Session: Metrology Meets Life Science

Session Chair: Prof. Dr. Gavin O´Connor
Englisch

Reference measurement procedures for intact proteins using inductively coupled plasma mass spectrometry (ICP-MS)

Claudia Swart, PTB
Metalloproteins, which represent around 30 % of the whole proteome, are involved in a wide range of important biological processes. Metalloproteins also play a central role in many severe diseases and, therefore, are used as biomarkers in medical diagnosis. They are defined as proteins whose function is induced by the metal bound to it. Some prominent examples are haemoglobin, transferrin or ceruloplasmin. [1] Different methods are used to detect and quantify proteins. In routine laboratories enzyme-linked immunosorbent assays (ELISA) or photometric methods are mainly used. For reference measurement procedures enzymatic digestion and quantification via protein specific peptides using isotope dilution mass spectrometry (IDMS) has been established. However, many of those methods involve a denaturing step which causes the protein to release its metal. This can result in the loss of valuable information concerning the biological activity of the protein. A more recent complementary approach is the use of inductively coupled plasma mass spectrometry (ICP-MS), in combination with a non-denaturing chromatographic separation, which enables the detection of proteins with the metal still in place. In combination with the information regained from complementary methods, conclusions can be drawn on possible modifications such as phosphorylation or truncation and the correct incorporation of the metal in the protein. [2, 3] To demonstrate the abilities and limitations of different methods and the advantage of a multi-method approach, a comparison of the quantification of haemoglobin using various IDMS methods, ID Raman spectroscopy as well as two optical methods used in routine laboratories is presented and discussed. [4]
Englisch

Reference measurement procedures for intact proteins using inductively coupled plasma mass spectrometry (ICP-MS)

Claudia Swart, PTB
Metalloproteins, which represent around 30 % of the whole proteome, are involved in a wide range of important biological processes. Metalloproteins also play a central role in many severe diseases and, therefore, are used as biomarkers in medical diagnosis. They are defined as proteins whose function is induced by the metal bound to it. Some prominent examples are haemoglobin, transferrin or ceruloplasmin. [1] Different methods are used to detect and quantify proteins. In routine laboratories enzyme-linked immunosorbent assays (ELISA) or photometric methods are mainly used. For reference measurement procedures enzymatic digestion and quantification via protein specific peptides using isotope dilution mass spectrometry (IDMS) has been established. However, many of those methods involve a denaturing step which causes the protein to release its metal. This can result in the loss of valuable information concerning the biological activity of the protein. A more recent complementary approach is the use of inductively coupled plasma mass spectrometry (ICP-MS), in combination with a non-denaturing chromatographic separation, which enables the detection of proteins with the metal still in place. In combination with the information regained from complementary methods, conclusions can be drawn on possible modifications such as phosphorylation or truncation and the correct incorporation of the metal in the protein. [2, 3] To demonstrate the abilities and limitations of different methods and the advantage of a multi-method approach, a comparison of the quantification of haemoglobin using various IDMS methods, ID Raman spectroscopy as well as two optical methods used in routine laboratories is presented and discussed. [4]
Englisch

Reference measurement procedures for intact proteins using inductively coupled plasma mass spectrometry (ICP-MS)

Claudia Swart, PTB
Metalloproteins, which represent around 30 % of the whole proteome, are involved in a wide range of important biological processes. Metalloproteins also play a central role in many severe diseases and, therefore, are used as biomarkers in medical diagnosis. They are defined as proteins whose function is induced by the metal bound to it. Some prominent examples are haemoglobin, transferrin or ceruloplasmin. [1] Different methods are used to detect and quantify proteins. In routine laboratories enzyme-linked immunosorbent assays (ELISA) or photometric methods are mainly used. For reference measurement procedures enzymatic digestion and quantification via protein specific peptides using isotope dilution mass spectrometry (IDMS) has been established. However, many of those methods involve a denaturing step which causes the protein to release its metal. This can result in the loss of valuable information concerning the biological activity of the protein. A more recent complementary approach is the use of inductively coupled plasma mass spectrometry (ICP-MS), in combination with a non-denaturing chromatographic separation, which enables the detection of proteins with the metal still in place. In combination with the information regained from complementary methods, conclusions can be drawn on possible modifications such as phosphorylation or truncation and the correct incorporation of the metal in the protein. [2, 3] To demonstrate the abilities and limitations of different methods and the advantage of a multi-method approach, a comparison of the quantification of haemoglobin using various IDMS methods, ID Raman spectroscopy as well as two optical methods used in routine laboratories is presented and discussed. [4]
Englisch

Metrology for support of nucleic acid analysis in health and food

Mojca Milavec, National Institute of Biology
Nucleic acid (NA) analysis represents one of a key measurements in various areas of basic life sciences such as biotechnology, cell biology, genetics, microbiology, and molecular biology, as well as in their applications in different fields such as medicine, veterinary, food safety, and environmental monitoring. NA analysis enables detection, identification and quantification of NA of different organisms in diverse matrices, however accurate, precise and comparable measurements of NA are crucial for providing reliable results in science as well as in its applications to support decision makers like doctors, inspectors and competent authorities. In a biological measurement context NA analysis includes but is not limited to analysis of chromosomes, DNA, nucleotides, oligonucleotides, modified DNA (e.g. DNA methylation and other epigenetic modifications), mRNA, and miRNA (and other short non-coding RNAs). Until now quantification of NA has been the most studied type of analysis with absolute copy number concentration (e.g. viral count) and ratios (e.g. % of genetically modified organism and mutation/wild type), within and between different samples as measurands. Currently in many areas comparability of results NA quantification is unsatisfactory among laboratories, also due to the absence of reference materials and reference measurement procedures. National metrology institutes are investigating different methods to understand the sources of variability and measurement uncertainty and to assess their potential for reference measurement procedures. Examples of international research for support of NA measurements in different areas such as advances in precision medicine [1], infectious diseases [2], and food safety [3] will be presented.
Englisch

Metrology for support of nucleic acid analysis in health and food

Mojca Milavec, National Institute of Biology
Nucleic acid (NA) analysis represents one of a key measurements in various areas of basic life sciences such as biotechnology, cell biology, genetics, microbiology, and molecular biology, as well as in their applications in different fields such as medicine, veterinary, food safety, and environmental monitoring. NA analysis enables detection, identification and quantification of NA of different organisms in diverse matrices, however accurate, precise and comparable measurements of NA are crucial for providing reliable results in science as well as in its applications to support decision makers like doctors, inspectors and competent authorities. In a biological measurement context NA analysis includes but is not limited to analysis of chromosomes, DNA, nucleotides, oligonucleotides, modified DNA (e.g. DNA methylation and other epigenetic modifications), mRNA, and miRNA (and other short non-coding RNAs). Until now quantification of NA has been the most studied type of analysis with absolute copy number concentration (e.g. viral count) and ratios (e.g. % of genetically modified organism and mutation/wild type), within and between different samples as measurands. Currently in many areas comparability of results NA quantification is unsatisfactory among laboratories, also due to the absence of reference materials and reference measurement procedures. National metrology institutes are investigating different methods to understand the sources of variability and measurement uncertainty and to assess their potential for reference measurement procedures. Examples of international research for support of NA measurements in different areas such as advances in precision medicine [1], infectious diseases [2], and food safety [3] will be presented.
Englisch

Metrology for support of nucleic acid analysis in health and food

Mojca Milavec, National Institute of Biology
Nucleic acid (NA) analysis represents one of a key measurements in various areas of basic life sciences such as biotechnology, cell biology, genetics, microbiology, and molecular biology, as well as in their applications in different fields such as medicine, veterinary, food safety, and environmental monitoring. NA analysis enables detection, identification and quantification of NA of different organisms in diverse matrices, however accurate, precise and comparable measurements of NA are crucial for providing reliable results in science as well as in its applications to support decision makers like doctors, inspectors and competent authorities. In a biological measurement context NA analysis includes but is not limited to analysis of chromosomes, DNA, nucleotides, oligonucleotides, modified DNA (e.g. DNA methylation and other epigenetic modifications), mRNA, and miRNA (and other short non-coding RNAs). Until now quantification of NA has been the most studied type of analysis with absolute copy number concentration (e.g. viral count) and ratios (e.g. % of genetically modified organism and mutation/wild type), within and between different samples as measurands. Currently in many areas comparability of results NA quantification is unsatisfactory among laboratories, also due to the absence of reference materials and reference measurement procedures. National metrology institutes are investigating different methods to understand the sources of variability and measurement uncertainty and to assess their potential for reference measurement procedures. Examples of international research for support of NA measurements in different areas such as advances in precision medicine [1], infectious diseases [2], and food safety [3] will be presented.
Englisch

A Reference System Approach for Cell and Particle Counting

Martin Hussels, PTB Berlin
PTB has developed (primary) reference measurement procedures for cell counting to determine erythrocyte, leukocyte and platelet concentrations in blood, which account for counting losses due to arbitrary coincidences and influencing quantities like sedimentation, evaporation and adhesion. In this presentation the state of the art for reference measurement procedures for cell counting is shown including instrumental characteristics and protocols required to establish a primary reference procedure for the determination of blood cell concentrations. Furthermore, the potential of flow cytometric measurement procedures as higher order methods for phenotyping of cells by antibody staining as well as for nanoparticle detection in flow are discussed.
Englisch

A Reference System Approach for Cell and Particle Counting

Martin Hussels, PTB Berlin
PTB has developed (primary) reference measurement procedures for cell counting to determine erythrocyte, leukocyte and platelet concentrations in blood, which account for counting losses due to arbitrary coincidences and influencing quantities like sedimentation, evaporation and adhesion. In this presentation the state of the art for reference measurement procedures for cell counting is shown including instrumental characteristics and protocols required to establish a primary reference procedure for the determination of blood cell concentrations. Furthermore, the potential of flow cytometric measurement procedures as higher order methods for phenotyping of cells by antibody staining as well as for nanoparticle detection in flow are discussed.
Englisch

A Reference System Approach for Cell and Particle Counting

Martin Hussels, PTB Berlin
PTB has developed (primary) reference measurement procedures for cell counting to determine erythrocyte, leukocyte and platelet concentrations in blood, which account for counting losses due to arbitrary coincidences and influencing quantities like sedimentation, evaporation and adhesion. In this presentation the state of the art for reference measurement procedures for cell counting is shown including instrumental characteristics and protocols required to establish a primary reference procedure for the determination of blood cell concentrations. Furthermore, the potential of flow cytometric measurement procedures as higher order methods for phenotyping of cells by antibody staining as well as for nanoparticle detection in flow are discussed.
Englisch

Development of certified reference materials for in–vitro diagnostics.

Liesbet Deprez, European Commission
The European Commission's Joint Research Centre (JRC) produces reference materials that are tailored to meet the needs of European policy in various fields including health applications. Certified Reference Materials (CRMs) for In-Vitro Diagnostics (IVD) are essential for the development of reference systems for the standardisation of routine IVD measurements and their use is required by the IVD Regulation (EU) 2017/746. Protein biomarkers are prime targets for standardisation efforts due to their importance in diagnostics and health care and the typically observed significant discrepancies in measurement results obtained without standardisation. The standardisation process is often complicated by the heterogeneous nature of the analyte in terms of fragmentation, modification, substitution, primary, secondary, tertiary and quaternary structure. The approach of the JRC starts with the assessment of the state of the art for a particular analyte. Correlation studies, in which sets of single patient samples are measured with the main routine methods, will show the degree of variability between the methods. These studies will also provide information on the degree of standardization that can be achieved for the existing routine methods. The development of a CRM for protein biomarkers requires investigations on several parameters such as the availability of raw materials, commutability, stability, the level of the analyte of interest, and the strategy for assigning a certified value. In the recent years, the JRC has produced CRMs for several protein biomarkers in various clinical fields. Examples are: - ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC are a panel of three pooled liquid frozen cerebrospinal fluid materials with different levels of amyloid-β 1-42 (Aβ1-42) which is a biomarker for the early detection of Alzheimer's disease. - ERM-DA483/IFCC was produced from plasmapheresis sample of a patient diagnosed with the auto-immune disorder called vasculitis. This material is certified for the mass concentration of the immunoglobin G proteinase 3 anti-neutrophil cytoplasmic autoantibodies (IgG PR3 ANCA). - ERM-AD456/IFCC was certified for the catalytic activity concentration of alphaamylase using the primary reference measurement procedure as established by the IFCC.
Englisch

Development of certified reference materials for in–vitro diagnostics.

Liesbet Deprez, European Commission
The European Commission's Joint Research Centre (JRC) produces reference materials that are tailored to meet the needs of European policy in various fields including health applications. Certified Reference Materials (CRMs) for In-Vitro Diagnostics (IVD) are essential for the development of reference systems for the standardisation of routine IVD measurements and their use is required by the IVD Regulation (EU) 2017/746. Protein biomarkers are prime targets for standardisation efforts due to their importance in diagnostics and health care and the typically observed significant discrepancies in measurement results obtained without standardisation. The standardisation process is often complicated by the heterogeneous nature of the analyte in terms of fragmentation, modification, substitution, primary, secondary, tertiary and quaternary structure. The approach of the JRC starts with the assessment of the state of the art for a particular analyte. Correlation studies, in which sets of single patient samples are measured with the main routine methods, will show the degree of variability between the methods. These studies will also provide information on the degree of standardization that can be achieved for the existing routine methods. The development of a CRM for protein biomarkers requires investigations on several parameters such as the availability of raw materials, commutability, stability, the level of the analyte of interest, and the strategy for assigning a certified value. In the recent years, the JRC has produced CRMs for several protein biomarkers in various clinical fields. Examples are: - ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC are a panel of three pooled liquid frozen cerebrospinal fluid materials with different levels of amyloid-β 1-42 (Aβ1-42) which is a biomarker for the early detection of Alzheimer's disease. - ERM-DA483/IFCC was produced from plasmapheresis sample of a patient diagnosed with the auto-immune disorder called vasculitis. This material is certified for the mass concentration of the immunoglobin G proteinase 3 anti-neutrophil cytoplasmic autoantibodies (IgG PR3 ANCA). - ERM-AD456/IFCC was certified for the catalytic activity concentration of alphaamylase using the primary reference measurement procedure as established by the IFCC.
Englisch

Development of certified reference materials for in–vitro diagnostics.

Liesbet Deprez, European Commission
The European Commission's Joint Research Centre (JRC) produces reference materials that are tailored to meet the needs of European policy in various fields including health applications. Certified Reference Materials (CRMs) for In-Vitro Diagnostics (IVD) are essential for the development of reference systems for the standardisation of routine IVD measurements and their use is required by the IVD Regulation (EU) 2017/746. Protein biomarkers are prime targets for standardisation efforts due to their importance in diagnostics and health care and the typically observed significant discrepancies in measurement results obtained without standardisation. The standardisation process is often complicated by the heterogeneous nature of the analyte in terms of fragmentation, modification, substitution, primary, secondary, tertiary and quaternary structure. The approach of the JRC starts with the assessment of the state of the art for a particular analyte. Correlation studies, in which sets of single patient samples are measured with the main routine methods, will show the degree of variability between the methods. These studies will also provide information on the degree of standardization that can be achieved for the existing routine methods. The development of a CRM for protein biomarkers requires investigations on several parameters such as the availability of raw materials, commutability, stability, the level of the analyte of interest, and the strategy for assigning a certified value. In the recent years, the JRC has produced CRMs for several protein biomarkers in various clinical fields. Examples are: - ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC are a panel of three pooled liquid frozen cerebrospinal fluid materials with different levels of amyloid-β 1-42 (Aβ1-42) which is a biomarker for the early detection of Alzheimer's disease. - ERM-DA483/IFCC was produced from plasmapheresis sample of a patient diagnosed with the auto-immune disorder called vasculitis. This material is certified for the mass concentration of the immunoglobin G proteinase 3 anti-neutrophil cytoplasmic autoantibodies (IgG PR3 ANCA). - ERM-AD456/IFCC was certified for the catalytic activity concentration of alphaamylase using the primary reference measurement procedure as established by the IFCC.