DE102017003312A1 (de) 2017-04-05 2017-04-05 Nachweisverfahren und Vorrichtung
PCT/EP2018/000144 WO2018184719A1 (fr) 2017-04-05 2018-04-04 Procédé de détection et dispositif

Publications (1)

Publication Number Publication Date
EP3607094A1 true EP3607094A1 (fr) 2020-02-12

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP18719437.8A Pending EP3607094A1 (fr) 2017-04-05 2018-04-04 Procédé de détection et dispositif

Country Status (4)

Country Link
US (1) US11629374B2 (fr)
EP (1) EP3607094A1 (fr)
DE (1) DE102017003312A1 (fr)
WO (1) WO2018184719A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102017003312A1 (de) 2017-04-05 2018-10-11 Rolf Günther Nachweisverfahren und Vorrichtung

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2097952C (fr) * 1993-06-08 2006-03-14 Alex D. Romaschin Diagnostic precoce de la sepsie au moyen des interactions antigene-anticorps amplifiees par chimiluminescence du sang total
WO2003022440A2 (fr) * 2001-08-16 2003-03-20 Millipore Corporation Support de sequençage a plusieurs puits / plaque de pcr
NZ590555A (en) * 2008-08-04 2012-11-30 Synmed Res Gmbh Method for characterizing, in particular for quantifying, molecular markers that are intracellularly absorbed from tissues by blood macrophages that are recirculated from the tissues into the circulatory system
ES2749351T3 (es) * 2011-12-21 2020-03-19 Geneohm Sciences Canada Inc Enriquecimiento y aislamiento de células microbianas y ácidos nucleicos microbianos a partir de una muestra biológica
WO2015200788A1 (fr) * 2014-06-26 2015-12-30 Corning Incorporated Microplaque renforcée
DE102017003312A1 (de) 2017-04-05 2018-10-11 Rolf Günther Nachweisverfahren und Vorrichtung

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIU ROBIN HUI ET AL: "Self-Contained, Fully Integrated Biochip for Sample Preparation, Polymerase Chain Reaction Amplification, and DNA Microarray Detection", ANALYTICAL CHEMISTRY, 25 February 2004 (2004-02-25), pages 1824 - 1831, XP093257416, ISSN: 0003-2700, Retrieved from the Internet <URL:https://pubs.acs.org/doi/full/10.1021/ac0353029?casa_token=sEzvJIMnwX4AAAAA%3AsRxbfVliyZQCXRmrvQzhv96yctbihlDb91DZgu8CwqzisDb5OEpt7Hml-K5cVettyrJhE8RDkVWOXnQ> [retrieved on 20250310], DOI: 10.1021/ac0353029 *

Also Published As

Publication number Publication date
US11629374B2 (en) 2023-04-18
WO2018184719A1 (fr) 2018-10-11
US20200032318A1 (en) 2020-01-30
DE102017003312A1 (de) 2018-10-11

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