METHOD FOR FUNCTIONAL CHARACTERISTICS OF PROTEINS FOR CLASSIFICATION OF PATIENTS TO BE ASSOCIATED TO A SPECIFIC DISEASE

Abstract

The invention describes a method for functional characteristics of proteins for classification of patients to be associated to a specific disease performed in a computing device by applying biophysical parameters as an input to a marker linear combination, wherein the marker linear combination comprises a predefined combination of at least two biophysical parameters, using the marker linear combination to determine the input parameters relating to one or more of said predetermined diseases, and outputting a result according to the marker linear combination, wherein the marker linear combination comprises at least one of the biophysical parameters: a binding constant of a spin probe, a polarity surrounding a spin probe, an order parameter of a spin probe, a rotation correlation time of a spin probe, a spectral component from free spin probe molecules, a spectral component from spin probe on lipid-fraction of serum, or a geometry factor.

Claims

1. A method for treatment of a hepatic disease in a subject, comprising: providing a sample from a subject comprising serum albumin; combining, in aliquots of the sample, a spin probe and a polar reagent comprising alcohol or dimethyl sulfoxide (DMSO), wherein at least one of the concentration of the spin probe and the concentration of the polar reagent varies between the aliquots; detecting a plurality of electron spin resonance spectroscopy (ESR) spectra from the aliquots; determining from the plurality of ESR spectra, a plurality of biophysical parameters selected from the group consisting of: a binding constant of a spin probe (KB), a polarity surrounding a spin probe (H), an order parameter of a spin probe (S), a rotation correlation time of a spin probe (T), a spectral component from free spin probe molecules (C3), a spectral component from spin probe on lipid-fraction of serum (C5), and a geometry factor (alpha); applying the determined biophysical parameters as an input to a marker linear combination, wherein a predefined combination of at least two biophysical parameters are input into the marker linear combination; determining a hepatic disease in a subject from the output of the marker linear combination, wherein the output of the marker linear combination is equal to or greater than a cut-off; and providing to the subject a treatment appropriate to the determined hepatic disease, wherein: (a) to prognose development of cirrhosis with acute decompensation (AD) to acute on chronic liver failure (ACLF), at least parameters T and H are input into the marker linear combination, (b) to discriminate between cirrhosis without acute decompensation (CC) and cirrhosis with acute decompensation (AD), at least one of: C3 and C5; C3 and alpha; and/or C5 and alpha are input into the marker linear combination; (c) to discriminate between cirrhosis with acute decompensation (AD) and acute on chronic liver failure (ACLF) at least one of: KB and H; KB and S; and/or H and S are input into the marker linear combination; and (d) to prognose the one-year mortality of the subject from a hepatic disease, at least H and C5 are input into the marker linear combination.

2. The method of claim 1, wherein to prognose development of cirrhosis with acute decompensation (AD) to acute on chronic liver failure (ACLF), the cut-off is a sensitivity between about 78% to about 97% and/or a specificity between about 78% to about 97%.

3. The method of claim 1, wherein to discriminate between cirrhosis without acute decompensation (CC) and cirrhosis with acute decompensation (AD), the cut-off is a sensitivity between about 78% to about 97% and/or a specificity between about 78% to about 97%.

4. The method of claim 1, wherein to discriminate between cirrhosis with acute decompensation (AD) and acute on chronic liver failure (ACLF), the cut-off is a sensitivity between about 78% to about 97% and/or a specificity between about 78% to about 97%.

5. The method of claim 1, wherein to prognose the one-year mortality of the subject from a hepatic disease, the cut-off is a sensitivity between about 78% to about 97% and/or a specificity between about 78% to about 97%.

6. The method of claim 1, wherein the subject is determined to have CC, and the treatment provided comprises optimization of nutrition and vitamin intake, treatment of inflammation and/or with antiviral medicaments in those subjects also diagnosed with viral hepatitis, and/or diuretic therapy.

7. The method of claim 1, wherein the subject is determined to have AD, and the treatment provided comprises optimization of nutrition and vitamin intake, treatment of inflammation and/or with antiviral medicaments in those subjects also diagnosed with viral hepatitis, diuretic therapy, and/or intervention with liver support systems.

8. The method of claim 1, wherein the subject is determined to have ACLF, and the treatment provided comprises optimization of nutrition and vitamin intake, treatment of inflammation and/or with antiviral medicaments in those subjects also diagnosed with viral hepatitis, diuretic therapy, intervention with liver support systems and/or haemofiltration, ventilation, and/or liver transplantation.

9. The method of claim 1, wherein the subject is determined to have a high risk of development of AD to ACLF and the treatment provided comprises optimized nutrition, vitamin supplementation, broad spectrum or specific antibiotics, lactulose, diuretics, albumin, haemofiltration and/or ventilation.

10. The method of claim 9, wherein the treatment further or alternatively comprises liver support systems and albumin dialysis, anti-inflammatory therapy, and/or liver transplantation.

11. The method of claim 1, wherein the subject is determined to have a high risk of one-year mortality, and the provided treatment comprises increasing liver support systems and albumin dialysis, haemofiltration, anti-inflammatory therapies, and/or liver transplantation.

12. The method of claim 2, wherein the cut-off is a sensitivity between about 81% to about 97% and/or a specificity between about 79% to about 97%.

13. The method of claim 3, wherein the cut-off is a sensitivity between about 80% to about 96% and/or a specificity between about 83% to about 95%.

14. The method of claim 4, wherein the cut-off is a sensitivity between about 79% to about 97% and/or a specificity between about 79% to about 96%.

15. The method of claim 5, wherein the cut-off is a sensitivity between about 79% to about 96% and/or a specificity between about 79% to about 96%.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0292] In the following, further features, advantages and embodiments of the present invention are explained with reference to the Figures, wherein

[0293] FIG. 1 shows a schematic diagram of the structure of human serum albumin for use in an embodiment,

[0294] FIG. 2 shows a 9.45 GHz ESR spectrum of human serum albumin,

[0295] FIG. 3 shows five sub-spectra of the spectrum shown in FIG. 4, i.e. five spectral components,

[0296] FIG. 4 shows an example of an acquired ESR spectrum, illustrating the relation between an ESR spectrum and the geometry factor alpha,

[0297] FIG. 5 shows a system of an embodiment comprising an ESR spectrometer and a computing device,

[0298] FIG. 6 shows the albumin concentration (panel A) and the detoxification efficiency (DTE) (panel B) for control, CC (stable), AD and ACLF samples,

[0299] FIG. 7 shows the binding efficiency (BE) for control, CC (stable), AD and ACLF samples, as a dot plot (panels A, C) and a box plot (panel B),

[0300] FIG. 8 shows values according to the marker linear combination according to formula (3) for CC (stable) samples and AD samples (panels A-C),

[0301] FIG. 9 shows values according to the marker linear combination according to formula (4) for CC (stable) samples and AD samples (panels A-C),

[0302] FIG. 10 shows the ROC curves (panel A) and comparison of AUROC (panel B) according to BE, DTE and marker linear combination according to formula 3,

[0303] FIG. 11 shows the ROC curves (panel A) and comparison of AUROC (panel B) according to BE, DTE and marker linear combination according to formula 4,

[0304] FIG. 12 shows the ROC curves (panel A) and comparison of AUROC (panel B) according to BE, DTE and marker linear combination according to formula 5,

[0305] FIG. 13 shows the ROC curves (panel A) and comparison of AUROC (panel B) according to BE, DTE and marker linear combination according to formula 6,

[0306] FIG. 14 shows the ROC curves (panels A, B) and comparison of AUROC (panel C) according to BE, DTE, CLIF-C-AD score and marker linear combinations according to formula 1 and 2,

[0307] FIG. 15 shows Kaplan-Meier survival curves of AD and ACLF patients, according to BE (panel A) and formula 7 (panel B)

[0308] FIG. 16 shows ROC curves related to KBA, S2B, combination of H and KB and marker linear combination according to formula 5,

[0309] FIG. 17 shows ROC curves related to H, combination of H and KB, combination of H, KB and S, and marker linear combination according to formula 6,

[0310] FIG. 18 shows ROC curves related to alpha, C3B, C5B, combination of alpha and C5, combination of alpha and C3 and marker linear combination according to formula 3 (panel A) and ROC curves related to C5, combination of alpha and C5, and marker linear combination according to formula 3 (panel B),

[0311] FIG. 19 shows ROC curves related to alpha, combination of alpha and C5, combination of alpha, C5 and C3, and marker linear combination according to formula 4 (panel A) and ROC curves related to C5, combination of alpha and C5, and combination of alpha, C5 and C3 (panel B),

[0312] FIG. 20 shows ROC curves related to alpha, combination of alpha and C5, combination of alpha, C5 and C3, and marker linear combination according to formula 4, for patients with bacterial infection, and

[0313] FIG. 21 shows ROC curves related to T, CLIF-C-AD, combination of H and T, and combination of H, T and CLIF-C-AD score.

DETAILED DESCRIPTION

[0314] FIG. 1 is a schematic diagram of the structure of human serum albumin (HSA) 1 for use in an embodiment. The structure of HSA 1 comprises three homologous domains 10, 20, 30 that fold into a heart-shaped albumin molecule 1. X-ray crystallographic analysis of HSA has identified at least seven distinct fatty acid binding sites (FA1-FA7) that are located in various parts of the protein (Bhattacharya et al. J Mol Biol. 2000 Nov. 10; 303(5):721-32) and which show relative affinities for fatty acids (Simard et al. J Mol Biol. 2006 Aug. 11; 361(2):336-51). The FA binding sites have certain common features: in each case, the hydrocarbon chain of the fatty acid is accommodated in a long and narrow hydrophobic well, while the carboxyl moiety is located near basic or polar residues (Curry et al. Nat Struct Biol. 1998 September; 5(9):827-35). These FA binding sites (FA1-FA7) represent specific (primary) hydrophobic binding sites for fatty acids in HSA. In addition, non-specific (secondary) hydrophobic binding sites for fatty acids on serum albumin have been identified. These non-specific hydrophobic binding sites are believed to be located in the hydrophobic area 40 between the albumin domains 10, 20, 30 (Gurachevsky et al. Biochem Biophys Res Commun. 2007 Sep. 7; 360(4):852-6). It is believed that there is migration of fatty acids between the non-specific binding sites in the hydrophobic area 40 of HSA and the specific FA binding sites (FA1-FA7) and vice versa and that this process is important for the transport properties of albumin.

[0315] Albumin is produced in the liver. In case of a hepatic disease, the albumin concentration is reduced. Further, albumin can have an abnormal functionality in case of a hepatic disease, for instance, its ability to bind a particular ligand can be altered.

[0316] The properties of albumin to bind a ligand can be determined by means of a plurality of ESR spectra, in particular be means of at least one characteristic biophysical parameter derived from the plurality of ESR spectra. This means that by means of a plurality of ESR spectra, a binding property of albumin can be determined that can be an indication of a particular hepatic disease.

[0317] The procedure of the evaluation of albumin by ESR spectroscopy is also described in international patent application WO 01/65270, the entirety of which is incorporated herein by reference and comprises the following steps:

[0318] In a first step, a sample aliquot containing albumin is placed into a container. In one embodiment, the sample is a serum sample. In one embodiment, the sample is a blood sample or a drug or product that contains albumin. For example, in one embodiment, the sample is a commercial solution comprising a human or bovine albumin preparation. In one embodiment, three sample aliquots are used. However, in an alternative embodiment, fewer or more sample aliquots are used; for example, 1, 2, 4, 5, 6, 7 or 8. In one embodiment, prior to step (1), a pre-analytical phase is conducted in order to preserve the original (native) conformational state of albumin as it is comprised within the sample to be evaluated. It is preferred that one or more of the following considerations are taken into account.

[0319] It is preferred that serum and EDTA-plasma samples are used. It is preferred that preparations with preservatives or anticoagulants (such as heparin), which can bind albumin and modify its native conformational state, are avoided. In embodiments where the sample is whole blood, it is preferred that the process of haemolysis is avoided. In one embodiment, to reduce haemolysis, centrifugation of whole blood for serum sampling is done within one hour of sampling at room temperature. In one embodiment, centrifugation is performed for 10 minutes at 1000 to 1500 g. In one embodiment, vacuum sampling systems are avoided during sampling of whole blood as it is believed that this can influence the stability of certain whole blood and serum samples over time. It is preferred that freezing of the whole blood sample before serum sampling is avoided as it is believed that this disturbs the native conformational state of the components of whole blood. In one embodiment, whole blood is stored or transported cooled for a maximum of 24 hours before centrifugation. In one embodiment, separated serum or EDTA-plasma is stored before analysis in a frozen state at a temperature not higher than −28° C. (due to on-going biochemical prepossess that are believed to occur even in frozen serum). It is preferred that samples are unfrozen only once and that this is shortly before use in the procedure. In one embodiment, the maximum time between defrosting the sample and measurement in the ESR spectrometer is 40 minutes. In embodiments where the sample is an albumin containing preparation such as a commercial albumin solution or a control sample, the recommendations of the manufacturer regarding the preparation of a control sample, an in particular the dilution of lyophilised albumin, are considered. It is preferred to have enough material of each sample for at least one controlling repetition of the measurement. Furthermore, it is preferred that samples from donors that fall within certain categories are excluded. In one embodiment, these categories comprise patient less than 21 days post surgery of a defined invasive procedure, as defined in the NCI dictionary (https://www.cancer.gov/publications/dictionaries/cancer-terms/def/invasive-procedure).

[0320] Secondly, each sample aliquot is mixed with a spin probe in a polar reagent. A spin probe (also known as a spin label) is an organic molecule that possesses an unpaired electron and has the ability to bind to another molecule. In the present embodiment, the spin probe is 16-doxyl stearic acid. In an alternative embodiment, the spin probe is an alternative spin-labelled fatty acid, preferably a doxyl stearic acid, and is one of 5-, 7-, 12- or 16-doxyl stearic acid or 16-doxyl stearate (Soduim). In one embodiment, it is a doxyl lauric acid, preferably 7-doxyl lauric acid. In one embodiment, any spin-labelled compound which can undergo specific binding to albumin (including a further spin-labelled fatty acid, a steroid hormone or a heterocyclic hydrocarbon) is used as a spin probe. Hydrophobic compounds labelled with nitroxyl radicals are used in one embodiment. In the present embodiment, the polar reagent is ethanol. In an alternative embodiment, an alternative alcohol or DMSO is used. It is preferred to use a C1-06 alcohol. In the present embodiment, the polar reagent acts as a solvent for the spin probe and acts to modify the polarity of the mixture. As will be described in further detail below, in the present embodiment, the three sample aliquots vary in the concentration of albumin and spin probe. In addition, the strength of hydrophobic interactions in the albumin-spin probe mixture is varied through use of differing amounts of polar reagent. In alternative embodiments, fewer or more than three different concentrations of albumin, spin probe and/or polar reagent are used; for example, 1, 2, 4, 5, 6, 7 or 8. Varying the concentration of spin label that is added to albumin and changing the ionic strength of the spin label mixture enables ESR spectra to be generated under different conditions. Suitable spin probes and polar reagents are known and some suitable spin probes and polar reagents are disclosed in WO 01/65270, the entirety of which is incorporated herein by this reference.

[0321] In an embodiment, three different concentrations of spin probe of 3.5, 5.8 and 7.5 mmol/l are mixed with 50 μl of each serum sample aliquot in the volumes of 10, 12 and 14 μl respectively. In one embodiment, the mean value of the ratio of spin probe concentration to albumin concentration is 2.5±0.5 and, starting from this mean value, at least two additional concentrations are selected whose deviation from this mean value is no less than 1.0. The concentrations of polar reagent to be added are selected in such a way that the mean value of the final concentration of polar reagent in the aliquots is (0.6±0.25)×Cp, wherein Cp represents the critical concentration of polar reagent, surpassing of which would result in denaturing of the albumin, and, starting from this mean value, at least two additional concentrations of polar reagent are selected, whose deviation from this mean value is at least 15%. Further details on the proportions of spin probe, albumin and polar reagent are described in US 2003/170912 A1/U.S. Pat. No. 7,166,474, which are incorporated herein by reference in their entirety. Without wishing to be bound by theory, by varying the concentration of spin probe and the concentration of polar reagent, it is believed that a combination of concentrations can be produced, which enables the transport properties of the albumin to be detected at different stages, namely, the physiological state during binding of hydrophobic compounds such as fatty acids (low concentration of spin probe and low concentration of polar reagent), the physiological state during transport of hydrophobic compounds through the vascular system (high concentration of spin probe and low concentration of polar reagent), and the physiological state during delivery (release) of hydrophobic compounds to the target cells (high concentration of spin probe and high concentration of polar reagent).

[0322] In a third step of the exemplary procedure, the mixture of the sample, spin probe and polar reagent is incubated. In the present embodiment, the incubation period is 10 minutes at 37° C. and at the physiological pH of blood. In an alternative embodiment, the incubation period is less or more than 10 minutes; for example, from 7 to 15 minutes. In an alternative embodiment, two or more different temperature values of the samples ranging between 15 and 45° C. and/or two or more different pH values of the serum samples ranging from 7.5 to 3.5 are used. In a following step, the mixture can be taken up by capillary tubes. In a subsequent step, an ESR spectrometer can be used to measure ESR spectra from each capillary tube. In a subsequent step, the ESR spectra can be analysed and results can be calculated.

[0323] To perform ESR spectroscopy the capillary tube can be inserted into an ESR spectrometer. Suitable ESR spectrometers are available from Medlnnovation GmbH (Berlin, Germany), for example models EPR 01-08, MS-400 and Espire-5000. ESR spectroscopy is a known technique that does not need to be discussed in detail in the present disclosure. Briefly, however, ESR spectra are acquired by exposing the sample to a strong static magnetic field. The application of the static magnetic field causes the separation of free electrons into two spin states. The application of microwave energy at the correct frequency causes spins to transition between the states. The microwave energy absorbed in this transition is measurable. It is either possible to maintain the microwave frequency constant and change the strength of the static magnetic field while monitoring the amount of microwave energy that is being absorbed or keeping the static magnetic field unchanged while sweeping the microware frequency over a range whilst monitoring energy absorption. The exact static magnetic field strength/microwave frequency combination that provides the correct amount of energy required by a spin to transition between the spin states depends on the chemical environment of the spin. It will be understood that a given spin probe consequently requires different amounts of energy to accomplish this transition, depending on the whether or not the spin probe is bound to albumin and indeed on the manner in which it is bound to albumin. Consequently ESR based methods can distinguish between unbound and bound spin probes. Moreover, ESR is able to distinguish between spin probes located at different binding sites on the albumin complex or between different spin probes in different unbound conditions. As such ESR is a powerful tool for assessing the binding conditions found on a molecule, in the embodiment on serum albumin.

[0324] An EPR spectrum can be generated by tracking the amount of microwave energy absorbed as the strength of the static magnetic field is ramped up or down over a predetermined range and by forming the first derivative of the tracked absorption spectrum. As the strength of the static magnetic field changes, so does the separation between the two spin states of the free electron. The separation between these two spin states does not only depend on the applied static magnetic field strength but also on the chemical environment within which the spin is located. By observing the amount of energy absorbed at each given static magnetic field strength conclusions can be drawn regarding the amount of spin probe present in a binding state that produces a separation in the energy.

[0325] Other X-Band EPR spectrometers (operating with a microwave frequency of approximately 9-10 GHz, can be also used in embodiments. The sample can be maintained at 37° C. during the measurement process to mimic physiologic conditions. Particular binding sites of spin probes on albumin produce spectral ESR patterns all signatures that can be detected. Analysis of such signatures allows determination of the amount of spin probe bound to the various binding sites. This said, it is not unusual for ESR patterns are generated by the influence different binding sites have on spin probes to overlap with each other.

[0326] FIG. 2 illustrates a 9.45 GHz ESR spectrum of human serum albumin. This spectrum consists of a number of overlapping sub-spectra. The measured spectrum shown in FIG. 2 can, for example, be decomposed into five sub-spectra, as shown in FIG. 3. The spectral components mentioned in FIG. 3 described in Table 1 and relate to spin probes respectively bound to albumin with high and low affinity or are present in the serum in the states mentioned in the table (Table 1).

TABLE-US-00001 TABLE 1 Spectral components C1 low motion albumin bound component with a high affinity C2 low motion albumin bound component with a lower affinity C3 free spin probe (16-doxyl stearic acid) molecules C4 free spin probe (16-doxyl stearic acid) in micelles C5 spin probe (16-doxyl stearic acid) bound on lipid-fraction of the serum

[0327] C1 represents the proportion of the fatty acid spin probe bound to the specific binding sites in albumin (i.e. at FA binding sites FA2, FA4 and FA5). C2 represents the proportion of fatty acid spin probe bound to the non-specific binding sites, binding sites 1, 3, 6 and 7 in the hydrophobic area of albumin. C3 to C5 represent unbound spin probe molecules (i.e. spin probes that are not bound to albumin). More specifically, C3 represents the proportion of spin probe molecules that are unbound and present free in the sample; C4 represents the proportion of spin probe molecules that are aggregated into clusters of fatty acid micelles; and C5 represents the proportion of spin probe molecules that are associated with or bound to lipoproteins in the serum sample. The contributions individual spectral components make to the measure spectrum can be determined by simulating the individual spectral components and then fitting the simulated components to the measured spectrum, adjusting the magnitude and phase of the individual spectral components until a fitting criterion is fulfilled. This criterion may, for example, be the minimisation of the root mean square error between the measured spectrum and the sum of the individual simulated spectral components as weighted by the above mentioned phase and magnitude term. The magnitudes and phases the spectral components have after this fitting indicate the concentration of spin probes in the various binding states set out in Table 1. In an embodiment the spectral components are simulated in the manner described by Andrey Gurachevsky, Ekaterina Shimanovitch, Tatjana Gurachevskaya, Vladimir Muraysky (2007) Intra-albumin migration of bound fatty acid probed by spin label ESR. Biochemical and Biophysical Research Communications 360 (2007) 852-856, the entirety of which is incorporated herein by this reference. The details of the method of simulation the spectral components need not be discussed in detail in the present disclosure. In an embodiment the simulated spectral lines are fitted to the measured ESR spectrum using a least squares fit. Alternatively, maximum likelihood estimation may be used. Based on the fitting of the five simulated components C1 to C5 as defined in Table 1, the relative concentrations of components C1 to C5 for each of the three above described aliquots (hereinafter also referred to by the short-hand A, B, C) are determined. This can be done in the manner described in WO 2000/004387A3, the entirety of which is incorporated herein by reference.

[0328] FIG. 4 illustrates the relation between an ESR spectrum and the geometry factor alpha. FIG. 4 shows an ESR spectrum, A, B or C. The geometry factor alpha is calculated as the ratio of the amplitude of the left-most spectral component of the ESR spectrum and the magnitude of the last but one spectral component on the right hand side of the ESR spectrum. These two peaks are related by their g-factors. Alpha is the ratio of intensities at the position of the high-field line of unbound 16-doxyl-stearic spin label relative to the spectral intensity at the position of low-field line of the bound 16-DSA in the albumin binding sites with high affinity. FIG. 4 illustrates that the geometry factor is determined by the raw spectra, such that it is not dependent on particular assumptions and/or simplifications of the model for simulation of EPR spectra.

[0329] FIG. 5 depicts a system comprising an ESR spectrometer 10 and a computing device 12. The ESR spectrometer comprises an experimental section in which spectra are acquired in the manner discussed above from samples entered into a sample chamber. Acquired spectra are output to the computing device 12 via the output interface 16 of the ESR spectrometer and the input interface 18 of the computing device 12. The computing device 12 comprises a processor 20 and a memory 22. The memory 22 stores program instructions for execution by the processor 20. The program instruction cause the processor 20 to perform the methods described herein when executed by the processor 20. The computing device 12 further comprises an output device 24. The output device 24 may be a display for displaying a determination result to the user or may be an electronic output interface that allows results to be transmitted to other devices. Any such transmission may take place using wireless or wired means.

[0330] Data associated to healthy patients (control), CC (stable; particularly stable cirrhosis), AD (cirrhosis with acute decompensation) and ACLF (acute on chronic liver failure) patients is analysed by means of an embodiment of the method. Data of 10 control samples, 18 stable samples (outpatients, CC), 241 AD samples and 78 ACLF samples is considered. Further information regarding CC (outpatient, stable), AD and ACLF patients, including demographic, biochemical and clinical characteristics, is provided in Table 2.

[0331] Table 2: demographic, biochemical and clinical characteristics of patients. Demographic data comprises age and sex. The aetiology of cirrhosis is characterised in terms of a viral, alcoholic, NASH (non-alcoholic steatohepatitis), a mixed aetiology or other causes. Biochemical and prognostic data comprises number of leucocytes, number of platelets, concentration of Na, concentration of bilirubin, concentration of creatinine, INR, MELD score, and Child-Pugh score. Data is reported as median and inter quartile range (IQR), absolute number and frequency. P-values indicate significance. Significant p-values are indicated in bold.

TABLE-US-00002 Outpatients AD ACLF p N 18 241 78 Demographic data Age (years) 59 (53-77) 63 (51-75) 62 (56-74) 0.800 Male sex 15 (83) 144 (60) 51 (65) 0.112 Etiology of cirrhosis Viral 8 (44) 118 (49) 28 (36) 0.131 Alcohol 5 (28) 41 (17) 17 (22) 0.383 NASH 0 (0) 17 (7) 7 (9) 0.409 Mixed etiology 0 (0) 35 (15) 10 (13) 0.215 Other 5 (28) 30 (12) 16 (21) 0.069 Biochemical and prognostic data Leucocyte (10.sup.9/L) 5.0 (3.8-6.7) 5.5 (3.6-8.5) 7.5 (5.1-9.6) 0.006 Platelets (10.sup.9/L) 139 (101-159) 95 (59-158) 86 (55-144) 0.102 Sodium (mmol/L) 141 (138-142) 137 (134-139) 136 (132-139) 0.001 Bilirubin (mg/dL) 0.9 (0.5-1.4) 2.2 (1.1-3.7) 6.8 (2.1-14.5) <0.001 Creatinine (mg/dL) 0.8 (0.7-0.9) 0.9 (0.7-1.2) 1.9 (1.4-2.5) <0.001 INR 1.1 (1.1-1.3) 1.4 (1.2-1.5) 1.6 (1.3-2.2) <0.001 MELD 13 (10-17) 14 (10-17) 26 (22-30) <0.001 Child-Pugh score 6 (5-6) 8 (7-10) 11 (10-12) <0.001

[0332] FIG. 6, panel A gives the albumin concentration related to samples of healthy patients (control), CC (stable), AD and ACLF patients as box-and-whisker plots. In the box-and-whisker plot, the central box represents the values from the lower to upper quartile (25 to 75 percentile). Hence, the box represents the IQR. The middle line represents the median. The horizontal line extends from the minimum to the maximum value, excluding outside and far out values, which are displayed as separate points. The median albumin concentration is highest in control samples and lowest in ACLF patients. The median albumin concentration in AD samples differs significantly with the median albumin concentration of control samples as well as with the median albumin concentration of samples of CC patients. However, the median albumin concentration of AD patients does not differ significantly with median albumin concentration associated to ACLF samples. The IQR related to AD samples overlap with the IQR related to the ACLF sample. Taken together, based on the albumin concentration a differentiation between AD and ACLF samples is impossible.

[0333] FIG. 6. panel B gives the detoxification efficiency (DTE) related to samples of healthy patients (control), CC (stable), AD and ACLF patients as box-and-whisker plots. The DTE in % of the control is given. The DTE in control sample is highest, the DTE in ACLF is lowest. The DTE of AD samples differs significantly with DTE in control, CC (stable) as well as ACLF. The IQR related to AD samples overlap with the IQR related to the ACLF sample.

[0334] FIG. 7 gives the binding efficiency (BE) related to samples of healthy patients (control), CC (stable), AD and ACLF patients as dot plots (A) and box-and-whisker plots (B). The BE in % of the control is given. The BE in control sample is highest, the BE in ACLF is lowest. The BE of AD samples differs significantly with BE in control, CC (stable) as well as ACLF. The IQR related to AD samples overlap with the IQR related to the ACLF sample.

[0335] In FIG. 7. panel C, data related to AD and CC (stable) patients is shown as dot plots, wherein additionally a cut-off (straight line) is shown. The cut-off is set such that a sensitivity of about 92% is set. At a sensitivity of 91.7%, a specificity of 61.1% is reached.

[0336] In FIG. 8. panel A, marker linear combination according to formula 3 related to AD and CC (stable) patients is shown as dot plots. Median values are indicated as lines. In FIG. 8, panel B, the data shown in FIG. 8A is presented and a cut-off which is set such that a sensitivity of about 92% is set. At a sensitivity of 92.5%, a specificity of 77.8% is reached. Comparing the specificity according to BE (FIG. 7, panel C) and marker linear combination according to formula 3 (FIG. 8, panel B) at about 92%, the specificity of marker linear combination according to formula 3 is increased considerably. In comparison to the specificity related to BE, the specificity according to the marker linear combination according to formula 3 is increased about 16.7%. This means that about 17% more patients are classified correctly as CC patients.

[0337] FIG. 8, panel C exemplifies a cut-off set at a specificity of 100%. At 100% specificity, the marker linear combination according to formula 3 has a sensitivity of 72.2%. A specificity of 100% implies that all CC patients are classified correctly as CC patients.

[0338] For further investigations, the ROC curves are calculated and analysed. In particular, the respective AUROC is determined and compared.

[0339] In FIG. 9, panel A, marker linear combination according to formula 4 related to AD and CC (stable) patients is shown as dot plots. Median values are indicated as lines. In FIG. 9. panel B, the data shown in FIG. 9. panel A is presented and a cut-off which is set such that the cut-off is set such that a sensitivity of about 92% is set. At a sensitivity of 92.4%, a specificity of 83.3% is reached. Comparing the specificity according to BE (FIG. 7. panel C) and marker linear combination according to formula 4 (FIG. 9. panel B) at about 92%, the specificity of marker linear combination according to formula 4 is increased considerably. In comparison to the specificity related to BE, the specificity according to the marker linear combination according to formula 4 is increased about 22.2%. This means that about 22% more patients are classified correctly as CC patients.

[0340] FIG. 9. panel C exemplifies a cut-off set at a specificity of 100%. At 100% specificity, the marker linear combination according to formula 4 has a sensitivity of 68.1%. A specificity of 100% implies that all CC (stable) patients are classified correctly as CC (stable) patients.

[0341] For further investigations, the ROC curves are calculated and analysed. In particular, the respective AUROC is determined and compared.

[0342] For comparison of CC and AD patients, the ROC curves associated to BE, DTE and marker linear combination according to formula 3 are shown in FIG. 10, panel A. In FIG. 10, panel B, the respective AUROC values are given, in particular the median. The 95% confidence interval is provided in brackets. Further, the p-values are given. A p-value is a measure for the significance. Typically with a p-value smaller than 0.05, significance is accepted. The closer the AUROC is to one, the better the performance.

[0343] The AUROC related to the marker linear combination according to formula 3 is 0.94 (0.9-0.96), while the AUROC related to BE is 0.87 (0.82-0.91) and the AUROC related to DTE is 0.85 (0.8-0.89). Hence, the AUROC related to the marker linear combination according to formula 3 is greater than the AUROC related to BE and is greater than the AUROC related to DTE. The improvement is significant (p=0.01). This means that the performance of the marker linear combination according to formula 3 (to distinguish between AD and CC samples) is increased in comparison to the performance of BE alone or DTE alone.

[0344] For comparison of CC and AD patients, the ROC curves associated to BE, DTE and marker linear combination according to formula 4 (“combination 1”) are shown in FIG. 11, panel A. In FIG. 11, panel B, the respective AUROC values are given, in particular the median. The 95% confidence interval is provided in brackets. Further, the p-values are given. A p-value is a measure for the significance. Typically with a p-value smaller than 0.05, significance is accepted. The closer the AUROC is to one, the better the performance.

[0345] The AUROC related to the marker linear combination according to formula 4 is 0.94 (0.9-0.96), while the AUROC related to BE is 0.87 (0.82-0.91) and the AUROC related to DTE is 0.85 (0.8-0.89). Hence, the AUROC related to the marker linear combination according to formula 4 is greater than the AUROC related to BE and is greater than the AUROC related to DTE. The improvement is significant (p=0.01). This means that the performance of the marker linear combination according to formula 4 (to distinguish between AD and CC samples) is increased in comparison to the performance of BE alone or DTE alone.

[0346] For comparison of AD patients and ACLF patients, the ROC curves associated to BE, DTE and marker linear combination according to formula 5 are shown in FIG. 12, panel A. In FIG. 12, panel B, the respective AUROC values are given as well as p-values.

[0347] The AUROC related to the marker linear combination according to formula 5 is 0.71 (0.65-0.76), while the AUROC related to BE is 0.64 (0.59-0.7) and the AUROC related to DTE is 0.66 (0.6-0.71). Hence, the median AUROC related to the marker linear combination according to formula 5 is greater than the median AUROC related to BE and is greater than the median AUROC related to DTE. The increase is significant. The performance of the marker linear combination according to formula 5 is increased in comparison to the performance of BE alone or DTE alone.

[0348] For comparison of AD patients and ACLF patients, the ROC curves associated to BE, DTE and marker linear combination according to formula 6 (“combination 2”) are shown in FIG. 13, panel A. In FIG. 13, panel B, the respective AUROC values are given as well as p-values.

[0349] The AUROC related to the marker linear combination according to formula 6 is 0.72 (0.67-0.77), while the AUROC related to BE is 0.64 (0.59-0.7) and the AUROC related to DTE is 0.66 (0.6-0.71). Hence, the median AUROC related to the marker linear combination according to formula 6 is greater than the median AUROC related to BE and is greater than the median AUROC related to DTE. The increase is partially significant. The performance of the marker linear combination according to formula 6 is increased in comparison to the performance of BE alone or DTE alone.

[0350] For prognostic information about the development of AD, in particular the characterisation of AD patients who developed ACLF within 30 days after admission, the ROC curves associated to BE, DTE, CLIF-C-AD score, linear combination comprising H and T, in particular H and TG, marker linear combination according to formula 1 (“combination 3”) and marker linear combination according to formula 2 (“combination 4”) are shown in FIG. 14, panel A and FIG. 14, panel B. In FIG. 14, panel C, the respective AUROC values are given as well as p-values.

[0351] The AUROC related to CLIF-C-AD score is 0.73 (0.67-0.79). The AUROC related to linear combination according to formula 1 (comprising H and T) is 0.76 (0.7-0.81). The CLIF-C-AD score and the linear combination according to formula 1 show a similar performance. The AUROC related to marker linear combination according to formula 2 is 0.81 (0.76-0.86). Hence, the performance of marker linear combination according to formula 2 is improved in comparison to the CLIF-C-AD score as well as in comparison to the linear combination comprising H and T according to formula 1. This shows that the combination of a diagnostic score, such as the CLIF-C-AD score, and biophysical parameters determined from ESR spectra can improve the performance in comparison to common medical practice that considers the CLIF-C-AD score only. In particular, the combination of a diagnostic score, such as the CLIF-C-AD score, and biophysical parameters determined from ESR spectra can improve the performance to predict the development of AD, in comparison to common medical practice that considers the CLIF-C-AD score only.

[0352] In FIG. 15, the Kaplan-Meier survival analysis is shown. It shows that the binding efficiency (A) has prognostic potential in predicting survival in AD patients and/or in ACLF patients. In FIG. 15B a marker linear combination according to formula 7 shows improved prognostic information in comparison to BE with respect to the survival in AD patients and/or in ACLF patients. Considering the median of formula 7 as a measure to predict the survival shows a better discrimination between the two sub-populations (greater/smaller than median) than considering BE as a measure to predict survival. After one year (365 days from admission) 16% more patients are alive in the group with BE higher than median, but 32% with parameter combination according to formula 7.

[0353] FIG. 16 is related to the classification of samples to be associated to AD or ACLF. FIG. 16 shows ROC curves related to KB, in particular KBA, S, in particular S2B, and related to a linear combination comprising H (particularly H1A) and KB, in particular KBA. Further ROC curves related to the marker linear combination related to formula 5 are shown. The respective characteristic values are provided in Table 3.

TABLE-US-00003 TABLE 3 AUROC values, 95% confidence interval (95% CI), p values, sensitivities (sensi.) as well as specificities (speci.) are given. KH: linear combination comprising H1A and KBA; combination formula 5: marker linear combination related to formula 5. AUROC 95% CI p sensi. speci. KBA 0.675 0.620 to 0.726 80% 38% S2B 0.614 0.558 to 0.667 0.16 80% 32% KH 0.694 0.641 to 0.744 0.22 80% 38% Combination 0.706 0.653 to 0.755 0.15 80% 49% formula 5

[0354] Previous analysis shows that KBA is the biophysical parameter having the highest AUROC. This implies that with respect to a single biophysical parameter, KBA provides the best performance to discriminate between AD samples and ACLF samples. Exemplarily it is shown that the AUROC related to S2B is lower than the AUROC related to KBA.

[0355] A combination of at least two biophysical parameters can increase the AUROC. This means that a combination of biophysical parameters can improve the performance.

[0356] The data illustrates that the performance of combination KH is improved in comparison to KBA and S2B alone. The AUROC related to KH is greater than the AUROC related to KBA. The AUROC related to KH is also greater than the AUROC related to S2B.

[0357] In comparison to the AUROC related to KBA, the AUROC associated to the marker linear combination related to formula 5 is increased. The AUROC associated to the marker linear combination related to formula 5 is also increased in comparison to the AUROC related to S2B and combination KH. The significance in the case of marker linear combination related to formula 5 is higher than the significance related to KH (KH: p=0.22; marker linear combination related to formula 5: p=0.15).

[0358] The data shows that at sensitivity of 80%, the specificity of marker linear combination related to formula 5 is greater than specificity related to KBA to classifying a sample to be associated to ACLF or to be associated to AD. In particular, the specificity is increased by 11%. This means that at sensitivity of 80%, 11% more patients get the correct diagnosis. This means 11% more patients are classified correctly. Hence, FIG. 16 and the associated Table 3 illustrate the improvement of the performance when considering marker linear combination related to formula 5, instead of KBA, S2B or combination KH for the classification of a sample to be associated to ACLF or to be associated to AD.

[0359] Taken together, a combination comprising the hydrophobicity, in particular hydrophobicity of high-affinity binding site, the binding constant and the order parameter can show an improved performance to classify AD and ACLF patients correctly. In particular, the marker linear combination according to formula 5 can be used to discriminate between AD and ACLF patients.

[0360] FIG. 17 is related to the classification of samples to be associated to AD or ACLF. FIG. 17 shows ROC curves related to H, in particular H1A, C5, in particular C5B, and related to a linear combination comprising H (particularly H1A) and KB, in particular KBB and KBC. Further ROC curves related to a linear combination comprising H (particularly H1A) and KB, in particular KBB and KBC, and S, in particular S1B is shown as well as ROC related to the marker linear combination related to formula 6 (“ACLF gg AD n”). The respective characteristic values are provided in Table 4.

TABLE-US-00004 TABLE 4 AUROC values, 95% confidence interval (95% CI), p values, sensitivities (sensi.) as well as specificities (speci.) are given. KH: linear combination comprising H1A, KBB and KBC; KHS: linear combination comprising H1A, KBB, KBC and S1B; combination formula 6: marker linear combination related to formula 6. AUROC 95% CI p sensi. speci. H1A 0.657 0.602 to 0.709 80% 34% C5B 0.637 0.582 to 0.690 0.69 80% 36% KH 0.694 0.641 to 0.744 0.29 80% 38% KHS 0.706 0.653 to 0.755 0.14 80% 49% Combination 0.723 0.671 to 0.772 0.07 80% 46% formula 6

[0361] Previous analysis shows that H1A is the biophysical parameter having the highest AUROC. This implies that with respect to a single biophysical parameter, H1A provides the best performance to discriminate between AD samples and ACLF samples. Exemplarily it is shown that the AUROC related to C5B is lower than the AUROC related to H1A.

[0362] A combination of at least two biophysical parameters can increase the AUROC. This means that a combination of biophysical parameters can improve the performance.

[0363] The data illustrates that the performance of combination KH is improved in comparison to H1A and C5B alone. The AUROC related to KH is greater than the AUROC related to H1A. The AUROC related to KH is also greater than the AUROC related to C5B.

[0364] The data further shows that the combination KHS shows an improvement of the performance in comparison to H1A, C5B or combination KH, in classifying a sample to be associated to ACLF or to be associated to AD.

[0365] In comparison to the AUROC related to H1A, the AUROC associated to the marker linear combination related to formula 6 is increased. The AUROC associated to the marker linear combination related to formula 6 is also increased in comparison to the AUROC related to C5B, combination KH, and combination KHS. The significance in the case of marker linear combination related to formula 6 is higher than the significance related to KHS (KHS: p=0.14; marker linear combination related to formula 6: p=0.07).

[0366] The data shows that at sensitivity of 80%, the specificity of marker linear combination related to formula 6 is greater than specificity related to H1A to classifying a sample to be associated to ACLF or to be associated to AD. In particular, the specificity is increased by 12%. This means that at sensitivity of 80%, 12% more patients get the correct diagnosis. This means 12% more patients are classified correctly. Hence, FIG. 17 and the associated Table 4 illustrate the improvement of the performance when considering marker linear combination related to formula 6, instead of H1A, C5B or combination KH for the classification of a sample to be associated to ACLF or to be associated to AD.

[0367] Taken together, a combination comprising the hydrophobicity, in particular hydrophobicity of high-affinity binding site, the binding constant and the order parameter can show an improved performance to classify AD and ACLF patients correctly. In particular, the marker linear combination according to formula 6 can be used to discriminate between AD and ACLF patients.

[0368] FIG. 18 is associated to the classification of CC and AD samples. FIG. 18. panel A shows ROC curves related to alpha, C3B, C5B, a combination comprising alpha and C5, in particular C5B, a combination comprising alpha and C3, in particular C3B, and the marker linear combination related to formula 3. The respective characteristic values are provided in Table 5.

TABLE-US-00005 TABLE 5 AUROC values, 95% confidence interval (95% CI), p values, sensitivities (sensi.) as well as specificities (speci.) are given. HS: linear combination of H1A and S2B; AC3: linear combination comprising alpha and C3B; AC5: linear combination comprising alpha and C5B; combination formula 3: marker linear combination related to formula 3. AUROC 95% CI p sensi. speci. Alpha 0.882 0.836 to 0.918 75% 83% C3B 0.872 0.825 to 0.910 0.30 77% 83% C5B 0.871 0.824 to 0.909 0.24 91% 83% HS 0.708 0.649 to 0.763 0.00 48% 83% AC3 0.889 0.844 to 0.925 0.38 76% 83% AC5 0.907 0.865 to 0.939 0.24 91% 83% Combination 0.938 0.902 to 0.964 0.02 90% 83% formula_3

[0369] Previous analysis shows that alpha is the biophysical parameter having the highest AUROC. This implies that with respect to a single biophysical parameter, alpha provides the best performance to discriminate between AD samples and CC samples. Exemplarily it is shown that the AUROC related to C3B and C5B is lower than the AUROC related to alpha.

[0370] A combination of at least two biophysical parameters can increase the AUROC. This means that a combination of biophysical parameters can improve the performance. For instance, the AUROC related to combination AC5 (0.907 (0.865-0.939)) is greater than the AUROC related to a single biophysical parameter such as C5B, C3B or alpha. The AUROC related to combination AC3 (0.889 (0.844-0.925)) is also greater than the AUROC related to a single biophysical parameter such as C5B, C3B or alpha.

[0371] The data further shows that not every combination of at least two biophysical parameters increases the AUROC. This means that not every combination of at least two parameters can provide an increased performance to classify samples to a particular disease, in particular to an increased performance to classify samples to be associated to AD or CC.

[0372] Exemplarily, the AUROC of the combination of H1A and S2B is given (HS). It is lower than the AUROC of alpha alone. Hence, the performance of the combination HS is worse than the performance of alpha (to classify samples to be associated to AD or CC).

[0373] The marker linear combination according to formula 3 shows an AUROC of 0.938 (0.902-0.964) that is significantly greater than the AUROC related to alpha.

[0374] Data shows exemplarily, that at specificity of 83% the sensitivity of the marker linear combination according to formula 3 is 90% and, hence, 15% larger than the sensitivity related to alpha alone. The marker linear combination according to formula 3 can improve the classification of samples to be related to CC or AD.

[0375] The ROC curves shown in FIG. 18B, further illustrate that the ROC curve related to C5B is serrated. In contrast, the ROC curve related to formula 3 is curved. The curved ROC curve is much more robust than the serrated ROC curve.

[0376] FIG. 19 is associated to the classification of CC and AD samples. FIG. 19, panel A and FIG. 19, panel B show ROC curves related to alpha, C5B, a combination comprising alpha and C5, in particular C5B, and related to a linear combination comprising alpha and C5, particularly C5B, and C3, particularly C3A and C3B. Further, ROC curves related to the marker linear combination related to formula 4 (“AD_vs_CC”) are shown. The respective characteristic values are provided in Table 6. Table 6 gives additional information about ROC curves associated to C3A, C3B, BE, and a combination of alpha and C3, in particular alpha, C3A and C3B.

TABLE-US-00006 TABLE 6 AUROC values, 95% confidence interval (95% CI), p values, sensitivities (sensi.) as well as specificities (speci.) are given. AC5: linear combination comprising alpha and C5B; AC3: linear combination comprising alpha, C3A and C3B; AC5C3: linear combination comprising alpha, C5B, C3A and C3B; combination formula 4: marker linear combination related to formula 4. AUROC 95% CI p sensi. speci. Alpha 0.865 0.809 to 0.910 72% 83% C3B 0.856 0.799 to 0.902 0.40 75% 83% C5B 0.863 0.806 to 0,.908 0.94 89% 83% BE 0.853 0.796 to 0.900 0.04 68% 83% C3A 0.853 0.795 to 0.899 0.03 68% 83% BE_DTE 0.862 0.805 to 0.907 0.83 76% 83% BE_C5B 0.876 0.821 to 0.919 0.71 88% 83% C3B_C5B 0.880 0.826 to 0.922 0.62 89% 83% AC5 0.893 0.841 to 0.933 0.24 89% 83% AC3 0.870 0.815 to 0.914 0.56 72% 83% AC3C5 0.925 0.878 to 0.958 0.02 88% 83% Combination 0.923 0.877 to 0.957 0.03 91% 83% formula 4

[0377] Previous analysis shows that alpha is the biophysical parameter having the highest AUROC. This implies that with respect to a single biophysical parameter, alpha provides the best performance to discriminate between AD samples and CC samples. Exemplarily it is shown that the AUROC related to C5B, C3A, C3B or BE is lower than the AUROC related to alpha.

[0378] A combination of at least two biophysical parameters can increase the AUROC. This means that a combination of biophysical parameters can improve the performance. For instance, the AUROC related to combination AC5 (0.893 (0.841-0.933)) is greater than the AUROC related to a single biophysical parameter such as C3A, BE, C5B, C3B or alpha. The AUROC related to combination AC3 (0.87 (0.815-0.914)) is also greater than the AUROC related to a single biophysical parameter such as C3A, BE, C5B, C3B or alpha.

[0379] The data further shows that not every combination of at least two biophysical parameters increases the AUROC. This means that not every combination of at least two parameters can provide an increased performance to classify samples to a particular disease, in particular to an increased performance to classify samples to be associated to AD or CC. Exemplarily, the AUROC of the combination of BE (binding efficiency) and DTE (detoxification efficiency) is given (BE_DTE). It is lower than the AUROC of alpha alone. Hence, the performance of the combination BE_DTE is worse than the performance of alpha (to classify samples to be associated to AD or CC). The combination comprising one of a spectral component from free spin probe molecules (C3), a spectral component from spin probe on lipid-fraction of serum (C5), or a geometry factor (alpha) increases the performance. In particular, the AUROC of the combination BE_C5B is greater than the AUROC of alpha. The AUROC of the combination BE_C5B is also greater than the AUROC of the combination BE_DTE. A combination comprising a plurality of a spectral component from free spin probe molecules (C3), a spectral component from spin probe on lipid-fraction of serum (C5), or a geometry factor (alpha) further increase the AUROC. This means that a combination comprising a plurality of a spectral component from free spin probe molecules (C3), a spectral component from spin probe on lipid-fraction of serum (C5), or a geometry factor (alpha) further increase the performance, in particular the performance to discriminate between a sample associated to AD and a sample associated to CC.

[0380] The linear combination comprising alpha, C3 and C5 increases partially significantly the AUROC (0.925 (0.878-0.958)) in comparison to the AUROC related to alpha (0.865 (0.809-0.91)). The marker linear combination according to formula 4 shows an AUROC of 0.923 (0.877-0.957) that is partially significantly greater than the AUROC related to alpha.

[0381] Data shows exemplarily, that at specificity of 83% the sensitivity of the marker linear combination according to formula 4 is 91% and, hence, 19% larger than the sensitivity related to alpha alone. The marker linear combination according to formula 4 can improve the classification of samples to be related to CC or AD.

[0382] The ROC curves shown in FIG. 19B, further illustrate that the ROC curve related to C5B is serrated. In contrast, the ROC curve related to combination AC5C3 is curved. The curved ROC curve is much more robust than the serrated ROC curve.

[0383] FIG. 20 and associated Table 7 show ROC curves and data to discriminate between CC and AD patients in case the AD is in some patients accompanied by a bacterial infection.

TABLE-US-00007 TABLE 7 AUROC values, 95% confidence interval (95% CI), p values, sensitivities (sensi.) as well as specificities (speci.) are given. AC5: linear combination comprising alpha and C5B; AC5C3: linear combination comprising alpha, C5B, C3A and C3B; combination formula 4: marker linear combination related to formula 4. AUROC 95% CI p sensi. speci. Alpha 0.882 0.836 to 0.918 75% 83% AC5 0.907 0.865 to 0.939 0.24 91% 83% AC5C3 0.938 0.902 to 0.964 0.02 90% 83% Combination 0.936 0.900 to 0.963 0.02 93% 83% formula 4

[0384] Also in case of a bacterial infection, a combination of biophysical parameters can improve the performance (to discriminate between AD and CC) in comparison to the performance of a single parameter, such as alpha. The combination AC5C3 as well as the marker linear combination according to formula 4 strongly increase the AUROC.

[0385] At a specificity of 83%, the sensitivity according to marker linear combination according to formula 4 (93%) is considerably increased in comparison to the sensitivity of alpha only (75%). Further, the sensitivity of combination AC5 (91%) as well as sensitivity of combination AC5C3 (90%) are increased compared to the sensitivity of alpha.

[0386] Hence, the marker linear combination according to formula 4 can be used to discriminate between AD and CC patients in case of a bacterial infection as well as in absence of an accompanied bacterial infection.

[0387] According to an embodiment of the method, it can be used to estimate the development of a disease, in particular to predict the development of AD, in particular to predict the development of AD to ACLF.

[0388] In FIG. 21, the ROC curves related to T, in particular TG, in particular TG2A, CLIF-C-AD score, a combination comprising H (particularly H1B) and T (particularly TG2A) are shown. Further, the ROC curve related to the marker linear combination according to formula 2 is shown. In Table 8, the associated characteristics are provided.

TABLE-US-00008 TABLE 8 AUROC values, 95% confidence interval (95% CI), p values, sensitivities (sensi.) as well as specificities (speci.) are given. HT: linear combination comprising H1B and TG2A; combination formula 2: marker linear combination related to formula 2. AUROC 95% CI p sensi. speci. TG2A 0.733 0.673 to 0.788 81% 57% CLIF-C-AD 0.733 0.672 to 0.787 0.99 81% 55% HT 0.761 0.703 to 0.814 0.34 81% 60% Combination 0.813 0.758 to 0.860 0.01 81% 76% formula 2

[0389] The performance of biophysical parameter TG2A is as good as the performance of the CLIF-C-AD score, to particularly predict the development of AD. Both show identical AUROCs and comparable specificity at a sensitivity of 81%. A combination of two appropriate biophysical parameters can improve the performance. In particular, the combination HT shows a greater AUROC (0.761 (0.703-0.814) in comparison to TG2A (0.733 (0.673-0.788)) as well as in comparison to CLIF-C-AD score (0.733 (0.672-0.787)). Hence, the performance, in particular the performance to predict the development of AD, of the combination HT is improved in comparison to the CLIF-C-AD score that is used for diagnosis and prognosis in clinical practice.

[0390] The marker linear combination according to formula 2 further increases the AUROC (0.813 (0.758-0.86)). Compared to the AUROC of TG2A, the increase is significant.

[0391] At a sensitivity of 81%, the specificity of TG2A is 57%. At sensitivity of 81%, the specificity according to the CLIF-C-AD score is 55%. The marker linear combination according to formula 2 shows a specificity of 76% (at sensitivity 81%). Hence, in comparison to TG2A, the specificity increases by 19% when considering the marker linear combination according to formula 2. In comparison to the CLIF-C-AD score, the specificity increases by 21% when considering the marker linear combination according to formula 2.

[0392] In particular, the marker linear combination according to formula 2 can be used to predict the development of AD.