Session: NMR in clinical applications

Session Chair: Prof. Dr. Astrid Petersmann
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English

Approaches to reference intervals for NMR spectra

Daniel Rosenkranz, Klinikum Oldenburg
Reference intervals are an important tool for the clinical interpretation of laboratory results. The usual approach for establishing reference intervals implies the calculation of designated percentiles after the measurement of a single measurand in a defined “healthy” reference population. For multi-measurand determinations within one analytical run, e.g. from NMR spectra, the calculation of reference intervals becomes more complex and is not well established. In this work, 600 MHz NMR spectra from approximately 400 human plasma samples out of the study of health in Pomerania (SHIP) serve as the data base for the calculation of a reference spectrum. The NMR-measurement provides 4000 measuring points per spectrum, which refers to a spectral resolution of 0.0025 ppm (1.5 Hz). Percentiles of intensities are calculated at each measuring point and the reference spectrum is depicted as color-coded intensities. At this time point, no diseasespecific exclusions were applied, so that the results are preliminary. The established reference spectrum represents the distribution of the intensity of a given ppm across a large population. The intensity percentiles at a given ppm are color-coded, so that we are able to describe regions at the spectra which are uniform up to regions, which are very variable between participants. According to the high standardization and comparability of the NMR spectra in human plasma samples, reference spectra are needed, to improve the medical value of this technology in the health care setting. Here we show a first approach to calculate a reference spectrum out of a large epidemiological study for plasma, measured by NMR spectroscopy.
23-Jun-2022 15:00 (30 Minutes) ICM/Hall 4a
English

Pitfalls in clinical applications of NMR analytics

Dr. Kathrin Budde, Universitätsmedizin Greifswald
The NMR technique has a high potential in patient care and is currently already a valuable tool for clinical research. Sample preparation is easy and the measurements works without material consumption. As there are, no reagents needed for the measurements, batch effects of reagents play no role and therefore we expect a better comparability between instruments and laboratories. A large number of results can be determined within minutes using one experiment. However, despite these relevant advantages of the NMR technique, every step of NMR analytic has to be validated. Here we describe some pitfalls of our daily work, which have to be avoided by the increasing number of applicants in future. According to well established Standard Operation Procedures (SOP) for sample preparation it is possible to use the biosamples from our daily clinical setting: at least plasma (serum) and urine. For the standard 5mm NMR tube a minimum sample volume of 300 µl for plasma/serum and 540 µl for urine are requested to receive high quality results. A decrease in the sample amount leads to a relevant decrease in the quality of the spectra and has to be avoided. The mentioned sample volume is usually no problem in regular patient care. The opposite is the case in the field of biobanking. Here, as a rule, much smaller sample volumes are available for investigations. To reduce sample volume needed there are also 3mm tubes available. To fill correctly a 3mm tube only 150 µl plasma/serum and 225 µl urine are needed. Because of the slime volume, mixing sample and buffer to get a homogenous sample is more challenging. Beneficial is the sample preparation applying pipetting robots. We established a pipetting and mixing scheme on a Tecan pipetting robot, which show at least comparable results to manual preparation but decreased handling time and hands on time significantly. However, all details and conditions have to be clearly defined and validated. For instance, sample properties like viscosity and pipetting speed have a huge impact of the quality and comparability of the NMR spectra. In order to ensure a high quality of NMR spectra at different instruments and sites, the measurement of pool samples is beneficial to detect systematic errors and an early stage. In addition, external quality testing through inter-laboratory proficiency tests is currently established for the NMR spectroscopy at least in Germany.
23-Jun-2022 15:30 (15 Minutes) ICM/Hall 4a
English

Pitfalls in clinical applications of NMR analytics

Dr. Ann-Kristin Henning, Universitätsmedizin Greifswald
The NMR technique has a high potential in patient care and is currently already a valuable tool for clinical research. Sample preparation is easy and the measurements works without material consumption. As there are, no reagents needed for the measurements, batch effects of reagents play no role and therefore we expect a better comparability between instruments and laboratories. A large number of results can be determined within minutes using one experiment. However, despite these relevant advantages of the NMR technique, every step of NMR analytic has to be validated. Here we describe some pitfalls of our daily work, which have to be avoided by the increasing number of applicants in future. According to well established Standard Operation Procedures (SOP) for sample preparation it is possible to use the biosamples from our daily clinical setting: at least plasma (serum) and urine. For the standard 5mm NMR tube a minimum sample volume of 300 µl for plasma/serum and 540 µl for urine are requested to receive high quality results. A decrease in the sample amount leads to a relevant decrease in the quality of the spectra and has to be avoided. The mentioned sample volume is usually no problem in regular patient care. The opposite is the case in the field of biobanking. Here, as a rule, much smaller sample volumes are available for investigations. To reduce sample volume needed there are also 3mm tubes available. To fill correctly a 3mm tube only 150 µl plasma/serum and 225 µl urine are needed. Because of the slime volume, mixing sample and buffer to get a homogenous sample is more challenging. Beneficial is the sample preparation applying pipetting robots. We established a pipetting and mixing scheme on a Tecan pipetting robot, which show at least comparable results to manual preparation but decreased handling time and hands on time significantly. However, all details and conditions have to be clearly defined and validated. For instance, sample properties like viscosity and pipetting speed have a huge impact of the quality and comparability of the NMR spectra. In order to ensure a high quality of NMR spectra at different instruments and sites, the measurement of pool samples is beneficial to detect systematic errors and an early stage. In addition, external quality testing through inter-laboratory proficiency tests is currently established for the NMR spectroscopy at least in Germany
23-Jun-2022 15:45 (15 Minutes) ICM/Hall 4a
English

Unravelling the NMR signals associated to glycosylated acute-phase proteins in serum

Dr. Alvaro Mallagaray de Benito, Uni Lübeck
Nuclear Magnetic Resonance (NMR) spectra of blood serum show two signals arising from acute phase glycoproteins termed GlycA and GlycB,[1] which are known to be good markers for cardiovascular disease and other inflammatory processes, including COVID19 acute disease.[2] However, the exact origin of these signals has never been rigorously examined. Previous work associated GlycA with N-acetyl glucosamine (GlcNAc) and Nacetyl galactosamine (GalNAc), and GlycB with neuraminic acid [3]. We have assigned spectra of blood serum using novel NMR and enzymatic approaches, showing that previous assignments are incorrect. Superimposed to GlycA/B are also hitherto unreported signals of methionine methyl groups. Here we illustrate how methionine signals can be selectively removed using NMR techniques and how the residual glycoprotein signals can be used as markers for inflammatory processes. Considering the immense diagnostic potential of GlycA/B signals which reflects a plethora of serum acute phase proteins, we have also examined correlations with the most abundant glycoproteins in blood sera. Using a novel NMR approach that selects proteins by their chemical shifts and diffusion rates, we were able to identify signal components that can be associated with specific acute phase proteins. This approach significantly enhances the diagnostic potential of GlycA/B signals in NMR spectra by providing more selective information for diverse disease processes. Our method allows to focus on individual acute phase proteins in spectra that can be acquired in less than 20 minutes.
23-Jun-2022 16:00 (30 Minutes) ICM/Hall 4a
English

Epidemiological approaches to clinical NMR applications

Prof. Dr. Johannes Hertel, Universitätsmedizin Greifswald
Urine NMR metabolomics allows for the quantification of a wide range of metabolites relevant to human and health and disease. In consequence, applied to large prospective cohorts, NMR metabolomics can be utilised in describing not only the metabolic patterns associated with common diseases, but it can also be employed for constructing individualised measures of health and disease in a data-driven way. Accordingly, we highlight the possibilities of large-scale NMR metabolomics on cohort data using the example of measuring biological age in the prospective populationbased cohort Study of Health in Pomerania [1]. On our way, we will discuss a generalised methodology to formalise the concept of individualisation in the context of Omics data [2]. As a second example of application of NMR metabolomics in epidemiological research, we will delve into the topic of microbiome-host metabolism, showcasing how microbiome and NMR metabolome data can be integrated with microbiome data. In particular, we demonstrate that NMR metabolomics can be successfully applied for understanding the metabolic traits mediating the beneficial effects of a heatlhy microbiome to human physiology [3]. In conclusion, the talk focusses on application of NMR metabolomics to large cohort data and corresponding analyses strategies to intersect NMR metabolomics with other Omics data in the task of individualisation.
23-Jun-2022 16:30 (30 Minutes) ICM/Hall 4a