Clinical immunoassay with early signal measurement

Abstract

The invention relates to an in vitro method for assessing whether a biomarker concentration in a sample is above a clinical threshold value, comprising signal measurement and reporting said signal, preferably within 5 minutes of initiating a homogeneous immunoassay in a liquid mixture, wherein the signal indicates whether the biomarker concentration in said sample is above a clinical threshold value, or provides a likelihood or risk of whether the biomarker concentration in said sample is above a clinical threshold value.

Description

[0001] CLINICAL IMMUNOASSAY WITH EARLY SIGNAL MEASUREMENT

[0002] DESCRIPTION

[0003] The invention is in the field of clinical diagnostics, medical risk assessment, and therapy guidance, and corresponding methods and products.

[0004] The invention relates to an in vitro method for assessing whether a biomarker concentration in a sample is above a clinical threshold value, comprising signal measurement and reporting said signal within 5 minutes of initiating said immunoassay, wherein the signal indicates whether the biomarker concentration in said sample is above a clinical threshold value, or provides a likelihood or risk of whether the biomarker concentration in said sample is above a clinical threshold value.

[0005] The invention relates to an in vitro method for assessing whether a biomarker concentration in a sample is above a clinical threshold value, or provides a likelihood or risk of whether the biomarker concentration in said sample is above a clinical threshold value, said method comprising providing a sample, providing a calibrator comprising a known concentration of said biomarker, initiating a homogeneous immunoassay in a liquid mixture in said sample and said calibrator, preferably comprising time-resolved fluorescence resonance energy transfer (TR- FRET) and / or a time-resolved amplified cryptate emission (TRACE) reaction, with non-radiative energy transfer between a donor label and an acceptor label coupled to first and second binding agents, respectively, that bind to said biomarker, wherein proximity of the donor and the acceptor labels in complex with said biomarker produces a signal, wherein a signal measurement is carried out in said sample and said calibrator within 5 minutes of initiating said immunoassay, and when a ratio of the signal of the sample and the signal of the calibrator (signal ratio sample / calibrator) is above a signal threshold value, reporting that said signal ratio is above said signal threshold value within 5 minutes of initiating said immunoassay, wherein a signal ratio above said signal threshold value indicates the biomarker concentration in said sample is above the clinical threshold value, or provides a likelihood or risk of whether the biomarker concentration in said sample is above a clinical threshold value.

[0006] In preferred embodiments the biomarker is selected from the group consisting of procalcitonin (PCT) or fragment(s) thereof, proadrenomedullin (proADM) or fragment(s) thereof, preferably MR- proADM, interleukin-6 (IL-6), copeptin, sFlt-1 and troponin, or another marker that is used in the acute care or emergency setting, such as in cases where fast risk prediction and clinical decision making is important for the clinical outcome of a patient.

[0007] The invention further relates to the method comprising a diagnosis, prognosis and / or risk stratification of a medical condition in a subject. In embodiments the medical condition is an infectious disease, such as sepsis, severe sepsis or septic shock, prenatal complications like preeclampsia, the HELLP syndrome or preterm birth, blood vessel embolism such as pulmonary embolism, stroke, or myocardial infarction and / or an acute medical condition such as an acute cardiac condition or other conditions related to acute life-threatening conditions, such as organ dysfunctions.

[0008] The invention further relates to a system for assessing whether a biomarker concentration in a sample is above a clinical threshold value and a method for determining a signal threshold value.

[0009] BACKGROUND OF THE INVENTION

[0010] Acute medical conditions such as sepsis and septic shock or cardiac conditions such as acute myocardial infarction typically exhibit rapid onset of severe symptoms often necessitate immediate medical intervention and intensive care to prevent complications, adverse events or death.

[0011] Numerous biomarkers have been established to diagnose acute medical conditions and assess disease severity and required treatment. These biomarkers include for example C-reactive protein (CRP), procalcitonin (PCT), mid-regional proadrenomedullin (MR-proADM) and IL-6 for sepsis diagnosis (see for example Yentis et al., Intensive Care Med. Jul 1995;21 (7):602-605; Matson A, et al., 1991 ; 19(2): 182-186; Assicot et al., Lancet. Feb 27, 1993;341 (8844):515-518; Al- Nawas B et al., Eur J Med Res. Apr 18, 1996;1 (7):331-333; Schuetz et al., BMC Medicine. 2011 ; 9:107 ; Albrich et al. BMC Infect Dis. 2011 ;11 :112 and EP2320237) and copeptin and troponin for diagnosis of acute myocardial infarction (see for example Wereski et al. (Corculation, 2021 , 144, 7 and Mockel et al.; Eur J Emerg Med 2013; 20: 103-108).

[0012] Methods for detecting such biomarkers have been described previously. Morgenthaler (Clinical Chemistry 51 :10; 1823-1829, 2005) for example describes the technical characterization of a sandwich immunoassay for the measurement of MR-proADM in human plasma, its reference interval in healthy individuals, and the finding of increased plasma concentrations in patients with cardiovascular disease or sepsis.

[0013] Further, the B R A H M S MR-proADM™ KRYPTOR™ system enables measurement of biomarker concentrations and provides a precise assessment of disease severity and patient risk management and can enhance clinical investigation and treatment decisions. The

[0014] B R A H M S™ KRYPTOR Analysers are fully automatic, closed laboratory analysis systems that can perform numerous analyses in random-access operations using a unique measuring principle called TRACE™ (Time Resolved Amplified Cryptate Emission). Background information on the KRYPTOR™ compact (CEZANNE SAS) is provided in Truchaud et al ("An Innovative Modular Approach in an Automated Compact Immunoassay System", JALA, Feb 2009, vol. 14, no. 1 , 41- 48), describing the system as an automated immunoassay system using a homogeneous technology.

[0015] Nevertheless, the KRYPTOR technology requires several minutes (such as 9 to 30 minutes) to accurately determine the concentration of a biomarker in a sample of a patient. In light of the often-severe symptoms patients with acute medical conditions, requiring immediate initiation of treatment, the exact determination of the biomarker concentration may not be necessary to determine further treatment guidance or initiation of therapy. Medical personnel would often prefer not needing to wait for exact biomarker concentration determination to initiate therapy, for example when a longer period of time is required before the measurement is completed and an accurate diagnosis of the medical condition of the patient has been made. Alternatively, medical personnel may initiate treatment before receiving a clear indication of biomarker levels, due to the urgency of the medical condition and the time required for analysis. This may disadvantageously lead to inappropriate or unnecessary treatment of the patient. Similar difficulties occur in patients with overlapping symptoms, e.g. with chest pain and shortness of breath, that can be a symptom of an acute myocardial infarction, a lung embolism or pneumonia. Making the wrong decision or waiting for further diagnostic procedures, like imaging of the lung or the heart, could cause a loss in critical time to initiate the most effective therapy. Such patients are often treated iteratively by “best guess” or “try and error” approaches, which is often highly inconsistent, depending on the experience and qualification of the medical personal.

[0016] Further methods for determining the concentration of a biomarker are for example disclosed in Alba-Patino et al. (Nanoscale Advances, 2020, 2, 1253-1260) testing nanoparticle-based mobile biosensors for the detection of sepsis biomarkers such as IL-6 in blood samples, however requiring 17 minutes for the determination of the IL-6 concentration in the sample. Bradley et al. (Biosensors and Bioelectronics, 2023, 2277, 115181) discloses different microfluidic technologies for Point-of-care diagnostics for sepsis, wherein clinical biomarkers such as CRP, PCT and IL-6 are determined. However, determination of the concentration of these biomarkers requires 8 minutes up to 3,5 h. Further the Elecsys immunoassay system by Roche and the PATHFAST system allow the detection of several biomarkers such as IL-6, PCT and troponin, but similarly requires approx. 17 to 18 minutes for biomarker measurement.

[0017] Further time-resolved homogenous analytical approaches are known in the art, such as those described by Hildebrandt ("Lanthanides and quantum dots - time resolved laser spectroscopy of biochemical Forster resonance energy transfer (FRET) systems", Dissertation, University of Potsdam), who describes homogenous FRET immunoassays, and Chen et al (Analytica Chimica Acta, vol. 741 , 2012, 100-105), who describe a quantum dot-based homogeneous time-resolved fluorescence assay used for detection of AFP, a marker for many cancers and diseases.

[0018] Despite some immunoassays and systems for the determination of biomarker concentration being available on the market, the need exists for alternative or improved methods that provide a fast, initial determination of the biomarker concentration in a sample, allowing a more rapid assessment of the clinical status of a patient to apply suitable treatment means, especially for patients with acute medical conditions.

[0019] SUMMARY OF THE INVENTION

[0020] In light of the prior art, the technical problem underlying the present invention is to provide improved and / or alternative means for assessing the concentration of a biomarker in a sample that overcome the disadvantages of the prior art.

[0021] Another problem of the present invention is to provide improved and / or alternative means for assessing the concentration of a biomarker in a sample that can be rapidly performed and allow an initial, semi-quantitative, assessment of the biomarker concentration within a few minutes. Another problem underlying the present invention is to provide improved and / or alternative means to determine whether the concentration of a biomarker is above a critical clinical threshold value and that allow the rapid assessment of the clinical condition of a patient and initiation of the respective treatment means within a few minutes.

[0022] Another problem underlying the present invention is to provide improved and / or alternative means to determine whether the concentration of a biomarker is above a critical clinical threshold value, wherein the means are non-destructive, thereby not destroying the sample upon analysis, thereby allowing additional determination of the exact concentration of a biomarker in the same sample after performing the early initial assessment of the biomarker concentration.

[0023] Another problem underlying the present invention is to provide an improved and / or alternative immunoassay system based on time-resolved fluorescence resonance energy transfer (TR- FRET) or time-resolved amplified cryptate emission (TRACE), that provides an initial, semi- quantitative, assessment of the biomarker concentration within a few minutes.

[0024] These problems are solved by the features of the independent claims. Preferred embodiments of the present invention are provided in the dependent claims.

[0025] In one aspect, the invention relates to an in vitro method for assessing whether a biomarker concentration in a sample is above a clinical threshold value, comprising signal measurement and reporting said signal within 5 minutes of initiating said immunoassay, wherein the signal indicates the biomarker concentration in said sample is above the clinical threshold value.

[0026] In one aspect, the invention relates to an in vitro method based on time-resolved fluorescence resonance energy transfer (TR-FRET) or time-resolved amplified cryptate emission (TRACE) for assessing whether a biomarker concentration in a sample is above a clinical threshold value, comprising signal measurement and reporting said signal within 5 minutes of initiating said immunoassay, wherein the signal indicates the biomarker concentration in said sample is above the clinical threshold value.

[0027] The invention therefore relates to an in vitro method for assessing whether a biomarker concentration in a sample is above a clinical threshold value, said method comprising: providing a sample, providing a calibrator comprising a known concentration of said biomarker, initiating a homogeneous immunoassay in a liquid mixture in said sample, and in said calibrator, wherein a signal measurement is carried out in said sample and said calibrator within 5 minutes of initiating said immunoassay, and when a ratio of the signal of the sample and the signal of the calibrator (signal ratio sample / calibrator) is above a signal threshold value, reporting that said signal ratio is above said signal threshold value within 5 minutes of initiating said immunoassay, wherein a signal ratio above said signal threshold value indicates the biomarker concentration in said sample is above the clinical threshold value. In other aspects, the invention relates to an in vitro method for assessing whether a biomarker concentration in a sample is above a clinical threshold value, said method comprising: providing a sample, providing a calibrator comprising a known concentration of said biomarker, initiating a homogeneous immunoassay in a liquid mixture in said sample, and in said calibrator, wherein a signal measurement is carried out in said sample and said calibrator within 5 minutes of initiating said immunoassay, and wherein said signal measurements indicate whether the biomarker concentration in said sample is above the clinical threshold value.

[0028] In other aspects, the invention relates to an in vitro method for assessing whether a biomarker concentration in a sample is above a clinical threshold value, said method comprising: providing a sample, providing a calibrator reference value, comprising a reference concentration of said biomarker, initiating a homogeneous immunoassay in a liquid mixture in said sample, wherein a signal measurement is carried out in said sample within 5 minutes of initiating said immunoassay, and wherein said signal measurement and reference concentration indicate whether the biomarker concentration in said sample is above the clinical threshold value.

[0029] In other aspects, the invention relates to an in vitro method for assessing whether a biomarker concentration in a sample is above a clinical threshold value, said method comprising: providing a sample, providing a calibrator reference value, comprising a reference concentration of said biomarker, initiating a homogeneous immunoassay in a liquid mixture in said sample, wherein a signal measurement is carried out in said sample within 5 minutes of initiating said immunoassay, and when a ratio of the signal of the sample to the reference concentration (signal ratio sample / reference) is above a threshold value, reporting that said ratio is above said threshold value within 5 minutes of initiating said immunoassay, wherein a ratio above said threshold value indicates the biomarker concentration in said sample is above the clinical threshold value.

[0030] As described in more detail below, each and any given embodiment of the description may be combined with any aspect of the invention, as outlined above. The aspects outlined above relate to variations of the same invention procedure, with some changes regarding signal measurement, calibration signal measurement, provision of a value as a reference concentration, and / or determining the ratio between sample and calibrator signals, or between sample signal and reference concentration.

[0031] In any of the above aspects, the term “ within 5 minutes” may preferably be “within 3 minutes”, more preferably within 2.5 minutes, 2 minutes, 1 .5 minutes or within 1 minute. Any of the alternative time indications provided herein may also be used in any one or more of the aspects or embodiments described above and herein. This may relate to e.g., signal measurement and / or reporting, in this time frame.

[0032] In one embodiment, the immunoassay comprises a time-resolved fluorescence resonance energy transfer (TR-FRET) and / or a time-resolved amplified cryptate emission (TRACE) reaction, preferably with non-radiative energy transfer between a donor label and an acceptor label coupled to first and second binding agents, respectively, that bind to said biomarker, wherein proximity of the donor and the acceptor label in complex with said biomarker produces a signal.

[0033] In embodiments, the invention therefore relates to an in vitro method for assessing whether a biomarker concentration in a sample is above a clinical threshold value, said method comprising: providing a sample, providing a calibrator comprising a known concentration of said biomarker, initiating an automated homogeneous immunoassay in a liquid mixture in said sample and said calibrator, preferably comprising time-resolved fluorescence resonance energy transfer (TR-FRET) and / or a time-resolved amplified cryptate emission (TRACE) reaction, with non-radiative energy transfer between a donor label and an acceptor label coupled to first and second binding agents, respectively, that bind to said biomarker, wherein proximity of the donor and the acceptor label in complex with said biomarker produces a signal, wherein a signal measurement is carried out in said sample and said calibrator within 5 minutes of initiating said immunoassay, and when a ratio of the signal of the sample and the signal of the calibrator (signal ratio sample / calibrator) is above a signal threshold value, reporting that said signal ratio is above said signal threshold value within 5 minutes of initiating said immunoassay, wherein a signal ratio above said signal threshold value indicates the biomarker concentration in said sample is above the clinical threshold value.

[0034] As demonstrated in the examples below, the method of the present invention surprisingly allows to indicate, determine, provide a likelihood, or risk, of whether the concentration of a biomarker in a sample is above a clinical threshold already within 10 minutes, 5 minutes, preferably within 4 minutes, more preferably within 3 minutes and even more preferably within 2.5 minutes, 2 minutes, 1.5 minutes or within 1 minute of assay initiation.

[0035] As used herein, the feature “within 5 minutes” may be replaced by another time frame, as disclosed herein. For example, other time frames, even those longer than 5 minutes, for example, “within 10 minutes”, may be employed in place of “within 5 minutes”. Other times, such as 10, 9, 8, 7, 6 minutes may also be employed. As used herein “within 5 minutes” means 5 minutes and less than 5 minutes, therefore also shorter timeframes like 3 or 2 minutes are covered, in addition to even shorter time frames, below 1 minute. In embodiments, the term “within 5 minutes” may be within 1 minute, within 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, or within 30 seconds, or within shorter time frames.

[0036] In embodiments, the term “within 5 minutes” may relate to a time frame with a lower limit, for example, within 1 and 5 minutes, or within 30 seconds and 5 minutes. In embodiments, the term “within 5 minutes” may relate to a range, that may be formed using any two time points described herein. For example, a range may be formed from two values from the values 10 minutes, 9, 8, 7, 6, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5 minutes, 1 minute, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, or 30 seconds.

[0037] The method of the present invention thus advantageously provides information on the disease and / or clinical status of a patient and the required treatment within a very short period of time after obtaining a sample from the patient and initiating measurement. In light of the prior art, disclosing methods to accurately determine the concentration of a biomarker in a sample, usually after 10 to 30 minutes or even after 1 -3 hours, there is no suggestion of how or at what time point measurement of the biomarker concentration allows such an initial assessment of the biomarker concentration with regards to a clinical threshold value.

[0038] In embodiments, the method comprises a signal measurement carried out in said sample and said calibrator within 5 minutes of initiating the immunoassay, and reporting that said signal ratio is above said signal threshold value within 5 minutes of initiating the immunoassay. In embodiments, signal measurement and / or reporting occurs within 1 to 7 minutes of initiating the immunoassay, preferably within 1.5 - 6 minutes, or within 2 - 5 minutes of initiating the immunoassay, such as within 1 , 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 minutes. In preferred embodiments, the signal measurement and reporting occur within 1.5, 2, 2.5 or 3 minutes of initiating the immunoassay. In embodiments, the time frame for signal measurement and reporting is the same, in other embodiments, the time frames for signal measurement and reporting are different.

[0039] The method of the present invention for example allows to determine whether a patient has a critical level of an acute care biomarker, such as PCT or proADM, or other markers disclosed herein, indicative of the patient having an acute severe medical condition, such as organ dysfunction or failure, sepsis or septic shock requiring a fast antibiotic treatment and hospitalization and / or intensive care, such as admission to the ICU.

[0040] The method of the present invention for example allows to determine whether a patient has a critical level of an acute care biomarker, such as Copeptin or troponin, indicative of the patient having an acute severe medical condition such as myocardial infarction requiring a fast treatment and hospitalization and / or intensive care, such as admission to the ICU.

[0041] The method of the present invention for example allows to determine whether a patient has a critical level of an acute care biomarker, such as IL-6, indicative of the patient having an acute severe inflammatory process requiring an anti-inflammatory treatment and hospitalization and / or intensive care, such as admission to the ICU. The method of the present invention for example allows to determine whether a patient has a critical level of an acute care biomarker, such as sFlt-1 is indicative of the patient having an acute severe medical pregnancy related condition such as preeclampsia, eclampsia or the HELLP syndrome requiring a fast treatment, treatment modifications, hospitalization and / or intensive care, such as admission to the ICU.

[0042] One advantage of the present invention is that even without final determination of biomarker concentration, immunoassay methods based on time-resolved fluorescence resonance energy transfer (TR-FRET) or time-resolved amplified cryptate emission (TRACE) may provide early signal measurements that correlate with a clinical threshold, thereby providing a very quick and reliable indication of whether a biomarker is above a clinical threshold value, without having to exactly determine biomarker concentration, which would typically require a longer analysis time.

[0043] Since the method according to the invention allows to determine whether the patient has a critical value of an acute care biomarker after just a few minutes, the acute severe medical condition of the patient can be recognised and assessed very quickly, and the necessary treatment can be initiated rapidly. This is particularly advantageous when considering that acute severe medical conditions, such as infectious disease, such as sepsis, severe sepsis or septic shock, prenatal complications like preeclampsia, the HELLP syndrome or preterm birth, blood vessel embolism such as pulmonary embolism, stroke, or myocardial infarction and / or an acute medical condition such as an acute cardiac condition or other conditions related to acute life-threatening conditions, such as organ dysfunctions, which usually require very rapid initiation of therapeutic measures.

[0044] An accurate measurement of the biomarker concentration by the methods of the prior art, which usually takes 10 to 30 minutes or even longer, requires too much time, and therefore can often not be waited upon before the first therapeutic measures are initiated. Therapeutic measures are therefore often initiated before the final biomarker concentration has actually been determined and an accurate diagnosis of the medical condition of the patient has been made. Therefore, often the first treatment and patient management is applied iteratively or by best guess or trial and error approach, leading to an increased rate of ineffective or insufficient patient management and treatment, compared to the treatment and patient management being initiated after having the final result of a biomarker concentration. The method of the present invention thus advantageously allows to obtain an initial assessment of acute biomarkers after only a few minutes and allows to initiate appropriate therapeutic measures based on this initial assessment.

[0045] The method and means of the invention are also beneficial for patients in which symptoms of a disease are unclear, or are symptoms common to multiple medical indications, such as in patients with non-specific complaints, whereby severity of a medical condition can be reliably determined, such as in cases where the biomarker is above a clinical threshold, without having to finally determine biomarker concentration. Thus, patients show often symptoms that are not specific enough to make a clear diagnosis but may provide an initial indication to a medical practitioner allowing an estimation (best guess approach). Additionally, the patient may not show specific disease related symptoms yet (asymptomatic) or have co-morbidities or primary disorders with overlapping symptoms. It is difficult for a clinician in the emergency department or acute care setting to evaluate the situation quickly and to initiate an effective treatment and patient management. The present invention thus addresses such cases. The present invention is therefore associated with various advantages in a clinical context, as the method advantageously provides information on the disease and / or clinical status of a patient (including diagnostic, prognostic, monitoring, therapy guidance or other clinically relevant statements) regarding the status and / or risk of a patient, within a very short period of time after obtaining a sample and initiating measurement.

[0046] By way of example, early rule-out of acute myocardial infarction (AMI), especially within the first few hours after chest pain onset, is enabled by early measurements of copeptin, which levels rise very quickly during acute stress (such as myocardial infarction). By setting appropriate clinical and ratio thresholds, copeptin can be used for rapid triage in emergency departments. The invention is therefore connected with huge clinical and economic benefits, such as shorter stays in emergency departments, reducing unnecessary admissions, and saving critical care resources. By way of further example, early determination of IL-6 shows strong performance at various clinical thresholds, and can effectively support rapid inflammatory risk assessment in critical care.

[0047] Since the method of the present invention is not a destructive measurement (i.e. does not lead to destruction of the sample under investigation upon signal measurement), it is also advantageously possible to continue the measurement after the initial assessment of the biomarker concentration and to determine the exact concentration of the biomarker in the sample. On the basis the exact biomarker concentration determination, for example, the therapy measures initiated on the basis of the initial assessment can be further adapted or continued.

[0048] To the knowledge of the inventors, such early semi-quantitative, non-destructive assessment of the biomarker concentration in a sample has neither been disclosed nor suggested by studies and approaches of the prior art.

[0049] In embodiments, the biomarker and immunoassay are selected such that the signal obtained in the homogeneous assay represents an increase in signal over time. The increase in signal from said assay is, in embodiments, provided by the TRACE or FRET dynamics in the liquid sample. In embodiments, the kinetics of the signal generation and measurement may follow any given curve or function, assuming an increase in signal over time, such as, without limitation, a linear, logarithmic, sigmoid, exponential, power or double exponential curve or function (when referring to a simple XY graph over time as a curve or function). In preferred embodiments, the function may follow - speaking generally - a logarithmic function, increasing in signal over time, with continuingly smaller increases in signal over time.

[0050] In one embodiment, the signal measurement is carried out at a first wavelength and a second wavelength, preferably at a first wavelength of 650 to 720 nm and at a second wavelength of 600 to 630 nm, more preferably at a first wavelength of 660 to 710 nm and at a second wavelength of 610 to 620 nm. In one embodiment the signal measurement is carried out at a first wavelength and a second wavelength, preferably at a first wavelength of 650 to 720 nm, such as 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715 or 720 nm, and at a second wavelength of 610 to 620 nm, such as 610, 611 , 612, 613, 614, 615, 616, 617, 618, 619 or 620 nm.

[0051] In one embodiment, the signal of the sample and the signal of the calibrator is a ratio of the signal measured at the first and the second wavelength for the sample and the calibrator, respectively. In one embodiment, the biomarker is an acute care biomarker. In one embodiment the biomarker is an acute care biomarker selected from the group consisting of procalcitonin (PCT) or fragment(s) thereof, proadrenomedullin (proADM) or fragment(s) thereof, interleukin-6 (IL-6), copeptin, troponin, pro brain natriuretic peptide (proBNP) or fragment(s) thereof, pro atrial natriuretic peptide (proANP) or fragment(s) thereof, C-reactive protein (CRP), proEndothelin-1 (proET-1), soluble fms-like tyrosine kinase-1 (sFlt-1) of fragment(s) thereof, Placental Growth Factor (PIGF), lactate and D-Dimer,

[0052] In one embodiment, the biomarker is an acute care biomarker selected from the group consisting of procalcitonin (PCT) or fragment(s) thereof, proadrenomedullin (proADM) or fragment(s) thereof, interleukin-6 (IL-6), copeptin and troponin.

[0053] In one embodiment, the biomarker is selected from the group consisting of procalcitonin (PCT) or fragment(s) thereof, proadrenomedullin (proADM) or fragment(s) thereof, interleukin-6 (IL-6), copeptin and troponin.

[0054] In one embodiment, the biomarker is procalcitonin (PCT) or fragment(s) thereof.

[0055] In one embodiment, the biomarker is proadrenomedullin (proADM) or fragment(s) thereof, preferably mid-regional proadrenomedullin (MR-proADM).

[0056] In one embodiment, the biomarker is interleukin-6 (IL-6).

[0057] In one embodiment, the biomarker is copeptin.

[0058] In one embodiment, the biomarker is troponin.

[0059] In one embodiment, the biomarker is sFlt-1 .

[0060] In one embodiment, the signal measurement and reporting occurs within 4 minutes of initiating the immunoassay.

[0061] In one embodiment, the signal measurement and reporting occurs within 3 minutes of initiating the immunoassay, preferably at about 1 .5, 2 or 2.5 minutes after initiating the immunoassay.

[0062] In one embodiment, when the biomarker is copeptin (or fragment thereof), the signal measurement and reporting occurs within 10 minutes of initiating the immunoassay. In one embodiment, when the biomarker is copeptin (or fragment thereof), the signal measurement and reporting occurs within 8 minutes of initiating the immunoassay, preferably at about 9, 7, or 6 minutes after initiating the immunoassay.

[0063] In one embodiment, the clinical threshold value of any given biomarker is the 90th, 95th, or 99thpercentile of a healthy population. In one embodiment, the clinical threshold value of any given biomarker is the 90th, 95th, or 99thpercentile of a total population. Any other percentile value above 90% of a healthy population or total population may be employed, such as 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 99.5th, or 99.9thpercentile.

[0064] In one embodiment, the biomarker is PCT or fragment(s) thereof and the clinical threshold value is 2 ng / mL ±10%. In one embodiment, the biomarker is proADM or fragments(s) thereof and the clinical threshold value is 2.25 nmol / l + / - 10% or 1.54 nmol / l ±10% or 0.87 nmol / l ±10%.

[0065] In one embodiment, the biomarker is IL-6 and the clinical threshold value is 60 pg / ml ±10%.

[0066] In other embodiments, the biomarker is IL-6 and the clinical threshold value is 150 pg / ml ±10%.

[0067] In other embodiments, the biomarker is IL-6 and the clinical threshold value is 1000 pg / ml ±10%.

[0068] In one embodiment, the biomarker is copeptin and the clinical threshold value is 10 pmol / L ±10%.

[0069] In one embodiment, the biomarker is troponin and the clinical threshold value is 99th percentile of healthy individuals.

[0070] In one embodiment, the biomarker is PCT or fragment(s) thereof, the clinical threshold value is 2 ng / mL ±10%, and the signal threshold value is 0.3 ±10%, preferably for 2 minutes.

[0071] In one embodiment, the biomarker is proADM or fragments(s) thereof, the clinical threshold value is 1.54 nmol / l ±10% and the signal threshold value is 1 ±10%, preferably for 2 minutes.

[0072] In one embodiment, the biomarker is proADM or fragments(s) thereof, the clinical threshold value is 0.87 nmol / l ±10% and the signal threshold value is 0.93 ±10%, preferably for 2 minutes.

[0073] In one embodiment, the biomarker is IL-6, the clinical threshold value is 150 pg / mL ±10% and the signal threshold value is 0.52 ±10%, preferably for 3 minutes.

[0074] In one embodiment, the biomarker is IL-6, the clinical threshold value is 1000 pg / mL ±10% and the signal threshold value is 1.78 ±10%, preferably for 3 minutes.

[0075] In one embodiment, the biomarker is copeptin or fragments(s) thereof, the clinical threshold value is 10 pmol / l ±10% and the signal threshold value is 0.33 or 0.34 ±10%, preferably for 3 minutes.

[0076] The above signal threshold values are non-limiting examples of those that may be employed in the present invention. This representation of signal threshold values is based on a ratio (sample / calibrator). Other values may be used or adjusting depending on the necessary or desired true positive or true negative values of the assessment.

[0077] The rapid semi-quantitative assessment of the biomarker level in a sample by the method of the present invention is particularly advantageous for acute care biomarkers such as proADM, PCT, IL-6, sFlt-1 , troponin and copeptin indicating the risk of acute and severe medical conditions of a patient such as acute inflammation, sepsis, septic shock, or myocardial events such as acute myocardial infarction and commonly assessed in in acute clinical care settings such as the emergency department (ED) or the intensive care unit (ICU). By the method of the present invention these acute care biomarkers can advantageously be determined in a semi-quantitative manner within a few minutes. Such rapid assessment whether the concentration of these biomarkers in a sample is above a critical clinical threshold level indicating the presence of acute and severe medical conditions allows a first assessment of the clinical status and the underlying medical condition and suitable treatment means can immediately be initiated. Clinical threshold values may be employed for any biomarker. Literature is available to a skilled person for determining appropriate biomarkers and clinical threshold values, for example Philipp Schuetz et al. (BMC Medicine. 2011 ; 9:107) discloses cut-off values for PCT, Wereski et al. (Corculation, 2021 , 144, 7) discloses clinical threshold values for troponin to differentiate between myocardial injury and myocardial infarction and (Mockel et al.; Eur J Emerg Med 2013; 20: 103- 108) discloses clinical threshold values for troponin and copeptin.

[0078] Further, EP4133274 discloses clinical threshold values for proADM in patients with severe acute respiratory syndrome (SARS), EP3577465 discloses clinical threshold values for proADM and PCT for the prognosis of adverse events in in patients with sepsis and organ failure, EP3928099 discloses clinical threshold values for proADM and proET-1 for the diagnosis and prognosis of adverse events in patients that have undergone surgery and EP3682245 discloses clinical threshold values for proADM for proADM and PCT for therapy monitoring of critically ill patients such as patients with infectious diseases, organ failure, sepsis, severe sepsis, or septic shock and posttraumatic or postsurgical patients. EP3578989 discloses clinical threshold values for proADM and PCT in polytrauma patients, wherein for example a proADM level equal or above 1.54 nmol / l ± 20% indicates subsequent trauma-related complications. EP2320237 discloses clinical threshold values for PCT for diagnosis of a bacterial infection in patients who suffered from an acute stroke (ischemic or hemorrhagic) or from a transient ischemic attack.

[0079] Terence Chan et al. (Expert Rev.Mol.Diagn.2011 ; 11 (5), 487.496) described that indicators such as the positive and negative likelihood ratios, which are calculated based on sensitivity and specificity, are also useful for assessing the strength of a diagnostic test. Values are commonly graphed for multiple cut-off values (CVs) as a receiver operating characteristic curve. The area under the curve value is used to determine the best diagnostically relevant CV. This literature describes the variation of CVs (cut-off values, that is dependent on the assay and study design), and suitable methods for determining cut-off values.

[0080] In embodiments, both the signal threshold value, which indicates the biomarker concentration in said sample is above the clinical threshold value, and the relevant clinical threshold value, may be adjusted or determined as is necessary. Different clinical values may be employed, and the signal threshold value can be determined appropriately to correspond with the clinical threshold value.

[0081] In one embodiment, the clinical threshold value for PCT may be a value in the range of 0.01 to 100.00 ng / mL in a serum or plasma sample, when using for example a Luminex MAC Pix E- Bioscience Assay or the B R A H M S PCT KRYPTOR Assay. In a preferred embodiment the clinical threshold value of PCT may be in the range of 0.01 to 100, 0.05 to 50, 0.1 to 20, or 0.1 to 2 ng / ml, or 2 to 10 ng / ml. Any value within these ranges may be considered as an appropriate clinical threshold value. For example 0.1 , 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 ng / ml may be employed. In some embodiments, PCT levels for healthy subjects are approximately 0.05 ng / ml. In preferred embodiments, established PCT clinical thresholds may be employed, for example 0.1 , 0.25, 0.5, 1 or 2 ng / ml.

[0082] In one embodiment of the invention, a clinical threshold value for proADM or fragment(s) thereof, preferably MR-proADM- for example for any given one or more diagnostic, prognostic or therapy guidance statements - may be a value in the range of 0.4 to 50 nmol / l in a plasma sample. Preferably for MR-proADM any value of 0.7, 0.8, 0.87, 0.88, 0.9, 1.0, 1.10, 1.20, 1.30, 1.33, 1.40, 1.50, 1.58, 1.60, 1.70, 1.80, 1.90, 2.00, 2.10, 2.20, 2.25, 2.30, 2.40, 2.50, or 3, 4, 5, 6, 7, 8, 9, or 10 nmol / l, or any value within these values, or any value within a range formed by any two endpoints from this list, may be employed as clinical threshold value. Any clinical threshold value may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. This possible variation in cut-off applies to any given embodiment in the present disclosure for the relevant biomarker.

[0083] In embodiments, MR-proADM can be generally used as risk marker being connected to an organ dysfunction and correlating with a mortality risk or the prognosis of survival. Different clinical cutoffs are known (e.g. for rule out / in hospitalization of a subject (0.87 or 0.88 nmol / l), 2.25 nmol / l for admission of patients to the ICU; or 1 .5 nmol / l for de-escalation of acute care patients or for the risk prediction of a progression of a disease, etc.). These cut-offs are established with the KRYPTOR instrument and may vary in other instruments. Thus, any given clinical value may be used, and the required signal that indicates whether the sample level is above the clinical threshold, may be determined appropriately.

[0084] Preferably for copeptin any value 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 20, 25 or 40 pmol / l (pM), or any value within these values, or any value within a range formed by any two endpoints from this list, may be employed as clinical threshold value. Any threshold value may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. Measurements using these clinical threshold values are preferably applicable for a serum or plasma sample in a B R A H M S KRYPTOR Assay. This possible variation in cut-off applies to any given embodiment in the present disclosure for the relevant biomarker. In embodiments, copeptin is used for clinical rule- out diagnostics, in other words ruling out the likelihood of risk or presence of a disease or condition. For example, a clinical value of below 10 nmol / L may rule out risk of a relevant medical condition, like myocardial infarction.

[0085] Preferably for troponin, especially cardiac Troponin (cTnl or cTnT), any value 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 25, 30, 34, 35, 40, 45 or 50 ng / l, or any value within these values, or any value within a range formed by any two endpoints from this list, may be employed as clinical threshold value. Any threshold value may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. Measurements using these clinical threshold values are preferably applicable for a serum or plasma sample e.g. B R A H M S KRYPTOR Assay. This possible variation in cut-off applies to any given embodiment in the present disclosure for the relevant biomarker. In embodiments, troponin is used for clinical diagnostics, for example, a clinical value of above about 35 ng / l may be employed, with is about the 99thpercentile of a healthy population.

[0086] Preferably for sFlt-1 any value of 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, or 20000 pg / ml, or any value within these values, or any value within a range formed by any two endpoints from this list, may be employed as clinical threshold value. Any clinical threshold value may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. Measurements using these clinical threshold values are preferably applicable for a serum or plasma sample in a B R A H M S KRYPTOR Assay. This possible variation in cut-off applies to any given embodiment in the present disclosure for the relevant biomarker. In embodiments, the 95th percentile in normal pregnancies is from 2500 - 4000 pg / ml in the second trimester, up to 8500 pg / ml in the 3rd trimester up to delivery (KRYPTOR data). Various values may be used as an sFItl cut-off for preeclampsia, as the marker levels change over time. In preferred embodiments, the precise cut-off is determined based on the 90th, 95thor 99th percentile of healthy pregnant woman.

[0087] Preferably for CT-proET-1 any value of 40, 45, 50, 55, 60, 65, 68, 70, 75, 80, 83, 83.6, 84, 85, 87, 87.4, 90, 91 , 91 .8, 95, 100, 102, 102.7, 105, 110, 115 or 120 pmol / l, or any value within these values, or any value within a range formed by any two endpoints from this list, may be employed as clinical threshold value. Any threshold value may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. Measurements using these clinical threshold values are preferably applicable for a serum or plasma sample in a B R A H M S KRYPTOR Assay. This possible variation in cut-off applies to any given embodiment in the present disclosure for the relevant biomarker.

[0088] Preferably for IL-6, any value of 1 , 2, 3, 4, 5, 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1300, 1300, 1400, 1500, 2000, 3000, 4000, 5000 or 10000 pg / ml, or any value within these values, or any value within a range formed by any two endpoints from this list, may be employed as clinical threshold value. Any threshold value may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. Measurements using these clinical threshold values are preferably applicable for a serum or plasma sample in a B R A H M S KRYPTOR Assay. This possible variation in cut-off applies to any given embodiment in the present disclosure for the relevant biomarker. In embodiments, the level of IL-6 in healthy people is low, generally not exceeding 7 pg / mL, but increases in the serum or plasma of patients with an infection, such as sepsis. Higher levels of IL-6 are associated with sepsis diagnosis, severity of organ dysfunction, and mortality. In embodiments, IL-6 levels in inflammations are typically >5 pg / ml, in severe infections like sepsis are between >40-100, for example 50 to 150 pg / ml. In embodiments septic shock patients have IL-6 levels above 100, 200, 300 or 1000 or 5000 pg / ml.

[0089] It is further to be understood that in embodiments of the invention, deviations from the disclosed possible threshold values below are also disclosed and claimed, such as deviations of ± 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% as well as the exact threshold value.

[0090] In combination with any one or more of the clinical threshold values above, a signal threshold value may be employed. In preferred embodiments, a signal threshold value may be employed that is compatible with the clinical threshold value. Examples of preferred combinations between clinical threshold values and signal threshold values are disclosed in the example below.

[0091] In embodiments, for PCT, any value of 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, or any value within these values, or any value within a range formed by any two endpoints from this list, may be employed as signal threshold value. Any signal threshold value may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. Measurements using these signal threshold values are preferably applicable for a serum sample in a B R A H M S KRYPTOR Assay. These values preferably enable analysis within 2 minutes. For PCT, preferred values are a clinical threshold value of 0.5, 1 .0, 1 .5, 2.0, 2.5, 3.0 or 3.5 ng / ml, combined with one or more of a signal threshold value of 0.2, 0.3, 0.33, 0.4, or 0.5. Preferred combinations for PCT relate to a signal threshold value of 0.3 or 0.33 with a clinical threshold of 2 ng / ml. These values preferably enable analysis within 2 minutes.

[0092] In embodiments, for MR-proADM, any value of 0.2, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1 .3, 1 .4, 1 .5, 2.0, 2.5 or 3.0, or any value within these values, or any value within a range formed by any two endpoints from this list, may be employed as signal threshold value. Any signal threshold value may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. Measurements using these signal threshold values are preferably applicable for a serum or plasma sample in a B R A H M S KRYPTOR Assay. These values preferably enable analysis within 2 minutes.

[0093] For MR-proADM, preferred values are a clinical threshold value of 0.5, 0.6, 0.7, 0.8, 0.87, 0.88, 0.9, 1.0, 1.10, 1.20, 1.30, 1.33, 1.40, 1.50, 1.54, 1.60, 1.70, 1.80, 1.90, 2.00, 2.25 nmol / l, combined with one or more of a signal threshold value of 0.7, 0.8, 0.9, 0.93, 1 .0, 1 .055, 1.1 , 1.2, 1 .3, 1 .4, or 1 .5. Preferred combinations for MR-proADM relate to a signal threshold value of 0.93 with a clinical threshold of 0.87 or 0.88 nmol / l, or a signal threshold value of 1 .0 with a clinical threshold of 1 .54 nmol / L, or a signal threshold value of 1 .055 with a clinical threshold of 1 .54 nmol / L. These values preferably enable analysis within 2 minutes.

[0094] In embodiments, for copeptin, any value of 0.1 , 0.2, 0.3, 0.33, 0.34, 0.35, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 .0, 1 .5, 2.0, 2.5, 3, 4, or 5, or any value within these values, or any value within a range formed by any two endpoints from this list, may be employed as signal threshold value. Any signal threshold value may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. Measurements using these signal threshold values are preferably applicable for a serum or plasma sample in a B R A H M S KRYPTOR Assay. These values preferably enable analysis within 3 minutes.

[0095] For copeptin, preferred values are a clinical threshold value of 1 , 5, 10, 15 or 20 pmol / l, combined with one or more of a signal threshold value of 0.1 , 0.2, 0.3, 0.33, 0.34, 0.35, 0.353, 0.4, 0.5. Preferred combinations for copeptin relate to a signal threshold value of 0.33 or 0.34 with a clinical threshold of 10 pmol / l.

[0096] In embodiments, for IL-6, any value of 0.1 , 0.2, 0.3, 0.4, 0.5, 0.52, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 , 1.2, 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .78, 1 .8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 3, 4, or 5, or any value within these values, or any value within a range formed by any two endpoints from this list, may be employed as signal threshold value. Any signal threshold value may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value. Measurements using these signal threshold values are preferably applicable for a serum or plasma sample in a B R A H M S KRYPTOR Assay. These values preferably enable analysis within 3 minutes.

[0097] For IL-6, preferred values are a clinical threshold value of 100, 150, 200, pg / mL, combined with one or more of a signal threshold value of 0.4, 0.5, 0.52, 0.6. For IL-6, preferred values are a clinical threshold value of 500, 1000, or 1500 pg / mL, combined with one or more of a signal threshold value of 1.5, 1.75, 1.78, 1.80 or 2.0. Preferred combinations for IL-6 relate to a signal threshold value of 0.52 with a clinical threshold of 150 pg / mL, or a signal threshold value of 1.78 with a clinical threshold of 1000 pg / mL.

[0098] Regarding the specific values above, any value from between these values, or any value within a range formed by any two points from this list, which may be designated as endpoints to a range, may be employed. Regarding the specific values above, any value may also employ variation by ± 1 , 2, 3, 4, 5, 10, 15, 20, 25, or 30%, from the specific value.

[0099] As shown in the examples below, when the ratio of the signal of the sample to the signal of the calibrator (signal ratio sample / calibrator) is above a signal threshold value, the measurement indicates that the sample concentration is above a clinical threshold value. The signal threshold value is therefore a threshold value, to which the ratio (sample / calibrator signal ratio) is compared. The signal threshold values may be used independent of the specific clinical threshold value, or the signal threshold value and clinical threshold value may be determined, set up, or measured in accordance with each other, for example to obtain a particular diagnostic or prognostic statement with a given statistical certainty.

[0100] In embodiments, the present invention enables a “rule-out” of a clinical condition. For example, if the level of biomarker is estimated to be below a particular threshold, this value may indicate the absence of the biomarker being above a clinical threshold, and thus the absence of the clinical condition. Any embodiment described herein, such as the threshold values disclosed, may also be employed to rule out the risk of a medical condition, for example when the signal indicates the biomarker is not above a clinical threshold value, this information may be used to infer a low likelihood or low risk or absence of a medical condition. Therefore, if the marker is NOT above the clinical threshold, there would be NO flag or signal reporting, or a report that the signal is below the threshold, and the risk is therefore low that the final concentration will be above the critical clinical cut-off.

[0101] In embodiments, the method may defined such that when a ratio of the signal of the sample and the signal of the calibrator (signal ratio sample / calibrator) is below a signal threshold value, reporting occurs that said signal ratio is below said signal threshold value within 5 minutes of initiating said immunoassay, wherein a signal ratio below said signal threshold value indicates the biomarker concentration in said sample is below the clinical threshold value.

[0102] In embodiments, the reporting that said signal ratio (sample / calibrator) is above or below a signal threshold value occurs by one or more various means, in essence indicating to a user or to an automated software, the result of the signal ratio. Any given means may be used, for example, an acoustic signal, a visual signal, lights or other such alerts, a flagging in the system, a pop-up window on a user interface of a computing device or measurement device, a report card, or printout, or any other communication means, either electronic or analogue.

[0103] In one embodiment, the method comprises a diagnosis, prognosis and / or risk stratification of a medical condition in a subject.

[0104] In one embodiment, the method comprises a diagnosis, prognosis and / or risk stratification of a medical condition in a subject, selected from, without limitation, an infectious disease, such as sepsis, severe sepsis or septic shock, prenatal complications like preeclampsia, the HELLP syndrome or preterm birth, blood vessel embolism such as pulmonary embolism, stroke, or myocardial infarction and / or an acute medical condition such as an acute cardiac condition or other conditions related to acute life-threatening conditions, such as organ dysfunctions or acute inflammation.

[0105] In one embodiment, the medical condition is an infectious disease, such as sepsis, severe sepsis or septic shock, and / or an acute medical condition such as an acute cardiac condition and / or an acute inflammation.

[0106] In one embodiment, the acute cardiac condition is an acute myocardial infarction, acute coronary syndrome, acute heart failure, unstable angina, cardiogenic shock, acute pericarditis or acute myocarditis, preferably acute myocardial infarction.

[0107] In one embodiment, the medical condition is a condition requiring frequent monitoring, hospitalization and / or critical care treatment.

[0108] In one embodiment, the medical condition is a condition requiring admission to the intensive care unit (ICU).

[0109] In one embodiment, the donor label comprises a rare earth cryptate or chelate, preferably comprising a lanthanide ion, and the acceptor label comprises a fluorescent or chemiluminescent dye.

[0110] Lanthanide luminescence is established in the art and offers several advantages for fluorescence-based biological assays, including large Stoke’s shifts (>150 nm) and multiple, narrow emission bands (typically <10 nm at half-maximum) and thus allows efficient spectral separation of emission signals. Lanthanide luminescence is also typically characterized by long luminescence lifetimes (micro- to millisecond) enabling time-resolved detection methods. Lanthanide ions (particularly Terbium and Europium) offer significant advantages over organic fluorescent dyes. Compared with traditional organic fluorescent materials, the lanthanide coordination compounds have a relatively long life-time, a large Stokes shift, and sharp fluorescent peak profiles. Due to these attractive properties, time-resolved reagents based on lanthanide fluorescence are used.

[0111] Terbium and europium probes typically incorporate the metal ion into an organic chelating ligand that contains a sensitizing chromophore. When excited with near-UV light in the absorption band, the chromophore transfers energy via intersystem crossing to the triplet excited state and intramolecular transfer to the emissive level of the chelated metal. Direct conjugation of lanthanide probes to antibodies thus enables the development of sensitive, time-resolved fluorescence resonance energy transfer (TR-FRET) or time-resolved amplified cryptate emission (TRACE) assays in various practical applications. The B R A H M S MR-proADM KRYPTOR system employs such TRACE technology, which is also employed in preferred embodiments of the present invention.

[0112] In one embodiment the donor label comprises a terbium cryptate or chelate and the acceptor label comprises a fluorescent dye with excitation and emission spectra compatible with the terbium excitation and emission spectra (in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplified cryptate emission (TRACE) reaction).

[0113] As used herein, the term “compatible with the terbium excitation and emission spectra”, or “compatible with the terbium excitation and emission spectra in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplified cryptate emission (TRACE) reaction”, refers to the election of suitable fluorophores with complementary (compatible) excitation and emission spectra that are receptive to effective energy transfer and subsequent detection in a time-resolved method (FRET or TRACE). By way of example, a terbium donor label is employed, and the acceptor label is selected to have a matching excitation spectrum to be excited by the donor upon binding, and an emission spectrum that can be detected at a suitable wavelength.

[0114] By way of example, the present method is based on and represents a further development of an older method using radiative energy transfer between a donor, the long-lived fluorophore Europium Cryptate, and an acceptor, such as a fluorophore emitting in the red wavelength, such as the Cyanine 5 (Cy5). The energy transfer depends on the proximity and the overlap of spectrum of the two fluorophores. In a sandwich immunoassay, the Europium and acceptor are bound to specific antibodies. In the older assay, the Europium is excited at 337 nm by a nitrogen laser and the energy is transferred to the Cy5 which reemits specifically at 665nm. This homogeneous assay uses time-resolved measurements and spectral selection to eliminate the background signal of the media and to select the specific signal.

[0115] In one embodiment, the donor label comprises a terbium cryptate or chelate.

[0116] In embodiments, a donor label may comprise terbium, terbium-derivatives, or a terbium N- hydroxy succinimide ester, for example CesHysN^OnTb.

[0117] In one embodiment, the donor label comprises a terbium cryptate or chelate with emission spectra at 485-495, 540-550, 585-595 and / or 615-625 nm, and the acceptor label comprises a fluorescent dye with a excitation spectrum corresponding to the donor label emission spectrum, and an emission spectrum distinct from the donor label emission spectra, preferably in a wavelength range of 500-570, preferably 510-560, or 515-555 nm (green), or 580-770, preferably 600-780, or 670-690 nm (red).

[0118] Suitable acceptor fluorophores may be elected based on compatible excitation and emission spectra, and are disclosed in more detail below.

[0119] In preferred embodiments, the method comprises a fully automated homogeneous assay, preferably a homogeneous sandwich fluoroimmunoassay, employing a Time Resolved Amplified Cryptate Emission (TRACE) technology with a non-radiative energy transfer between a donor and an acceptor, wherein the energy transfer depends on the proximity and the overlap of spectrum of the two fluorophores.

[0120] In one embodiment, of the method described herein, the method additionally comprises comparing the determined signal ratio to a signal threshold value, wherein said comparing is carried out in a computer processor using computer executable code. The methods of the present invention may in part be computer-implemented. For example, the step of comparing the signal ratio with a signal threshold value and the calculation of the signal threshold value can be performed in a computer system. In the computer-system, the determined signal ratio, the signal threshold value and the clinical threshold value can be combined with one or more additional biomarkers, other marker levels and / or parameters of the subject in order to calculate a score, which is indicative for the diagnosis, prognosis, prediction, risk assessment and / or risk stratification. For example, the determined values may be entered (either manually by a health professional or automatically from the device(s) in which the respective marker level(s) has / have been determined) into the computer-system. The computer-system can be directly at the point-of-care (e.g. primary care, ICU or ED) or it can be at a remote location connected via a computer network (e.g. via the internet, or specialized medical cloud-systems, optionally combinable with other IT-systems or platforms such as hospital information systems (HIS)).

[0121] Typically, the computer-system will store the values (e.g. marker level or parameters such as age, blood pressure, weight, sex, temperature, etc. or clinical scoring systems such as SOFA, qSOFA, BMI etc.), standard lab parameter like lactate, CRP, blood cell counts or shape, the clinical history, co-morbidities and primary disease, or symptoms of a patient on a computer-readable medium and calculate the score based-on pre-defined and / or pre-stored reference levels or reference values. The resulting score will be displayed and / or printed for the user (typically a health professional such as a physician). Alternatively or in addition, the associated prognosis, diagnosis, assessment, treatment guidance, patient management guidance or stratification will be displayed and / or printed for the user (typically a health professional such as a physician).

[0122] In embodiments, the computer-implementation may additionally comprise one or more machine learning or artificial intelligence software approaches, for example, by considering additional patient characteristics, including, without limitation, marker levels, parameters such as age, blood pressure, weight, sex, temperature, etc. or clinical scoring systems such as SOFA, qSOFA, BMI, or other lab parameters like lactate, CRP, blood cell counts or shape, clinical history, comorbidities and primary disease, and - having been preferably trained on such factors - including additional patient data in the determination of relevant clinical thresholds. Thus, determining a relevant clinical threshold may employ a known or established threshold, or a relevant threshold determined in light of additional patient data. Design and employment of machine learning or artificial intelligence software approaches based on suitable training sets are known to a skilled person.

[0123] In one aspect the invention relates to a system for assessing whether a biomarker concentration in a sample is above a clinical threshold value, wherein said system is configured for:

[0124] Initiating an automated homogeneous immunoassay in a liquid mixture in said sample and a calibrator comprising a known concentration of said biomarker, said immunoassay preferably comprising time-resolved fluorescence resonance energy transfer (TR-FRET), with non-radiative energy transfer between a donor label and an acceptor label coupled to first and second binding agents, respectively, that bind to said biomarker, wherein proximity of the donor and the acceptor label in complex with said biomarker produces a signal, wherein the system is configured to carry out a signal measurement in said sample and said calibrator within 5 minutes of initiating said immunoassay, and when a ratio of the signal of the sample and the signal of the calibrator (signal ratio sample / calibrator) is above a signal threshold value, the system reports that said signal ratio is above said signal threshold value within 5 minutes of initiating said immunoassay, wherein a signal ratio above said signal threshold value indicates the biomarker concentration is above the clinical threshold value.

[0125] The clinical threshold values and / or signal threshold values can be provided on a computer executable code configured for comparing the determined signal ratios with said signal threshold values.

[0126] In one embodiment, the system comprises a user interface for reporting that said signal ratio is above said signal threshold value.

[0127] In one aspect or embodiment, the invention relates to a method for determining a signal threshold value of the invention, for use in the method disclosed herein, said method comprising: providing a sample from a subject having a known (or pre-determined) biomarker concentration, wherein said known concentration corresponds to (or preferably is) a clinical threshold value (±10%), providing a calibrator comprising a known concentration of said biomarker, initiating an automated homogeneous immunoassay in a liquid mixture in said sample and said calibrator, preferably comprising time-resolved fluorescence resonance energy transfer (TR-FRET) and / or a time-resolved amplified cryptate emission (TRACE) reaction, with non-radiative energy transfer between a donor label and an acceptor label coupled to first and second binding agents, respectively, that bind to said biomarker, wherein proximity of the donor and the acceptor label in complex with said biomarker produces a signal, wherein a signal measurement is carried out in said sample and said calibrator within 5 minutes of initiating said immunoassay, and the signal threshold value is determined by calculating a ratio of the signal of the sample and the signal of the calibrator (signal ratio sample / calibrator).

[0128] In embodiments, the signal threshold value is the ratio of (a) the signal of the sample to (b) the signal of the calibrator (signal ratio sample / calibrator). By employing samples with a known (predetermined) biomarker concentration, the signal at an early time point (i.e. the inventive early time point described at length herein) can be determined and compared to the calibrator, thus establishing a ratio between the signal of the sample and the signal of the calibrator, wherein said ratio is employed as the signal threshold value.

[0129] In embodiments, a signal threshold value can be determined using the above approach for any given clinical threshold and / or for any given biomarker. Thus, the method enables analysis of a biomarker without finally determining the biomarker concentration, rather a signal threshold value (signal ratio sample / calibrator) can be employed as a “proxy” of a clinical threshold value, to semi-quantitatively assess (or estimate) whether a biomarker level at an early time point is above a clinical threshold value. In embodiments, the method comprises providing samples from two or more subjects having a known (predetermined) biomarker concentration in said samples corresponding to (or of) a clinical threshold value (±10%). In preferred embodiments, the signal threshold value is determined from multiple patient samples, thereby providing a higher accuracy to said signal threshold value or stronger correlation between the signal threshold value and clinical threshold value. When multiple patient samples are employed, an average of the signals may be used to determine the signal threshold value. In embodiments, multiple patient samples may be employed with known concentrations, for example from different studies, different locations, different instruments etc., to increase accuracy. In the examples provided for PCT, more than 1000 samples were used to determine a threshold with a beneficial outcome (target 100% true positive and / or 100% true negative).

[0130] All features described in the present specification may be employed to define any other embodiment or aspect of the invention. For example, features used to describe the in vitro method for assessing whether a biomarker concentration is above a clinical threshold value may be used to describe the system and the method for determining a signal threshold value, and vice versa. Despite covering various embodiments or aspects, these various means of the invention are preferably unified by their unique and related ability to effectively and accurately determining within 5 minutes whether a biomarker concentration is above a clinical threshold value as described herein.

[0131] DETAILED DESCRIPTION

[0132] The invention relates broadly to a method for determining whether a biomarker concentration in a sample is above a clinical threshold within 5 minutes. The method comprises multiple steps, each of which may be used to define the invention, without necessary limitation to all other method steps disclosed herein. The method comprises initiating an automated homogeneous immunoassay in a liquid mixture, preferably comprising time-resolved fluorescence resonance energy transfer (TR-FRET) and / or a time-resolved amplified cryptate emission (TRACE) reaction. A skilled person is aware of such homogeneous immunoassays. Suitable non-limiting examples are disclosed herein.

[0133] General terms:

[0134] As used herein, the terms “comprising” and “including” or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof. This term encompasses the terms “consisting of’ and “consisting essentially of’.

[0135] Thus, the terms “comprising” / “including” / ”having” mean that any further component (or likewise features, integers, steps and the like) can / may be present. The term “consisting of’ means that no further component (or likewise features, integers, steps and the like) is present.

[0136] The term “consisting essentially of’ or grammatical variants thereof when used herein are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof but only if the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, device or method.

[0137] Thus, the term “consisting essentially of’ means those specific further components (or likewise features, integers, steps and the like) can be present, namely those not materially affecting the essential characteristics of the composition, device or method. In other words, the term "consisting essentially of' (which can be interchangeably used herein with the term "comprising substantially"), allows the presence of other components in the composition, device or method in addition to the mandatory components (or likewise features, integers, steps and the like), provided that the essential characteristics of the device or method are not materially affected by the presence of other components.

[0138] The term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, biological and biophysical arts. As used herein, the “patient” or "subject" may be a vertebrate. In the context of the present invention, the term "subject" includes both humans and animals, particularly mammals, and other organisms.

[0139] Assays and time-resolved energy transfer:

[0140] In embodiments, the binding agents may be conjugated to one or more labels, such as fluorescent labels, preferably two separate fluorescent labels suitable for application in immunoassays and their related instruments. In embodiments, the B R A H M S KRYPTOR based assays are based on the TRACE technology, which is a non-radiative energy transfer from a donor [a cage-like structure with a europium ion in the center (cryptate)] to an acceptor. The proximity of donor (cryptate) and acceptor in a formed immunocomplex and the spectral overlap between donor emission and acceptor absorption spectra on the one hand intensifies the fluorescent signal and on the other hand extends the life span of the acceptor signal, allowing for the measurement of temporally delayed fluorescence.

[0141] After the sample to be measured has been excited with a nitrogen laser at a suitable wavelength, the donor (cryptate) emits a long-life fluorescent signal in the millisecond range, while the acceptor generates a short-life signal in the range of nanoseconds. When both components are bound in an immunocomplex, both the signal amplification and the prolonged life span of the acceptor signal occurs, and the life is in the microsecond range. This delayed acceptor signal is proportional to the concentration of the analyte to be measured. The specific fluorescence which is proportional to the antigen concentration is obtained through a double selection: spectral (separation depending on wavelength) and temporal (time resolved measurement). This enables an exclusive measurement of the signal emitted by the immunological complex and the ratio between the two wavelengths allows a real-time correction of the variations in optic transmission from the medium.

[0142] In embodiments, the assay is homogenous and does not require separation or washing steps. It is thus possible to obtain data without interrupting the immunological reaction.

[0143] In one embodiment the method is a homogeneous method, wherein the sandwich complexes formed by the binding agents and the biomarker, which is to be detected remains suspended in the liquid phase. In this case it is preferred, that when two binding agents are used, both binding agents are labelled with parts of a detection system, which leads to generation of a signal or triggering of a signal if both binding agents are integrated into a single sandwich with the biomarker.

[0144] Such techniques are to be embodied in particular as fluorescence enhancing or fluorescence quenching detection methods. A particularly preferred aspect relates to the use of detection reagents which are to be used pairwise, such as for example the ones which are described in US 4 882 733 A, EP-B1 0 180 492 or EP-B1 0 539 477 and the prior art cited therein. In this way, measurements in which only reaction products comprising both labelling components in a single immune-complex directly in the reaction mixture are detected, become possible.

[0145] For example, such technologies are offered under the brand names TRACE™ (Time Resolved Amplified Cryptate Emission) or KRYPTOR™, implementing the teachings of the above-cited applications. Therefore, in particular preferred aspects, a diagnostic device is used to carry out the herein provided method. For example, the level of a biomarker such as PCT, and / or the level of any further marker of the herein provided method are determined. In particular preferred aspects, the diagnostic device is a KRYPTOR instrument.

[0146] In one embodiment of the method described herein the method is an immunoassay and wherein the assay is performed in homogeneous phase. In one embodiment of the method described herein, a first binding agent and a second binding agent are present dispersed in a liquid reaction mixture, and wherein a first labelling component which is part of a labelling system based on fluorescence or chemiluminescence extinction or amplification is bound to the first binding agent, and a second labelling component of said labelling system is bound to the second binding agent so that, after binding of both antibodies to said biomarker to be detected, a measurable signal which permits detection of the resulting sandwich complexes in the measuring solution is generated.

[0147] In one embodiment of the method described herein the labelling system comprises a rare earth cryptate or chelate in combination with a fluorescent or chemiluminescent dye, in particular of the cyanine type.

[0148] By way of example, two types of cages have been developed to complex lanthanides and enable the labeling of the receptor of interest: (i) chelates display high affinity for europium and terbium ions but the complexation is reversible and can be impacted by the presence of other ions such as Mn2+ , Mg2+ , or Ca2+ ; (ii) cryptates, by contrast, offer a greater stability since terbium and europium cannot be released after complexation.

[0149] An example of structure of cryptate, Terbium cryptate, is illustrated below:

[0150]

[0151] Upon excitation, lanthanide fluorescence half time is in the range of 1 ms while it is in the range of few nanoseconds for classic fluorophores. TR-FRET and TRACE take advantage of this property: the introduction of a time delay (typically around 50 ps) between the excitation and the fluorescence signal detection allows discriminating between short-lived and longer-lasting fluorescence. Therefore, all short-lived fluorescence provided by the medium, the biological preparation or the direct excitation of the acceptor will be eliminated by the time delay. Only the long-lived fluorescence resulting from the donor or the acceptor engaged in a FRET process will be measured after the time delay. Both europium and terbium cryptates are excited at 300-350 nm. They both exhibit an important Stoke shift and complex emission spectra with multiple fluorescent peaks. For example, europium cryptate trisbipyridine [TBP(Eu)] exhibits four major fluorescent peaks at 585, 605, 620, and 700 nm, while the europium pyridine bisbipyridine (Eu-PBP) has two major peaks at 595 and 615 nm, and two minor peaks at 680 and 705 nm. Terbium cryptate also displays four emission peaks around 490, 550, 585, and 620 nm (Figure 2C). This makes europium and terbium cryptates compatible with deep red Cy5-or dy647-like fluorophores. Moreover because of the emission peak around 490 nm, terbium-cryptate is also compatible with fluorescein-like fluorophore as acceptor.

[0152] Non-limiting examples of acceptor fluorophores are (derived from www.abcam.com / technical):

[0153] Immunoassays:

[0154] An immunoassay is a biochemical test that measures the presence or concentration of a macromolecule / polypeptide in a solution through the use of a binding agent such as an antibody or antibody binding fragment or immunoglobulin. Exemplary immunoassays can be luminescence immunoassay (LIA), radioimmunoassay (RIA), chemiluminescence- and fluorescenceimmunoassays, fluorescence polarization (FPIA), enzyme immunoassay (EIA), Enzyme-linked immunoassays (ELISA), luminescence-based bead arrays, magnetic beads-based arrays, protein microarray assays, rapid test formats, rare cryptate assay. Further, assays suitable for point-of- care testing and rapid test formats such as for instance immune-chromatographic strip tests can be employed. Automated immunoassays are also intended, such as the B R A H M S KRYPTOR™ instrument related immunoassays. In embodiment the immunoassay is a homogeneous immunoassay in a liquid mixture. In embodiments, the immunoassay is not a lateral flow assay, or other immunoassay with immobilized detection reagents.

[0155] In certain aspects of the invention, the method is an immunoassay comprising the steps of: a) contacting the sample with i. a first binding agent specific for a first epitope of said biomarker, and ii. a second binding agent specific for a second epitope of said biomarker; and b) detecting the binding of the two binding agents to said biomarker.

[0156] In embodiments, the first binding agent and the second binding agent can be present dispersed in a liquid reaction mixture, and wherein a first labeling component which is part of a labeling system based on fluorescence or chemiluminescence extinction or amplification is bound to the first binding agent, and a second labeling component of said labeling system is bound to the second binding agent so that, after binding of both binding agents to said biomarker or fragments thereof to be detected, a measurable signal which permits detection of the resulting sandwich complexes in the measuring solution is generated. The labeling system can comprise a rare earth cryptate or chelate in combination with a fluorescent or chemiluminescent dye, in particular of the cyanine type.

[0157] As used herein, the term “binding agent” “detection reagent”, “binder” or the like include any reagents that are suitable to determine the herein described marker(s). Such exemplary detection reagents are, for example, ligands, e.g. antibodies or fragments thereof, which specifically bind to the peptide or epitopes of the herein described biomarker(s). Such ligands might be used in immunoassays as described herein.

[0158] Determination of the one or more biomarkers based on antibody recognition is a preferred embodiment of the invention. As used herein, the term, "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immuno reacts with) an antigen. According to the invention, the antibodies may be monoclonal as well as polyclonal antibodies.

[0159] An antibody is considered to be specific, if its affinity towards the molecule of interest, is at least 50-fold higher, preferably 100-fold higher, most preferably at least 1000-fold higher than towards other molecules comprised in a sample containing the molecule of interest. It is well known in the art how to develop and to select antibodies with a given specificity. In the context of the invention, monoclonal antibodies are preferred. The antibody or the antibody binding fragment binds specifically to the herein defined markers or fragments thereof. In particular, the antibody or the antibody binding fragment binds to the herein defined biomarkers.

[0160] Alternatively, instead of antibodies, other binding agents that specifically and / or selectively recognize the one or more biomarkers of the present invention may be encompassed by the scope of the present invention. Herein, the term “binding agent”, “capture molecules” or “molecular scaffolds” comprises molecules which may be used to bind target molecules or molecules of interest, i.e. biomarkers from a sample. Capture molecules must thus be shaped adequately, both spatially and in terms of surface features, such as surface charge, hydrophobicity, hydrophilicity, presence or absence of lewis donors and / or acceptors, to specifically bind the target molecules or molecules of interest. Hereby, the binding may, for instance, be mediated by ionic, van-der-Waals, pi-pi, sigma-pi, hydrophobic or hydrogen bond interactions or a combination of two or more of the aforementioned interactions or covalent interactions between the capture molecules or molecular scaffold and the target molecules or molecules of interest. In the context of the present invention, capture molecules or molecular scaffolds may for instance be selected from the group consisting of a nucleic acid molecule, a carbohydrate molecule, a PNA molecule, a protein, a peptide and a glycoprotein. Capture molecules or molecular scaffolds include, for example, aptamers, DARpins (Designed Ankyrin Repeat Proteins). Affimers and the like are included.

[0161] Diagnosis, prognosis, and therapy monitoring:

[0162] In embodiments, the method of the present invention comprises a diagnosis, prognosis and / or risk stratification of a medical condition in a subject.

[0163] As used herein, “diagnosis” in the context of the present invention relates to the recognition and (early) detection of a clinical condition. Also, the assessment of the severity may be encompassed by the term “diagnosis”. In embodiments, the present invention enables an indication, or likelihood of a diagnosis, by enabling an alert, warning, or signal, should the measured signal indicate that the sample value is above a clinical threshold value. Thus, diagnosis may also refer to indication of a likely diagnosis, based on the early measurement of the present invention.

[0164] “Prognosis” relates to the prediction of an outcome or a specific risk for a subject. This may also include an estimation of the chance of recovery or the chance of an adverse outcome for said subject. In embodiments, the present invention enables an indication, or likelihood of a prognosis, by enabling an alert, warning, or signal, should the measured signal indicate that the sample value is above a clinical threshold value. Thus, diagnosis may also refer to indication of a likely prognosis, based on the early measurement of the present invention.

[0165] The methods of the invention may also be used for monitoring, therapy monitoring, therapy guidance and / or therapy control. “Monitoring” relates to keeping track of a patient and potentially occurring complications, e.g. to analyze the progression of the healing process or the influence of a particular treatment or therapy on the health state of the patient.

[0166] The term “therapy monitoring” or “therapy control” in the context of the present invention refers to the monitoring and / or adjustment of a therapeutic treatment of said patient, for example by obtaining feedback on the efficacy of the therapy. As used herein, the term “therapy guidance” refers to application of certain therapies, therapeutic actions or medical interventions based on the value / level of one or more biomarkers and / or clinical parameter and / or clinical scores. This includes the adjustment of a therapy or the discontinuation of a therapy. As described at length herein, the present invention enables an early measurement of biomarker levels, indicating, or providing a likelihood, of a certain medical condition, also including potentially a suggested treatment. Thus, the early measurement of the invention enables improved therapy guidance compared to longer conventional methods.

[0167] In the present invention, the terms “risk assessment” and “risk stratification” relate to the grouping of subjects into different risk groups according to their further prognosis. Risk assessment also relates to stratification for applying preventive and / or therapeutic measures. The term “therapy stratification” in particular relates to grouping or classifying patients into different groups, such as risk groups or therapy groups that receive certain differential therapeutic measures depending on their classification. The term “therapy stratification” also relates to grouping or classifying patients with infections or having symptoms of an infectious disease into a group that are not in need to receive certain therapeutic measures. As described herein, such risk assessment or stratification relates in essence to a prognosis, and the present invention enables an early indication or assessment of prognosis, as described above.

[0168] As used herein, the term “sample” is a biological sample that is obtained or isolated from the patient or subject. “Sample” as used herein may, e.g., refer to a sample of bodily fluid or tissue obtained for the purpose of analysis, diagnosis, prognosis, or evaluation of a subject of interest, such as a patient. Preferably herein, the sample is a sample of a bodily fluid, such as blood, serum, plasma, capillary blood, venous blood, interstitial fluid (ISF), cerebrospinal fluid (CSF), urine, saliva, sputum, pleural effusions, cells, a cellular extract, a tissue sample, any tissue sample from the upper or lower respiratory tract, a tissue biopsy, a stool sample and the like. Particularly, the sample is blood, blood plasma, blood serum.

[0169] “Plasma” in the context of the present invention is the virtually cell-free supernatant of blood containing anticoagulant obtained after centrifugation. Exemplary anticoagulants include calcium ion binding compounds such as EDTA or citrate and thrombin inhibitors such as heparinates or hirudin. Cell-free plasma can be obtained by centrifugation of the anticoagulated blood (e.g. citrated, EDTA or heparinized blood), for example for at least 15 minutes at 2000 to 3000 g. “Serum” in the context of the present invention is the liquid fraction of whole blood that is collected after the blood is allowed to clot. When coagulated blood (clotted blood) is centrifuged serum can be obtained as supernatant.

[0170] As used herein, “urine” is a liquid product of the body secreted by the kidneys through a process called urination (or micturition) and excreted through the urethra.

[0171] Biomarkers:

[0172] As used herein, terms such as “marker”, “surrogate”, “prognostic marker”, “factor” or “biomarker” or “biological marker” are used interchangeably and relate to measurable and quantifiable biological markers (e.g., specific protein or enzyme concentration or a fragment thereof, specific hormone concentration or a fragment thereof, or presence of biological substances or a fragment thereof) which serve as indices for health- and physiology-related assessments, such as a disease / disorder / clinical condition risk, preferably an adverse event. A marker or biomarker is defined as a characteristic that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Biomarkers may be measured in a sample (as a blood, plasma, urine, or tissue test).

[0173] As used herein, “procalcitonin” or “PCT” relates to a peptide spanning amino acid residues 1-116, 2-116, 3-116, or fragments thereof, of the procalcitonin peptide. PCT is a peptide precursor of the hormone calcitonin. Thus, the length of procalcitonin fragments is at least 12 amino acids, preferably more than 50 amino acids, more preferably more than 110 amino acids. PCT may comprise post-translational modifications such as glycosylation, liposidation or derivatization. Procalcitonin is a precursor of calcitonin and katacalcin. Thus, under normal conditions the PCT levels in the circulation are very low (< about 0.05 ng / ml).

[0174] The level of PCT in the sample of the subject can be determined by immunoassays as described herein. As used herein, the level of ribonucleic acid or deoxyribonucleic acids encoding “procalcitonin” or “PCT” can also be determined. Methods for the determination of PCT are known to a skilled person, for example by using products obtained from Thermo Fisher Scientific I B R A H M S GmbH.

[0175] It is understood that in the context of the present invention, proADM or fragment(s) thereof’ or the like refers to any means of determining proADM or a fragment thereof. The fragment can have any length, e.g. at least about 5, 10, 20, 30, 40, 50 or 100 amino acids, so long as the fragment allows the unambiguous determination of the level of proADM or fragment thereof. In particular preferred aspects of the invention, “determining the level of proADM” refers to determining the level of midregional proadrenomedullin (MR-proADM). MR-proADM is a fragment and / or region of proADM.

[0176] The peptide adrenomedullin (ADM) was discovered as a hypotensive peptide comprising 52 amino acids, which had been isolated from a human phenochromocytome (Kitamura et al., 1993). Adrenomedullin (ADM) is encoded as a precursor peptide comprising 185 amino acids (“preproadrenomedullin” or “pre proADM”). An exemplary amino acid sequence is given in SEQ ID NO: 1.

[0177] SEQ ID NO:1 : amino acid sequence of pre-pro-ADM: 1 MKLVSVALMY LGSLAFLGAD TARLDVASEF RKKWNKWALS RGKRELRMSS

[0178] 51 SYPTGLADVKAGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN

[0179] 101 NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR

[0180] 151 RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

[0181] ADM comprises the positions 95-146 of the pre-proADM amino acid sequence and is a splice product thereof. “Proadrenomedullin” (“proADM”) refers to pre-proADM without the signal sequence (amino acids 1 to 21), i.e. to amino acid residues 22 to 185 of pre-proADM.

[0182] “Midregional proadrenomedullin” (“MR-proADM”) refers to the amino acids 45 to 92 of pre- proADM.

[0183] An exemplary amino acid sequence of MR-proADM is given in SEQ ID NO: 2.

[0184] SEQ ID NO:2: amino acid sequence of MR-pro-ADM (AS 45-92 of pre-pro-ADM):

[0185] ELRMSSSYPT GLADVKAGPA QTLIRPQDMK GASRSPEDSS PDAARIRV

[0186] It is also envisaged herein that a peptide and fragment thereof of pre-proADM or MR-proADM can be used for the herein described methods. For example, the peptide or the fragment thereof can comprise the amino acids 22-41 of pre-proADM (PAMP peptide) or amino acids 95-146 of pre- proADM (mature adrenomedullin, including the biologically active form, also known as bio-ADM).

[0187] SEQ ID NO:3: amino acid sequence of mature ADM (AS 95-146 of pre-pro-ADM):

[0188] YRQSMNNFQGLRSFGCRFGTCTVQKLAHQIYQFTDKDKDNVAPRSKISPQGY

[0189] A C-terminal fragment of proADM (amino acids 153 to 185 of pre proADM) is called adrenotensin.

[0190] Fragments of the proADM peptides or fragments of the MR-proADM can comprise, for example, at least about 5, 10, 20, 30 or more amino acids. Accordingly, the fragment of proADM may, for example, be selected from the group consisting of MR-proADM, PAMP, adrenotensin and mature adrenomedullin, preferably herein the fragment is MR-proADM. Accordingly, the fragment of proADM may, for example, be selected from the group consisting of MR-proADM, PAMP and adrenotensin, preferably herein the fragment is MR-proADM.

[0191] In embodiments, the fragments described herein may vary from the exact amino acid sequences indicated above, for example, may vary with respect to the numbering of amino acids and / or may vary with respect to the length of the fragment. For example, the N terminal signal sequence may be described as 20-22 amino acids in length. The PAMP fragment may therefore also vary in length depending on cleavage of the N terminal signal sequence. PAMP may in embodiments have amino acids 21 onwards with respect to SEQ ID NO 1 , or amino acids 22 onwards. In other embodiments, PAMP may have amino acids up to amino acid 41 of SEQ ID NO 1 , or up to amino acid 44 of SEQ ID NO 1. Some variation has been described previously, and any fragment may therefore vary in length. Similarly, the other proADM fragments described herein may vary in their length depending on cleavage of the peptide.

[0192] The determination of these various forms of ADM or proADM and fragments thereof also encompass measuring and / or detecting specific sub-regions of these molecules, for example by employing antibodies or other affinity reagents directed against a particular portion of the molecules. Any one or more of the “ADM peptides or fragments” described herein may be employed in the present invention. Methods for the determination of proADM, in particular MR- proADM, are known to a skilled person, for example by using products obtained from Thermo Fisher Scientific / B R A H M S GmbH.

[0193] In ebodiments, the method may comprise "determining a level of a proADM fragment that is equimolar to MR-proADM (equimolar proADM fragment)". An equimolar fragment refers to determining a proADM fragment other than MR-proADM, provided that said equimolar fragment is present in the same or similar molar concentration as MR-proADM, and / or exhibits similar expression and / or similar digestion kinetics and / or similar behavior to MR-proADM.

[0194] This ensures that the chosen fragment accurately reflects the amount of MR-proADM, thereby maintaining similar diagnostic and predictive capabilities. Additionally, this equimolar fragment is preferably characterized by a comparable in vivo half-life (digestion kinetics) to MR-proADM. This means that the fragment should exhibit similar stability and clearance in the body, ensuring its measured levels accurately reflect the underlying physiological state. Consequently, the invention allows flexibility in the specific proADM fragment used as long as it in essence mirrors the behavior and concentration of MR-proADM. A skilled person is capable of obtaining or developing means for the identification, measurement, determination, and / or quantification of any one of the above proADM molecules or fragments or variants thereof, as well as the other markers of the present invention, according to standard assay development practice. By way of example, the biomarkers PAMP, ADM-Gly and adrenotensin are considered as proADM fragments that are equimolar to MR-proADM.

[0195] D-Dimer is not normally present in human blood plasma. This biomarker is one of the Fibrinogen degradation products (FDP) which is released after a thrombus or blood clot is enzymatically degraded by Plasmin. Below a concentration range (e.g. 0,5 mg / l in a blood sample), D-Dimer can help to rule out clinical conditions that are characterized by inappropriate blood clot formation such as deep vein thrombosis, pulmonary embolism or disseminated intravascular coagulation. In case of increased levels of D-Dimer, further testing (such as ultrasound, scintigraphy, CT scanning) is required (see Adam SS, Key NS, Greenberg CS (March 2009). "D-dimer antigen: current concepts and future prospects". Blood. 113 (13): 2878-87. doi: 10.1182 / blood-2008-06- 165845).

[0196] C-reactive protein (CRP) is a pentameric protein, which can be found in bodily fluids such as blood plasma. CRP levels can rise in response to inflammation. Measuring and charting CRP values can prove useful in determining disease progress or the effectiveness of treatments.

[0197] Interleukin 6 (IL-6) is promptly and transiently produced in response to infections and tissue injuries, and contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions. Although its expression is controlled by transcriptional and posttranscriptional mechanisms, dysregulated continual synthesis of IL-6 plays a pathological effect on chronic inflammation and autoimmunity. IL-6 expression is associated with coronavirus infection and may predict the severity of COVID-19.

[0198] Brain Natriuretic Peptide (BNP) is a polypeptide originally isolated from porcine brain by T. Sudoh and coworkers (Nature 1988; 332: 78-81). After cloning and sequence analysis of CDNA coding for the peptide (T. Sudoh et al. 1989) human BNP was shown to be produced in the human heart. Heart ventricles produce B-type natriuretic peptide (BNP) in response to increased mechanical load and wall stretch. BNP protects the heart from adverse consequences of overload by increasing natriuresis and diuresis, relaxing vascular smooth muscle, inhibiting the renin- angiotensin-aldosterone system, and by counteracting cardiac hypertrophy and fibrosis. BNP is synthesized by human cardiac myocytes as a 108-amino acid prohormone (proBNP), which is cleaved to the 32-residue BNP and the 76-residue N-terminal fragment of proBNP (NT-proBNP).

[0199] BNP plasma concentration is increased in patients suffering from heart disease leading to heart failure. The cardiac monocytes secrete another factor, namely atrial natriuretic factor (ANF) but the secretory response to heart failure or incipient heart failure seems to be much larger in the BNP system compared to the ANF system (Mukoyama et al, J Clin Invest 1991 ; 87: 1402-12). Nowadays, BNP is acknowledge as a versatile biomarker for cardiac dysfunctions, in particular regarding left ventricular dysfunction and a predictor of myocardial infarction or heart failure (Vuolteenaho et al. 2005).

[0200] "Atrial natriuretic peptide (ANP)", a member of the natriuretic peptide family, regulates several physiological parameters including diuresis and natriuresis, and lower arterial blood pressure (BP). It is predominantly produced in the atrium of the heart and comprises 98% of natriuretic peptides in the circulation (Vesely DL. Life 2002;53:153-159). ANP is derived from the cleavage of its precursor pro-hormone, which is significantly more stable in the circulation than the mature peptide. Atrial natriuretic polypeptide (ANP) is mainly secreted from the atria of healthy adult humans and from the left ventricle of patients with left ventricular dysfunction. Clinical application of ANP is limited by a short half-life; however, its precursor NT-proANP is more stable in plasma and has a longer half-life. A midregional fragment of the precursor hormone (amino acids 53-90 of NT-proANP), called midregional-proANP (MR-proANP), may be relatively resistant to degradation by exoproteases, unlike epitopes in the N- or C-terminals of proANP used in previous immunoassays (Morgenthaler NG et al. Clin Chem 2004;50:234-236; Gerszten RE et al. 2008. Am J Physiol Lung Cell Mol Physiol).

[0201] It is understood that in the context of the present invention “determining the level of proANP or fragment(s) thereof’ or the like refers to any means of determining proANP or a fragment thereof. The fragment can have any length, e.g. at least about 5, 10, 20, 30, 40, 50 or 100 amino acids, so long as the fragment allows the unambiguous determination of the level of proANP or fragment thereof. In embodiments of the invention, “determining the level of proANP” refers to determining the level of mid-regional proANP (MR-proANP). MR-proANP is a fragment and / or region of proANP.

[0202] The amino acid sequence of the "atrial natriuretic peptide (ANP)" is given in SEQ ID NO:6. The sequence of the 153 amino acid pre-pro-ANP is shown in SEQ ID NO:4. Upon cleavage of an N- terminal signal peptide (25 amino acids) and the two C-terminal amino acids (127 / 128) proANP (SEQ ID NO:5) is released. ANP comprises residues 99-126 from the C-terminus of the precursor prohormone pro-ANP. This prohormone is cleaved into the mature 28 amino acid peptide ANP, also known as ANP (1-28) or a-ANP, and the amino terminal fragment ANP (1-98) (NT-proANP, SEQ ID NO 7). Mid-regional proANP (MR-proANP) is defined as NT-proANP or any fragments thereof comprising at least amino acid residues 53-90 (SEQ ID NO:8) of proANP. The C-terminal two arginine residues (positions 152 and 153 in pre-pro-ANP, SEQ ID NO:4, are not present in another allele of the gene encoding pre-pro-ANP, thus pre-pro-ANP may comprise only residues 1 to 151 . This of course is also true for the respective fragments of pre-pro-ANP, particularly pro- ANP.

[0203] “pro-atrial natriuretic peptide” or“proANP” refers to the pro-hormone comprising 128 amino acids. As used herein, a peptide comprising 28 amino acids (99-126) of the C-terminal section of a pro- hormone comprising 128 amino acids (proANP) is referred to as the actual hormone ANP. Upon release of ANP from its pro-hormone proANP, an equimolar amount of the remaining larger partial peptide of proANP, the N-terminal proANP, consisting of 98 amino acids (NT-proANP; proANP (1- 98)) is released into circulation. As NT-proANP possesses a significantly greater half life time and stability NT-proANP can be used as laboratory parameter for diagnosis, follow-up and therapy control; see, for example, Lothar Thomas (Editor), Labor und Diagnose, 5th expanded ed., subchapter 2.14 of chapter 2, Kardiale Diagnostik, pages 116-118, and WO 2008 / 135571 . The level of proANP is preferably measured in the plasma or serum of a subject.

[0204] SEQ ID NO:4: (amino acid sequence of pre-proANP, 153 AS ):

[0205] MSSFSTTTVS FLLLLAFQLL GQTRANPMYN AVSNADLMDF KNLLDHLEEK

[0206] MPLEDEVVPP QVLSEPNEEA GAALSPLPEV PPWTGEVSPA QRDGGALGRG

[0207] PWDSSDRSAL LKSKLRALLT APRSLRRSSC FGGRMDRIGA QSGLGCNSFR

[0208] YRR

[0209] SEQ ID NO:5: (amino acid sequence of proANP):

[0210] NPMYNAVSNA DLMDFKNLLD HLEEKMPLED EWPPQVLSE PNEEAGAALS

[0211] PLPEVPPWTG EVSPAQRDGG ALGRGPWDSS DRSALLKSKL RALLTAPRSL RRSSCFGGRM DRIGAQSGLG CNSFRY

[0212] SEQ ID NO:6: (amino acid sequence of ANP, AS 124- 151 of pre-proANP):

[0213] SLRRSSCFGG RMDRIGAQSG LGCNSFRY

[0214] SEQ ID NO:7: (amino acid sequence of NT-proANP, AS 26- 123 of pre-proANP):

[0215] NPMYNAVSNA DLMDFKNLLD HLEEKMPLED EWPPQVLSE PNEEAGAALS

[0216] PLPEVPPWTG EVSPAQRDGG ALGRGPWDSS DRSALLKSKL RALLTAPR

[0217] SEQ ID NO:8: (amino acid sequence of MR-proANP, AS 53-90 of proANP):

[0218] PEVPPWT GEVSPAQRDG GALGRGPWDS SDRSALLKSK L

[0219] (Atrial natriuretic peptide Uniprot Number: P01160)

[0220] The determination of these various forms of proANP and fragments thereof also encompass measuring and / or detecting specific sub-regions of these molecules, for example by employing antibodies or other affinity reagents directed against a particular portion of the molecules, or by determining the presence and / or quantity of the molecules by measuring a portion of the protein using mass spectrometry. Any one or more of the “proANP peptides or fragments” described herein may be employed in the present invention.

[0221] The level of proANP in the sample of the subject can be determined by immunoassays as described herein. As used herein, the level of ribonucleic acid or deoxyribonucleic acids encoding “Atrial natriuretic peptide” or “proANP” can also be determined. Methods for the determination of proANP and fragment(s) thereof are known to a skilled person, for example by using products obtained from Thermo Fisher Scientific / B R A H M S GmbH.

[0222] Endothelin-1 (ET-1), a 21 amino acid peptide, is a strong vasoconstrictor. Since its discovery in 1988, its biosynthesis, mode of action and association with disease has been extensively studied. There are three different genes encoding isoforms of endothelin (endothelin-1 , Endothelin-2, endothelin-3), of which endothelin-1 is found in the highest concentrations and is most effective. Endothelin-1 is found in endothelial cells, in the lungs, in the heart, synthesized in the kidney and brain. The primary translation product of the human endothelin-1 gene is a 212 amino acidcomprising peptide, termed prepro-endothelin-1 (preproendothelin). In the secretion process, a short N-terminal signal sequence (amino acids 1-17) of the preproendothelin is removed by the signal peptidase. The proendothelin obtained is then processed by the protease furin on dibasic amino acid pairs to give a biologically inactive peptide comprising 38 amino acids, big endothelin, from which finally the mature, biologically active endothelin-1 is formed by means of endothelin- converting enzymes (ECEs). In addition, the C-terminal proendothelin fragments (CT-proET-1) has the amino acid sequences 93-212 or 168-212 of the preproendothelin.

[0223] Endothelin typically acts via the bond to specific receptors which are localized on muscle cells, myocytes and fibroblasts. This bond leads to efflux of calcium, activation of phospholipase C and inhibition of Na / K ATPase. In addition to the vasoconstrictive effect, endothelin also has growthregulating properties. Elevated plasma concentrations of endothelin-1 and big endothelin have been described for various clinical indications. These include cardiovascular disease (inter alia pulmonary hypertension, atherosclerosis, congestive heart failure, myocardial infarction), sepsis and septic shock, and cancer.

[0224] Methods for the determination of CT-proET-1 are known to a skilled person, for example by using products obtained from Thermo Fisher Scientific / B R A H M S GmbH.

[0225] The neuropeptid arginine vasopressin (also denoted as vasopressin, vasopressin argipressin or antidiuretic hormone (ADH)) is a nonapeptide which is produced in the paraventricular nucleus of the hypothalamus and the supraoptic nucleus.

[0226] Copeptin is a 39-amino acid glycopeptide derived from the C-terminal portion of the precursor pre-provasopressin, also known as pre-pro-arginine vasopressin (pre-pro-AVP). It is produced in equimolar amounts with arginine vasopressin (AVP, also known as antidiuretic hormone or ADH) during the processing of pre-provasopressin in the hypothalamus. Copeptin is released into the bloodstream alongside AVP from the posterior pituitary gland. Copeptin serves as a stable surrogate marker for AVP due to its longer half-life and more stable nature, making it easier to measure in clinical settings. AVP plays a critical role in water homeostasis, blood pressure regulation, and the body's response to stress. Because of its stability and close correlation with AVP levels, copeptin is increasingly used in medical diagnostics as a biomarker for various conditions, including cardiovascular conditions such as acute myocardial infarction, cardiogenic shock and stroke, infectious diseases such as sepsis and septic shock and diabetes insipidus (Mockel M et al. Eur J Emerg Med 2013; 20: 103-108, Retard et al., Clin Endocrinol (Oxf). 2019 Jul; 91 (1): 22-32).

[0227] Troponin is a complex of three regulatory proteins integral to the contraction of skeletal and cardiac muscle. These proteins, namely troponin C (Tnc), troponin I (Tnl), and troponin T (TnT), are located on the thin filament of the sarcomere and are essential for muscle contraction in response to calcium binding. Toponin C (Tnc) binds calcium ions initiating the conformational change in the troponin complex necessary for muscle contraction. Troponin I (Tnl) inhibits actomyosin ATPase, which is responsible for muscle contraction. Tnl undergoes conformational changes upon calcium binding to Tnc, relieving its inhibitory effect. Troponin T (TnT) binds the troponin complex to tropomyosin, anchoring the troponin complex to the actin filament. Troponins, particularly cardiac-specific isoforms of troponin I (cTnl) and troponin T (cTnT), are highly specific and sensitive biomarkers for myocardial injury. When cardiac muscle is damaged, troponins are released into the bloodstream. Elevated levels of cardiac troponins are a key diagnostic indicator of acute myocardial infarction (Mockel M et al. Eur J Emerg Med 2013; 20: 103-108).

[0228] As used herein, the term "soluble Flt-1 (sFlt-1)" (soluble fms-like tyrosine kinase 1 , also known as sVEGF-RI) refers to the soluble form of the Flt-1 receptor, that is homologous to the protein defined by GenBank accession number U01134 or UniProt P17948 or entry name VGFR1 _HUMAN and that has sFlt-1 biological activity. The biological activity of an sFlt-1 polypeptide may be assayed using any standard method, for example, by assaying sFlt-1 binding to VEGF. sFlt-1 lacks the transmembrane domain and the cytoplasmic tyrosine kinase domain of the Flt-1 receptor. sFlt-1 can bind to VEGF and PIGF bind with high affinity, but it cannot induce proliferation or angiogenesis and is therefore functionally different from the Flt-1 and KDR receptors. sFlt-1 was initially purified from human umbilical endothelial cells and later shown to be produced by trophoblast cells in vivo. As used herein, sFlt-1 includes any sFlt-1 family member or isoform.

[0229] The at least one biomarker of said subject can be selected from the group consisting of procalcitonin (PCT) or fragment(s) thereof, proadrenomedullin (proADM) or fragment(s) thereof, interleukin-6 (IL-6), copeptin, troponin, pro brain natriuretic peptide (proBNP) or fragment(s) thereof, pro atrial natriuretic peptide (proANP) or fragment(s) thereof, C-reactive protein (CRP), proEndothelin-1 (proET-1), soluble fms-like tyrosine kinase-1 (sFlt-1) of fragment(s) thereof, lactate, D-Dimer, Histone H2A, Histone H2B, Histone H3, Histone H4, calcitonin, Endothelin-1 (ET-1), Arginine Vasopressin (AVP), Atrial Natriuretic Peptide (ANP), Neutrophil Gelatinase- Associated Lipocalin (NGAL), Brain Natriuretic Peptide (BNP), Pancreatic Stone Protein (PSP), Triggering Receptor Expressed on Myeloid Cells 1 (TREM1), Interleukin-24 (IL-24), Interleukin-22 (IL-22), Interleukin (IL-20) other ILs, Presepsin (sCD14-ST), Lipopolysaccharide Binding Protein (LBP), Alpha-1 -Antitrypsin, Matrix Metalloproteinase 2 (MMP2), Metalloproteinase 2 (MMP8), Matrix Metalloproteinase 9 (MMP9), Matrix Metalloproteinase 7 (MMP7, Placental growth factor (PIGF), Chromogranin A, S100A protein, SWOB protein and Tumor Necrosis Factor a (TNFa), Neopterin, Alpha-1-Antitrypsin, pro-arginine vasopressin (AVP, proAVP or Copeptin), CCL1 / TCA3, CCL11 , CCL12 / MCP-5, CCL13 / MCP-4, CCL14, CCL15, CCL16, CCL17 / TARC, CCL18, CCL19, CCL2 / MCP-1 , CCL20, CCL21 , CCL22 / MDC, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL3L3, CCL4, CCL4L1 / LAG-1 , CCL5, CCL6, CCL7, CCL8, CCL9, CX3CL1 , CXCL1 , CXCL10, CXCL11 , CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CXCL2 / MIP-2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 / Ppbp, CXCL9, IL8 / CXCL8, XCL1 , XCL2, FAM19A1 , FAM19A2, FAM19A3, FAM19A4, FAM19A5, CLCF1 , CNTF, IL11 , IL31 , IL6, Leptin, LIF, OSM, IFNA1 , IFNA10, IFNA13, IFNA14, IFNA2, IFNA4, IFNA7, IFNB1 , IFNE, IFNG, IFNZ, IFNA8, IFNA5 / IFNaG, IFNw / IFNWI , BAFF, 4-1 BBL, TNFSF8, CD40LG, CD70, CD95L / CD178, EDA-A1 , TNFSF14, LTA / TNFB, LTB, TNFa, TNFSF10, TNFSF11 , TNFSF12, TNFSF13, TNFSF15, TNFSF4, TRAIL, IP-10, IL18, IL18BP, IL1A, IL1 B, IL1 F10, IL1 F3 / IL1 RA, IL1 F5, IL1 F6, IL1 F7, IL1 F8, IL1 RL2, IL1 F9, I L33 or a fragment thereof, SCD14-ST, prothrombinase, antithrombin, cationic protein 18 (CAP18), von Willebrand factor (vWF)-cleaving proteases, lipoproteins in combination with CRP, fibrinogen, fibrin, B2GP1 , GPIIb-llla, and platelet factor 4.

[0230] Other biomarkers or parameters:

[0231] Accordingly, the methods and kits of the present invention can also comprise determining at least one further biomarker, marker, clinical score and / or parameter in addition to a first biomarker.

[0232] As used herein, a parameter is a characteristic, feature, or measurable factor that can help in defining a particular system. A parameter is an important element for health- and physiology- related assessments, such as a disease / disorder / clinical condition risk, preferably organ dysfunction(s). Furthermore, a parameter is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. An exemplary parameter can be selected from the group consisting of Pneumonia Severity Index (PSI), Acute Physiology and Chronic Health Evaluation II (APACHE II), the simplified acute physiology score (SAPSII score), sequential organ failure assessment score (SOFA score), quick sequential organ failure assessment score (qSOFA), body mass index, weight, age, gestational age in pregnant woman, sex, IGS II, liquid intake, white blood cell count, proteinuria, sodium, potassium, temperature, blood pressure, dopamine, bilirubin, respiratory rate, partial pressure of oxygen, World Federation of Neurosurgical Societies (WFNS) grading, Glasgow Coma Scale (GCS), CURB-65 pneumonia severity score, Pneumonia Severity Index (PSI), age, gender, clinical history, primary chronic medical condition, comorbidities and related regular medication intake, family history, ethnicity, body weight, body mass index (BMI), cystoscopy report, white blood cell count, lymphocyte count, imaging methods as such as CT scan, PET imaging or X-ray, ultrasound results, blood pressure, hypertension, heart rate, antihypertensive treatment, liquid intake, wheezing, body temperature, presence of diabetes mellitus, blood gluocose levels, and (current) smoking habits.

[0233] Such parameters may additionally be assessed in combination with the methods described herein in order to improve assay implementation, diagnostic statements and therapy guidance.

[0234] Medical conditions: As used herein, the “patient” or "subject" may be a vertebrate. In the context of the present invention, the term "subject" includes both humans and animals, particularly mammals, and other organisms.

[0235] In the present invention "treatment" or “therapy” generally means to obtain a desired pharmacological effect and / or physiological effect. The effect may be prophylactic in view of completely or partially preventing a disease and / or a symptom, for example by reducing the risk of a subject having a disease or symptom or may be therapeutic in view of partially or completely curing a disease and / or adverse effect of the disease.

[0236] In the present invention, "therapy" includes arbitrary treatments of diseases or conditions in mammals, in particular, humans, for example, the following treatments (a) to (c): (a) Prevention of onset of a disease, condition or symptom in a patient; (b) Inhibition of a symptom of a condition, that is, prevention of progression of the symptom; (c) Amelioration of a symptom of a condition, that is, induction of regression of the disease or symptom.

[0237] The term “acute medical condition” refers to a medical condition characterized by a sudden onset such a within minutes, hours or a few days such as 1 to 3 days, and typically short duration such as a few days or weeks such as 1 day to 4 weeks. These diseases typically exhibit rapid onset of symptoms, which can range from mild to severe. The symptoms often necessitate immediate medical intervention to prevent complications or death. Examples for acute medical conditions include, without limitation, infectious disease, such as sepsis, severe sepsis or septic shock, prenatal complications like preeclampsia, the HELLP syndrome or preterm birth, blood vessel embolism such as pulmonary embolism, stroke, or myocardial infarction and / or an acute medical condition such as an acute cardiac condition or other conditions related to acute life-threatening conditions, such as organ dysfunctions, or acute cardiac conditions such as acute myocardial infarction, acute heart failure, unstable angina, cardiogenic shock, acute pericarditis and acute myocarditis.

[0238] The term “chronic medical condition” refers to long-lasting medical conditions that typically progress slowly over time and may persist for a long time such as several weeks, months, years or even for duration of a subject’s life. These conditions often require ongoing management and treatment to control symptoms and prevent complications. Examples for acute medical conditions include without limitation diabetes, hypertension, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, and chronic kidney disease.

[0239] In embodiments the method of the present invention may comprise a diagnosis, prognosis and / or risk stratification of a medical condition in a subject, wherein the medical condition is an infectious disease, such as sepsis, severe sepsis or septic shock, and / or an acute medical condition such as an acute cardiac condition or an acute inflammation.

[0240] In embodiments, the method may employ diagnosis, prognosis, therapy management, and / or risk stratification for the severity assessment of patients in a clinical setting (general practitioners, ED, ICU, hospital ward), including, without limitation, rule-in / out survival (mortality), complications, companion diagnostics to monitor a treatment (responder / non-responder etc.), and / or to monitor or predict disease progression. “Sepsis” in the context of the invention refers to a systemic response to infection. Alternatively, sepsis may be seen as the combination of SIRS with a confirmed infectious process or an infection. Sepsis may be characterized as clinical syndrome defined by the presence of both infection and a systemic inflammatory response (Levy MM et al. 2001 SCCM / ESICM / ACCP / ATS / SIS International Sepsis Definitions Conference. Crit Care Med. 2003 Apr;31 (4): 1250-6). The term “sepsis” used herein includes, but is not limited to, sepsis, severe sepsis, and septic shock.

[0241] The term “sepsis” used herein includes, but is not limited to, sepsis, severe sepsis, and septic shock. Severe sepsis in refers to sepsis associated with organ dysfunction, hypoperfusion abnormality, or sepsis-induced hypotension. Hypoperfusion abnormalities include lactic acidosis, oliguria and acute alteration of mental status. Sepsis-induced hypotension is defined by the presence of a systolic blood pressure of less than about 90 mm Hg or its reduction by about 40 mm Hg or more from baseline in the absence of other causes for hypotension (e.g. cardiogenic shock). Septic shock is defined as severe sepsis with sepsis-induced hypotension persisting despite adequate fluid resuscitation, along with the presence of hypoperfusion abnormalities or organ dysfunction (Bone et al., CHEST 101 (6): 1644-55, 1992).

[0242] The term sepsis may alternatively be defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. For clinical operationalization, organ dysfunction can preferably be represented by an increase in the Sequential Organ Failure Assessment (SOFA) score of 2 points or more, which is associated with an in-hospital mortality greater than 10%. Septic shock may be defined as a subset of sepsis in which particularly profound circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than with sepsis alone. Patients with septic shock can be clinically identified by a vasopressor requirement to maintain a mean arterial pressure of 65 mm Hg or greater and serum lactate level greater than 2 mmol / L (>18 mg / dL) in the absence of hypovolemia.

[0243] The term “sepsis” used herein relates to all possible stages in the development of sepsis. The term “sepsis” also includes severe sepsis or septic shock based on the SEPSIS-2 definition (Bone et al., 2009). The term “sepsis” also includes subjects falling within the SEPSIS-3 definition (Singer et al., 2016). The term “sepsis” used herein relates to all possible stages in the development of sepsis.

[0244] As used herein, “infection” within the scope of the invention means a pathological process caused by the invasion of normally sterile tissue or fluid by pathogenic or potentially pathogenic agents / pathogens, organisms and / or microorganisms, and relates preferably to infection(s) by bacteria, viruses, fungi, and / or parasites.

[0245] Furthermore, the infection-related complication can be a “nosocomial” infection. Nosocomial infections are also called hospital-acquired infections (HAI) are infections that are acquired in a hospital or other health care facility. To emphasize both hospital and nonhospital settings, it is sometimes instead called a health care-associated infection (HAI or HCAI). Such an infection can be acquired in hospital, nursing home, rehabilitation facility, outpatient clinic, or other clinical settings. Other infections may be community acquired infections. Nosocomial infection may be spread to the susceptible patient in the clinical setting by various means. Health care staff can spread infection, in addition to contaminated equipment, bed linens, or air droplets. The infection can originate from the outside environment, another infected patient, staff that may be infected, or in some cases, the source of the infection cannot be determined. In some cases the microorganism originates from the patient's own skin microbiota, becoming opportunistic after surgery or other procedures that compromise the protective skin barrier. Though the patient may have contracted the infection from their own skin, the infection is still considered nosocomial since it develops in the health care setting.

[0246] Further, the subject suffering from an infection can suffer from more than one source(s) of infection simultaneously. For example, the subject suffering from an infection can suffer from a bacterial infection and viral infection; from a viral infection and fungal infection; from a bacterial and fungal infection, and from a bacterial infection, fungal infection and viral infection, or suffer from a mixed infection comprising one or more of the infections listed herein, including potentially a superinfection, for example one or more bacterial infections in addition to one or more viral infections and / or one or more fungal infections.

[0247] A "cardiovascular disease" also termed “cardiac condition” as used herein is characterized by the dysfunction of the heart muscle or the blood vessel system supplying the heart, brain and other vital organs. The term “cardiovascular disease” covers a wide array of disorders including arteriosclerosis, coronary artery disease, heart valve disease, arrhythmia, heart failure, hypertension, orthostatic hypotension, shock, endocarditis, diseases of the aorta and its branches, disorders of the peripheral vascular system, congenital heart disease or stroke.

[0248] An "acute cardiovascular disease" also termed “acute cardiac condition” refers to severe and sudden-onset heart-related medical conditions that usually require immediate initiation of therapeutic means. These conditions include, but are not limited to acute myocardial infarction, acute heart failure, unstable angina, cardiogenic shock, acute pericarditis or acute myocarditis, preferably acute myocardial infarction, as well as arteriovascular events such as blood clots of the leg, aneurysms, stroke and other arteriovascular ischemic events where arteriovascular blood flow and oxygenation is interrupted.

[0249] "Acute heart failure (AHF)" also termed “acute decompensated heart failure” is defined as the rapid onset of symptoms and signs secondary to abnormal cardiac function. It may occur with or without previous cardiac disease. The cardiac dysfunction can be related to systolic or diastolic dysfunction, abnormalities in cardiac rhythm, or to pre-load and after-load mismatch. AHF can present itself as acute de novo (new onset of AHF in a patient without previously known cardiac dysfunction) or acute decompensation of chronic heart failure (Nieminen et al. 2005. Eur Heart J 26: 384-416; Dickstein et al. 2008. Eur Hear J 29: 2388-442).

[0250] The cardiac dysfunction may be related to systolic or diastolic dysfunction, to abnormalities in cardiac rhythm, or to preload and afterload mismatch. It is often life threatening and requires urgent treatment. According to established classification, AHF includes several distinct clinical conditions of presenting patients: (I) acute decompensated congestive heart failure, (II) AHF with hypertension / hypertensive crisis, (III) AHF with pulmonary oedema, (IVa) cardiogenic shock / low output syndrome, (IVb) severe cardiogenic shock, (V) high output failure, and (VI) right-sided acute heart failure. For detailed clinical description, classification and diagnosis of AHF, and for summary of further AHF classification systems including the Killip classification, the Forrester classification and the ‘clinical severity’ classification, refer inter alia to Nieminen et al. 2005 (“Executive summary of the guidelines on the diagnosis and treatment of acute heart failure: the Task Force on Acute Heart Failure of the European Society of Cardiology”. Eur Heart J 26: 384- 416) and references therein.

[0251] “Acute myocardial infarction (AMI)” refers to a medical condition characterized by the sudden blockage of blood flow to a part of the heart muscle, resulting in tissue damage or death of the heart muscle cells. This condition is a critical and urgent medical emergency requiring immediate intervention to restore blood flow and minimize heart damage. AMI typically occurs due to the rupture of atherosclerotic plaques within the coronary arteries, leading to the formation of a blood clot (thrombus) that obstructs blood flow. Common symptoms include severe chest pain or discomfort, which may radiate to the arm, shoulder, back, neck, or jaw, along with shortness of breath, sweating, nausea, and lightheadedness. Immediate treatment aims to restore blood flow to the affected heart muscle, which can include medications (like thrombolytics), percutaneous coronary intervention (PCI) such as angioplasty and stenting, or coronary artery bypass grafting (CABG) surgery.

[0252] As used herein, organ dysfunctional or organ failure, relates to its ordinary meaning in the art. Organ dysfunction is a condition where an organ does not perform its expected function. Organ failure is organ dysfunction to a degree that normal homeostasis (a state of balance among all the body systems needed for the body to survive and function correctly) cannot be maintained, or cannot be maintained without external clinical intervention and / or life support. Multiple organ failure can be associated with sepsis and is often fatal.

[0253] “Acute inflammation” refers to the body’s immediate and early response to tissue injury or e.g. infection, aiming to eliminate the cause and initiate healing. It is characterized by the rapid activation of immune cells, increased blood flow, and the release of inflammatory mediators, leading to redness, heat, swelling, pain, and sometimes loss of function. An acute inflammation requiring hospital treatment is a severe, rapidly progressing response to infection, injury, or tissue damage that poses a risk to vital functions or may lead to complications without medical intervention. It often involves high fever, elevated inflammatory markers (e.g. CRP, IL-6), systemic symptoms (like hypotension, tachycardia, or confusion), and / or organ involvement (e.g. lungs in pneumonia, abdomen in peritonitis). Conditions such as Systemic Inflammatory Response Syndrome (SIRS), sepsis, acute appendicitis, pneumonia, or pancreatitis are common examples.

[0254] As used herein, "preeclampsia" (PE) is used in its ordinary meaning. PE can be defined according to well established criteria, such as a blood pressure of at least 140 / 90 mm Hg and urinary excretion of at least 0.3 grams of protein in a 24-hour urinary protein excretion (or at least +1 or greater on dipstick testing), each on two occasions 4-6 hours apart.

[0255] As used herein, preeclampsia is considered a multi-system disorder that is characterized by hypertension with proteinuria or edema, or both, glomerular dysfunction, brain edema, liver edema, or coagulation abnormalities due to pregnancy or the influence of a recent pregnancy. Preeclampsia generally occurs after the 20th week of gestation. Preeclampsia is generally defined as some combination of the following symptoms: (1) a systolic blood pressure (BP) > 140 mmHg and a diastolic BP > 90 mmHg after 20 weeks gestation (generally measured on two occasions, 4-168 hours apart), (2) new onset proteinuria (1+ by dipstick on urinalysis, > 300mg of protein in a 24-hour urine collection, or a single random urine sample having a protein / creatinine ratio > 0.3), and (3) resolution of hypertension and proteinuria by 12 weeks postpartum. Severe pre- eclampsia is generally defined as (1) a diastolic BP > 110 mmHg (generally measured on two occasions, 4- 168 hours apart) or (2) proteinuria characterized by a measurement of 3.5 g or more protein in a 24-hour urine collection or two random urine specimens with at least 3+ protein by dipstick.

[0256] In preeclampsia, hypertension and proteinuria generally occur within seven days of each other. In severe preeclampsia, severe hypertension, severe proteinuria and HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) or eclampsia can occur simultaneously or only one symptom at a time. Occasionally, severe preeclampsia can lead to the development of seizures. This severe form of the syndrome is referred to as eclampsia. "Eclampsia” can also include dysfunction or damage to several organs or tissues such as the liver (e.g., hepatocellular damage, periportal necrosis) and the central nervous system (e.g., cerebral edema and cerebral hemorrhage). The etiology of the seizures is thought to be secondary to the development of cerebral edema and focal spasm of small blood vessels in the kidney.

[0257] "Severe preeclampsia" or “high severity of preeclampsia” is also defined in accordance with established criteria, as a blood pressure of at least 160 / 110 mm Hg on at least 2 occasions 6 hours apart and greater than 5 grams of protein in a 24- hour urinary protein excretion or persistent +3 proteinuria on dipstick testing.

[0258] Severe preeclampsia may include HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count). Other elements of severe preeclampsia may include in-utero growth restriction (IUGR) in less than the 10 % percentile according to the US demographics, persistent neurologic symptoms (headache, visual disturbances), epigastric pain, oliguria (less than 500 mL / 24 h), serum creatinine greater than 1 .0 mg / dL, elevated liver enzymes (greater than two times normal), thrombocytopenia (< 100,000 cells / [mu]L).

[0259] As used herein “preterm birth” is defined as delivery before 37 weeks of gestation. In embodiments, early onset of preeclampsia refers to cases with delivery before 34 weeks of gestation.

[0260] Kits:

[0261] The invention further relates to kits, the use of the kits and methods wherein such kits are used.

[0262] The kit may additionally comprise items useful for obtaining a sample, such as a blood sample, for example the kit may comprise a container, wherein said container comprises a device for attachment of said container to a cannula or syringe, is a syringe suitable for blood isolation, exhibits an internal pressure less than atmospheric pressure, such as is suitable for drawing a pre-determined volume of sample into said container, and / or comprises additionally detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, such as guanidinium isothiocyanate, guanidinium hydrochloride, sodium dodecylsulfate, polyoxyethylene sorbitan monolaurate, RNAse inhibitor proteins, and mixtures thereof, and / or A filter system containing nitro-cellulose, silica matrix, ferromagnetic spheres, a cup retrieve spill over, trehalose, fructose, lactose, mannose, polyethylene-glycol, glycerol, EDTA, TRIS, limonene, xylene, benzoyl, phenol, mineral oil, anilin, pyrol, citrate, and mixtures thereof. As used herein, the “detection reagent” or the like are reagents that are suitable to determine the herein described marker(s). Such exemplary detection reagents are, for example, ligands, e.g. antibodies or fragments thereof, which specifically bind to the peptide or epitopes of the herein described marker(s). Such ligands might be used in immunoassays as described above. Further reagents that are employed in the immunoassays to determine the level of the marker(s) may also be comprised in the kit and are herein considered as detection reagents. Detection reagents can also relate to reagents that are employed to detect the markers or fragments thereof by MS based methods. Such detection reagent can thus also be reagents, e.g. enzymes, chemicals, buffers, etc, that are used to prepare the sample for the MS analysis. A mass spectrometer can also be considered as a detection reagent. Detection reagents according to the invention can also be calibration solution(s), e.g. which can be employed to determine and compare the level of the marker(s).

[0263] The methods of the present invention may in embodiments be partially computer-implemented. For example, the step of comparing the detected level of a biomarker to any reference level can be performed in a computer system. In the computer-system, the determined level of the biomarker(s) can be combined with other biomarker levels and / or clinical parameters of the subject in order to calculate a score, which is indicative for the prognosis, risk assessment and / or risk stratification. For example, the determined values may be entered (either manually by a health professional or automatically from the device(s) in which the respective marker level(s) has / have been determined) into the computer-system. The computer-system can be directly at the point-of-care (e.g. primary care, hospital or home setting) or it can be at a remote location connected via a computer network (e.g. via the internet, or specialized medical cloud-systems, optionally combinable with other IT-systems or platforms such as hospital information systems (HIS)). Typically, the computer-system will store the values (e.g. biomarker level or clinical parameters such as age, blood pressure, weight, BMI, sex or Scores (e.g. The SOFA score, qSOFA, Covid-19 ordinal scale, the infection grade) etc. on a computer-readable medium and calculate the score based-on pre-defined and / or pre-stored reference levels or reference values. The resulting score will be displayed and / or printed for the user (typically a health professional such as a physician or the patient). Alternatively or in addition, the associated prognosis, assessment, treatment guidance, patient management guidance or stratification will be displayed and / or printed for the user (typically a health professional such as a physician or the patient).

[0264] In one embodiment of the invention, a software system can be employed, in which a machine learning algorithm is evident, preferably to identify patients at risk for PE using data from electronic health records (EHRs). A machine learning approach can be trained on a random forest classifier using EHR data (such as labs, biomarker expression, vitals, and demographics) from patients. Machine learning is a type of artificial intelligence that provides computers with the ability to learn complex patterns in data without being explicitly programmed, unlike simpler rulebased systems. Earlier studies have used electronic health record data to trigger alerts to detect clinical deterioration in general. In one embodiment of the invention the processing of biomarker levels may be incorporated into appropriate software for comparison to existing data sets, and / or proADM or PCT levels or any other biomarker disclosed herein, may also be processed in machine learning software to assist in prognosing the risk of adverse outcomes.

[0265] Statistical measures: Various statistical measures may be employed to provide a level of statistical significance or statistical likelihood or certainty for any given correlation observed in the data, for example demonstrating that the observation is not the result of chance but based on an underlying biological correlation. For example, the positive and negative predictive values (PPV and NPV respectively) are the proportions of positive and negative results in statistics and diagnostic tests that are true positive and true negative results, respectively. The PPV and NPV describe the performance of a diagnostic test or other statistical measure. A high result can be interpreted as indicating the accuracy of such a statistic.

[0266] Positive predictive value: According to Emerg (Tehran). 2015 Summer; 3(3): 87-88, the positive predictive value (PPV) is the proportion of cases giving positive test results who are already patients. It is the ratio of patients truly diagnosed as positive to all those who had positive test results (including healthy subjects who were incorrectly diagnosed as patient). This characteristic can predict how likely it is for someone to truly be patient, in case of a positive test result.

[0267] Positive predictive value =TP / TP+FP

[0268] Negative predictive value: According to Emerg (Tehran). 2015 Summer; 3(3): 87-88, the negative predictive value is the proportion of the cases giving negative test results who are already healthy. It is the ratio of subjects truly diagnosed as negative to all those who had negative test results (including patients who were incorrectly diagnosed as healthy). This characteristic can predict how likely it is for someone to truly be healthy, in case of a negative test result.

[0269] Negative predictive value = TN / TN+FN

[0270] Therefore a 100% NPV shows no false negative rate. A biomarker or a ratio of marker values with a NPV of 100% differentiate all patients without an adverse outcome correctly in the low risk group.

[0271] A “rule-in” typically identifies a minimal proportion of subjects that with a high certainty will develop the disease or disorder and ensure that this true positive group has a sufficiently large proportion of the subjects testing positive. The test must reach a minimal sensitivity and minimal positive predictive value (PPV).

[0272] A “rule-out” typically identifies a minimal proportion of subjects that will with a high certainly not develop the disease or disorder and ensure that of the subjects testing negative, sufficiently few will develop the disease (false negatives). Such a test must therefore reach a minimal specificity and minimal negative predictive value (NPV).

[0273] The sensitivity and specificity of a diagnostic and / or prognostic test depends on more than just the analytical "quality" of the test, they also depend on the definition of what constitutes an abnormal result. Sensitivity is equal to the true positive rate and specificity is equal to the true negative rate. In practice, Receiver Operating Characteristic curves (ROC curves), are typically calculated by plotting the value of a variable versus its relative frequency in "normal" (i.e. apparently healthy individuals not having an adverse outcome and suffered from an adverse outcome, e.g. subjects who died within 28 days, had thrombotic events, were in the need of supplemental oxygen, had additional infection with the need of antimicrobials and hospitalization, were hospitalized or admitted to the ICU for more than 4 days etc.. For any particular marker (like proADM, IL-6 or PCT), a distribution of marker levels for subjects with and without a disease / condition will likely overlap. Under such conditions, a test does not absolutely distinguish normal from disease with 100% accuracy, and the area of overlap might indicate where the test cannot distinguish normal from disease. A threshold (for example a clinical threshold) may be selected, below which the test is considered to be abnormal and above which the test is considered to be normal or below or above which the test indicates a specific condition, e.g. infection. The area under the ROC curve is a measure of the probability that the perceived measurement will allow correct identification of a condition. ROC curves can be used even when test results do not necessarily give an accurate number. As long as one can rank results, one can create a ROC curve. For example, results of a test on "disease" samples might be ranked according to degree (e.g. 1=low, 2=normal, and 3=high). This ranking can be correlated to results in the "normal" population, and a ROC curve created. These methods are well known in the art; see, e.g., Hanley et al. 1982. Radiology 143: 29-36. Preferably, a clinical threshold is selected to provide a ROC curve area of greater than about 0.5 (<= 0.5 means a test without discriminative power, more preferably greater than about 0.7, still more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9 (up to 1.0 means a test with perfect discrimination). The term "about" in this context refers to + / - 5% of a given measurement.

[0274] The horizontal axis of the ROC curve represents (1 -specificity), which increases with the rate of false positives. The vertical axis of the curve represents sensitivity, which increases with the rate of true positives. Thus, for a particular cut-off selected, the value of (1 -specificity) may be determined, and a corresponding sensitivity may be obtained. The area under the ROC curve is a measure of the probability that the measured marker level will allow correct identification of a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

[0275] Receiver Operating Characteristic (ROC) curves and the Concordance (C) statistic (also termed C index) are often used to assess the ability of a risk factor to predict outcome. For example, often a biomarker or risk factor is included in a logistic regression model to predict the likelihood a patient will develop a disease of interest. These predictive probabilities or risks can be examined to see how accurate they are at identifying patients who will develop the disease or not. For example, if the predicted probabilities for the diseased individuals are all higher than the predicted probabilities for the healthy individuals, then we say that the model has perfect discrimination. Discrimination is commonly measured using ROC curves. To construct an ROC curve, the predicted probabilities of the outcome of interest are repeatedly dichotomized into above vs. below a cutoff. For each cutoff, one can estimate the sensitivity (probability that the predicted risk is above the cutoff among patients with the disease) and the specificity (probability that the predicted risk is below the cutoff among patients without the disease). One can vary the cutoff to show a range of sensitivities vs. specificities. If the model has perfect discrimination, the ROC curve should hit the upper left corner of the plot (100% sensitivity and 100% specificity) (Logan, Medical College of Wisconsin, Biostatistics).

[0276] The area under the ROC curve is a useful measure for summarizing the ROC curve. If a curve is close to the upper left corner (Sensitivity=100%, Specificity=100%), then the area under the ROC curve should be close to 1 . The area under the ROC curve is equivalent to another statistic commonly used to summarize model discrimination, the C statistic or Concordance statistic. The C statistic is interpreted as the probability that a randomly selected subject who experienced the outcome will have a higher predicted probability of having the outcome occur than a randomly selected subject who did not experience the outcome. In addition to computing the area under the ROC curve, this probability can be estimated by taking all pairs of observations where one patient experienced the event and the other did not, and computing the proportion of those pairs where the patient experiencing the event had the higher predicted risk. The C statistic can also be interpreted as the rank correlation between predicted probabilities of the outcome occurring and the observed response (Logan, Medical College of Wisconsin, Biostatistics).

[0277] In other embodiments, a ratio data, positive likelihood ratio, negative likelihood ratio, odds ratio, or hazard ratio is used as a measure of a test's ability to predict risk or diagnose a disease. These are established statistical terms and used herein with their ordinary meaning.

[0278] The “ratio data” is a quantitative data with same properties as interval data, with an equal and definitive ratio between each data. A negative ratio is not possible. A ratio of one explains no change between two values (100% equal). A ratio smaller 1 is connected to a decrease and be expressed in percent or as total number.

[0279] In the case of a "positive likelihood ratio", a value of 1 indicates that a positive result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group. In the case of a "negative likelihood ratio", a value of 1 indicates that a negative result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a negative result is more likely in the test group; and a value less than 1 indicates that a negative result is more likely in the control group. In certain preferred embodiments, markers and / or marker panels are preferably selected to exhibit a positive or negative likelihood ratio of at least about 1 .5 or more or about 0.67 or less, more preferably at least about 2 or more or about 0.5 or less, still more preferably at least about 5 or more or about 0.2 or less, even more preferably at least about 10 or more or about 0.1 or less, and most preferably at least about 20 or more or about 0.05 or less. The term "about" in this context refers to + / - 5% of a given measurement.

[0280] As described herein, the method of the invention also employs ratios between the signal of the sample and the signal of the calibrator. This ratio is termed “signal ratio sample / calibrator” or “signal ratio (sample / calibrator)”. For example, when a ratio of the signal of the sample and the signal of the calibrator (signal ratio sample / calibrator) is above a signal threshold value, the method reports that said signal ratio is above a signal threshold value within 5 minutes of initiating said immunoassay, wherein a signal ratio above said signal threshold value indicates the biomarker concentration in said sample is above the clinical threshold value. Also used herein is the term “signal threshold value”, which is a threshold value to which the signal ratio (sample / calibrator) is compared. This threshold value may therefore also be a ratio, to which the signal ratio is compared.

[0281] Medical treatments: The term “treatment” comprises various treatments and therapeutic strategies, which comprise, without limitation, acute care methods or treatments to prevent a disease progression or the expected disorder as quickly as possible, anti-inflammatory strategies, administration of biomarker-antagonists such as therapeutic antibodies, siRNA or DNA, the extracorporeal blood purification or the removal of harmful substances via apheresis, dialyses, adsorbers to prevent the cytokine storm, removal of inflammatory mediators, plasma apheresis, administration of vitamins such as vitamin C, surgery, emergency surgery, ventilation like mechanical ventilation and non-mechanical ventilation, to provide the body with sufficient oxygen, for example, focus cleaning procedures, transfusion of blood products, infusion of colloids, organ replacement, such as renal or liver replacement, antibiotic treatment, invasive mechanical ventilation, non-invasive mechanical ventilation, vasopressor use, fluid therapy, apheresis and measures for organ protection, restoring blood flow to the affected heart muscle, angioplasty and stenting, or coronary artery bypass grafting (CABG) surgery, low dose aspirin, statins, delivery of the baby.

[0282] Further treatments of the present invention comprise the administration of cells or cell products like stem cells, blood or plasma, and the stabilization of the patients circulation and the protection of endothelial glycocalyx, for example via optimal fluid management strategies, for example to reach normovolemia and prevent or treat hypervolemia or hypovolemia. Moreover, vasopressors or e.g. catecholamine as well as albumin or heparanase inhibition via unfractionated heparin or N-desulfated re-N-acetylated heparin are useful treatments to support the circulation and endothelial layer.

[0283] Additionally, medical treatments of the present invention comprise, without limitation, stabilization of the blood clotting, anti-fibrinolytic treatment, iNOS inhibitors, anti-inflammatory agents like hydrocortisone, sedatives and analgetics as well as insuline.

[0284] Artificial and mechanical ventilation are effective approaches to enhance proper gas exchange and ventilation and aim to save life during severe hypoxemia. Artificial ventilation relates to assisting or stimulating respiration of the subject. Artificial ventilation may be selected from the group consisting of mechanical ventilation, manual ventilation, extracorporeal membrane oxygenation (ECMO) and noninvasive ventilation (NIV). Mechanical ventilation relates to a method to mechanically assist or replace spontaneous breathing. This may involve a machine called a ventilator. Mechanical ventilation may be High-Frequency Oscillatory Ventilation or Partial Liquid Ventilation.

[0285] In a preferred embodiment, the term “medical treatment” or “treatment” comprises antibiotic treatment such as intravenous antibiotic, oral antibiotics or topical antibiotics. In a more preferred embodiment, the term “medical treatment” or “treatment” comprises intravenously applied antibiotic treatment.

[0286] Medical treatment also comprises methods to avoid hypothermia which includes the use of warmed intravenous fluids and warm air blankets.

[0287] Medical treatment also comprises wound management including haemorrhage control, bleeding control techniques with or without tourniquets, wound cleaning, local anaesthetic application, wound closure techniques where skin adhesive strips, tissue adhesive glue, sutures, staples and wound dressings can be used. A medical treatment of the present invention may be an antibiotic treatment, wherein one or more “antibiotics” or “antibiotic agents” may be administered if an infection has been diagnosed or prognosed by the method of the invention. Antibiotics or antibiotic agents according to the present invention also encompass potentially the anti-fungal or anti-viral compounds used to treat a diagnosed infection or sepsis.

[0288] A skilled person is capable of determining the suitable treatment depending on the medical condition of the patient.

[0289] A skilled person is also capable of determining which medical conditions, and which degrees of severity of such medical conditions, require treatments only (or primarily) available in hospital settings, for example in the ED or ICU.

[0290] FIGURES

[0291] The invention is demonstrated by way of the example through the figures disclosed herein. The figures provided represent particular, non-limiting embodiments and are not intended to limit the scope of the invention.

[0292] Brief description of the figures

[0293] Fi g.1 : Early assessment of a likelihood of whether level of PCT is above a clinical threshold of 2 ng / mL in a sample.

[0294] Detailed description of the figures

[0295] Fi g.1 : Early assessment of whether level of PCT is above a clinical threshold of 2 ng / mL in a sample. The level of PCT was determined in samples with different concentrations of PCT (below 2 ng / mL, 2 ng / mL and above 2 ng / mL) by performing an automated homogeneous immunoassay comprising TR-FRET using the B R A H M S KRYPTOR systems. The signal produced by TR- FRET was measured at two different wavelengths at different timepoints (signal shown on Y-axis as ratio of the signals measured at the two different wavelengths). The signal at 120 seconds (120 s or 2 minutes) after initiation of the assay is calculated by interpolation of the signal measured at different timepoints. It can be seen that the signal at 120 seconds is substantially higher in the sample with a concentration of PCT above 2 ng / mL compared to the samples with a PCT concentration equal or below the clinical threshold of 2 ng / mL.

[0296] EXAMPLES

[0297] The invention is demonstrated through the examples disclosed herein. The examples provided represent particular embodiments and are not intended to limit the scope of the invention. The examples are to be considered as providing a non-limiting illustration and technical support for carrying out the invention.

[0298] Early assessment of whether a biomarker concentration in a sample is above a clinical threshold value by usinq an automated homoqeneous immunoassay Example 1: General principle of the automated homogeneous immunoassay

[0299] The method was performed by using a B R A H M S KRYPTOR analyzer.

[0300] In the measurement process on a KRYPTOR analyzer, the samples are analysed by employing a homogeneous phase immunoassay, particularly a sandwich immunoassay. Therein, two specific antibodies binding to a biomarker such as PCT are labelled with matching fluorescent tracers (the first being termed doner, the second being termed acceptor). The labelled antibodies are termed “conjugates”.

[0301] Within the measurement process the two conjugates are mixed with the sample comprising the biomarker, leading to the conjugates progressively binding to the biomarker over time. When the two conjugates are bound to a biomarker molecule, the labels off the conjugates are in close proximity resulting in a FRET-mechanism and fluorescence emission of the acceptor when illuminated by a pule laser with a suitable wavelength. Thereby, the intensity of the fluorescence emitted is proportional to the concentration of the number of complexes of the biomarker and the two antibodies formed at the time of measurement and thus increases over time due to the formation of further biomarker-antibody complexes. The precise concentration of the biomarker (actual concentration, within the limit of accuracy of the device upon complete concentration measurement) can be determined when a plateau in fluorescence intensity reached, usually occurring after 10 to 30 minutes, depending on the biomarker measured.

[0302] The measurement of the fluorescence intensity is a non-destructive measurement and does not influence the further course of the formation of further biomarker-antibody complexes over time. It is thus possible to perform multiple measurements of fluorescence intensity over time to assess the kinetics of the formation of the biomarker-antibody complexes.

[0303] With the method of the present invention a signal measurement is carried out within 5 minutes in order to provide an early initial assessment whether the concentration of a biomarker in a sample is above a clinical threshold value. This is exemplarily demonstrated below for the biomarkers PCT and proADM.

[0304] Example 2: Early assessment of whether a level of PCT is above a clinical threshold of 2 nq / mL in a sample

[0305] The level of PCT was determined in samples with different concentrations of PCT (below 2 ng / mL, 2 ng / mL and above 2 ng / mL) by performing an automated homogeneous immune assay comprising TR-FRET using a B R A H M S KRYPTOR instrument (Figure 1 , Tables 1 to 3).

[0306] A value of 2 ng / ml of PCT is a clinical important cut-off in the case of sepsis and established for the diagnosis and management of sepsis. A value above 2ng / ml suggests a high likelihood of sepsis. Further a healthcare professional would distinguish a patient from sepsis vs. a noninfection related systemic inflammatory response syndrome (SIRS). Clinicians would immediately start to administer antibiotics. In patients that are already under antibiosis the doctor could conclude that the treatments are not effective enough and would amend the treatment regime (dose, additional or other antibiotic drug, application (oral, invasive). Beside this the clinician could fast differentiate a severe infection with sepsis compared to a non-septic infection (mild or local) or the origin of the infection, because PCT is specifically upregulated in patients with a bacterial infection and is normally low in viral or fungal infections.

[0307] The fluorescence intensity (signal intensity) emitted by a sample comprising PCT and by a calibrator comprising a known concentration of PCT is measured at different time points (see Tables 1 to 3). For example, the calibrator contains a known concentration of the analyte (such as 27.5 pg / L for PCT). By interpolating the signals measured at the different timepoint a ratio of the signal of the sample 2 minutes after initiating the immunoassay (Rs2min) and the signal of the calibrator 2 minutes after initiating the immunoassay is calculated (Rc2min) (signal ratio Rs2min / Rc2min). This signal ratio (Rs2min / Rc2min) is compared a signal threshold value correlating to a clinical threshold value of 2 ng / mL of PCT. If the ratio is above the signal threshold value, the sample comprises a concentration of PCT higher than a clinical threshold value of 2 ng / mL.

[0308] Rs and Rc refer to a ratio of signals determined at a channel A (665 nm or 707 nm) and channel B (620 nm). This ratio is proportional to the level of a biomarker such as PCT in the sample or calibrator and considered as “signal” of the sample and “signal” of the calibrator according to the present invention.

[0309] Table 1 : Time-resolved measurement (kinetics) of the signal (Rc) of the calibrator.

[0310] Table 2: Time-resolved measurement (kinetics) of a sample (Rs) with a PCT concentration of 0.2 ng / mL (below the clinical threshold value of 2 ng / mL) and calculation of a signal ratio (Rs2min / Rc2min).

[0311] Table 3: Time-resolved measurement (kinetics) of a sample (Rs) with a PCT concentration of 6 ng / mL (above the clinical threshold value of 2 ng / mL) and calculation of a signal ratio (Rs2min / Rc2min). The signal (Rs) at 120 s is substantially higher in the sample with a concentration of PCT above 2 ng / mL (6 ng / mL; see Table 3) compared to the samples with a PCT concentration equal or below the clinical threshold of 2 ng / mL (0.2 ng / mL and 2 ng / mL; see Figure 1 and Table 2). Further, the signal ratio (Rs2min / Rc2min) is substantially higher in the sample with a concentration of PCT above 2 ng / mL compared to the samples with a PCT concentration equal or below the clinical threshold 2 ng / mL (see Tables 2 and 3, Figure 1).

[0312] Example 3: Determination of the signal threshold value for PCT and MR-proADM:

[0313] To determine the signal threshold value several samples of patients with acute medical conditions with a concentration of the biomarkers equal to or substantially equal to the relevant clinical threshold value are employed. The concentration of biomarkers is determined in these samples by using the method of the present invention and calculating the signal ratio (Rs2min / Rc2min). This refers to a ratio between the signal of the sample to the signal of the calibrator, after two minutes. Based on these signal ratios determined, the threshold is set (Tables 4 to 8). Specificity, sensitivity false negative value (of the determined ratios is considered when determining the signal threshold value. Preferably the false negative value of the signal threshold value set is equal or below 5 %.

[0314] Table 4: Signal threshold value of 0.333 for a clinical threshold value for PCT of 2 ng / ml

[0315] Table 5: Signal threshold value of 0.3 for a clinical threshold value for PCT of 2 ng / ml

[0316] Table 6: Signal threshold value 1 .055 for a clinical threshold value for proADM of 1 .54 nmol / L

[0317] Table 7: Signal threshold value 1 for a clinical threshold value for proADM of 1 .54 nmol / L

[0318] Table 8: Signal threshold value of 0.93 for a clinical threshold value for proADM of 0.87 nmol / L

[0319] It is exemplary shown for the biomarkers PCT and proADM that by using a homogeneous immunoassay and a signal measurement within 5 minutes, in particular within 2 minutes, it can be easily and precisely assessed whether the concentration of a biomarker is above a clinical threshold value. If the signal ratio determined is above a signal threshold value, thus indicating that the biomarker concentration in the sample is above a clinical threshold vale, an alert is displayed to the user by the measurement system. Whereas, if the ratio determined is below the signal threshold value thus indicating that the biomarker concentration in the sample is below a clinical threshold vale, no alert is displayed to the user by the measurement system.

[0320] Subsequent to this early semi-quantitative assessment of the biomarker concentration the measurement within the homogeneous immunoassay is continued and the exact concentration of the biomarker is determined when the plateau in fluorescence intensity reached, e.g., 10 to 30 minutes, depending on the biomarker measured. The method of the present invention thus advantageously allows to perform both early semi-quantitative assessment of the biomarker concentration and exact determination of the concentration.

[0321] Such rapid semi-quantitative assessment of the biomarker level in a sample is particularly advantageous in acute clinical care settings such as the emergency department (ED) or the intensive care unit (ICU), as within only a few minutes a first assessment of the clinical status, the underlying medical condition (e.g. an infection such as sepsis or an acute cardiac condition) and the required treatment means can be assessed. For example, it may be determined whether the level of acute care biomarkers such as proADM, PCT, IL-6, troponin, sFlt-1 and copeptin is above a critical clinical threshold level indicating an acute and severe medical condition requiring immediate treatment. In the contrary the exact determination of the biomarker concentration in a patient sample such as by homogeneous immunoassays known in the prior art usually requires 10 to 30 minutes or even longer, often taking too much time in acute care situation such as in the ED or ICU and cannot be waited for before treatment is initiated.

[0322] Example 4: Early assessment of whether a level of Copeptin is above a clinical threshold in a sample:

[0323] Further validation studies for Copeptin were performed using clinical cut-off 10 pmol / l, focusing on minimizing false negatives for safe rule-out of myocardial infarction. An automated homogeneous immune assay comprising TR-FRET using a B R A H M S KRYPTOR instrument was carried out (Table 9).

[0324] Table 9: Copeptin, clinical cut-off (10 pmol / l), rule out Ml, within 3 minutes

[0325] The ratio of 0.33 achieves an extremely low false negative rate (0.7%), suitable for clinical applications where missing an acute myocardial infarction must be avoided. For example, such an approach is nearly ideal for critical care settings (where false negatives are much riskier than false positives). In other examples, a True Negative Rate (~80%) might be acceptable depending on your clinical priorities and limitation:

[0326] If patient safety and no-missed-MI are more important, this may be acceptable.

[0327] Despite a decrease in specificity (true negative rate ~80%), this trade-off is acceptable in emergency care triage settings.

[0328] In clinical practice, copeptin is used primarily for early rule-out of acute myocardial infarction (AMI), especially within the first few hours after chest pain onset. Copeptin levels rise very quickly (within minutes) during acute stress (such as myocardial infarction). Combined with cardiac troponin, copeptin allows very early and safe exclusion of myocardial infarction.

[0329] This combination of markers represents a preferred embodiment, and may improve negative predictive value (NPV), meaning if both copeptin and troponin are low, a heart attack can be ruled out with very high confidence. If copeptin <10 pmol / l and troponin is normal, acute myocardial infarction can be ruled out safely.

[0330] Copeptin alone (early phase) can be used for rapid triage in emergency departments, especially for faster decisions when Troponin is still negative. The 3-minute Copeptin measurement strategy therefore fits very well into the rule-out application.

[0331] In some embodiments, if the inventive early signal measurements for copeptin and troponin would be combined, it would enable rapid discharge or safe triage in e.g., chest pain units. The new approach could transform chest pain triage in hospitals. This example demonstrates that the invention is connected with huge clinical and economic benefits, such as shorter stays in emergency departments, reducing unnecessary admissions, and saving critical care resources.

[0332] Example 5: Early assessment of whether a level of Interleukin 6 (IL-6) is above a clinical threshold in a sample:

[0333] Validation was performed on 323 samples from one clinical site. An automated homogeneous immune assay comprising TR-FRET using a B R A H M S KRYPTOR instrument was carried out (Table 10-12). Two different clinical cut-offs for IL-6 were analyzed at the time of 3 minutes after reaction initiation:

[0334] Table 10: IL-6, Clinical threshold 150 pg / ml, signal threshold ratio 0.52 (after 3 minutes)

[0335] Using a clinical threshold of 150 pg / ml and a signal ratio threshold of 0.52, after 3 minutes after assay initiation, showed very good sensitivity (96%) and high specificity (85%). False negatives were detected at only 4%, which is considered clinically acceptable for early inflammatory risk detection, e.g. systemic inflammation (SIRS, or sepsis). When assessing a clinical threshold of 150 pg / ml, an early warning system according to the present invention would catch almost all relevant patients. In the case of no PCT-alert but an IL-6 determination (example 10-11) with an alert for IL-6, the patient could be identified as having a high likelihood of an acute inflammation such as SIRS, but not as having a high likelihood of suffering from sepsis.

[0336] Table 11 : Clinical threshold 1000 pg / ml, signal threshold ratio 1.78 (after 3 minutes)

[0337] Using a clinical threshold of 1000 pg / ml and a ratio threshold of 1 .78, after 3 minutes after assay initiation, showed excellent specificity (100%), seen by no false positives. A slightly higher false negative rate (6%) was observed but is still acceptable for high thresholds, for example 1000 pg / mL. This threshold is often used for severe systemic inflammation or infection / sepsis, as high critical risk situation. At a clinical threshold of 1000 pg / ml, the system becomes extremely specific (no false alarms) but at the cost of missing a few critical cases. The early detection method of the present invention therefore shows strong performance at both clinical thresholds, with particularly high specificity at the 1000 pg / ml threshold. This supports the utility of the invention for rapid inflammatory risk assessment in critical care.

[0338] Table 12: Summary of Copeptin, IL-6.

[0339] In summary, when using the present invention, the early detection of Copeptin enables a safe and rapid rule-out of myocardial infarction, allowing immediate discharge of low-risk patients from emergency departments. The early detection of IL-6 provides a critical tool for rapid identification of inflammatory syndromes, such as sepsis and cytokine storms. Thus, the invention directly addresses major unmet clinical needs in emergency medicine, improving patient outcomes while reducing healthcare costs. The very low numbers of false negatives means high clinical confidence, which is especially important for emergency and critical care.

Claims

CLAIMS1 . An in vitro method for assessing whether a biomarker concentration in a sample is above a clinical threshold value, said method comprising: providing a sample, providing a calibrator comprising a known concentration of said biomarker, initiating a homogeneous immunoassay in a liquid mixture, in said sample, and in said calibrator, wherein a signal measurement is carried out in said sample and said calibrator within 5 minutes of initiating said immunoassay, and when a ratio of the signal of the sample and the signal of the calibrator (signal ratio sample / calibrator) is above a signal threshold value, reporting that said signal ratio is above said signal threshold value within 5 minutes of initiating said immunoassay, wherein a signal ratio above said signal threshold value indicates the biomarker concentration in said sample is above the clinical threshold value.

2. The method according to claim 1 , wherein said immunoassay comprises a time-resolved fluorescence resonance energy transfer (TR-FRET) and / or a time-resolved amplified cryptate emission (TRACE) reaction, preferably with non-radiative energy transfer between a donor label and an acceptor label coupled to first and second binding agents, respectively, that bind to said biomarker, wherein proximity of the donor and the acceptor label in complex with said biomarker produces a signal.

3. The method according to any one of the preceding claims, wherein the signal measurement is carried out at a first wavelength and a second wavelength, preferably at a first wavelength of 650 to 720 nm and at a second wavelength of 600 to 630 nm, more preferably at a first wavelength of 660 to 710 nm and at a second wavelength of 610 to 620 nm, and the signal of the sample and the signal of the calibrator is a ratio of the signal measured at the first and the second wavelength for the sample and the calibrator, respectively.

4. The method according to any one of the preceding claims, wherein the biomarker is selected from the group consisting of procalcitonin (PCT), proadrenomedullin (proADM), interleukin-6 (IL-6), copeptin and troponin, or fragment(s) thereof.

5. The method according to any one of the preceding claims, wherein the biomarker is procalcitonin (PCT) or fragment(s) thereof.

6. The method according to any one of the preceding claims, wherein the biomarker is mid- regional proadrenomedullin (MR-proADM).

7. The method according to any one of the preceding claims, wherein the signal measurement and reporting occurs within 3 minutes of initiating the immunoassay, preferably at about 2 minutes after initiating the immunoassay.

8. The method according to any one of the preceding claims, wherein one or more of said first and second binding agents are monoclonal or polyclonal antibodies or fragments thereof that bind different epitopes of said biomarker.

9. The method according to any one of the preceding claims, wherein the biomarker is PCT or fragment(s) thereof and the clinical threshold value is 2 ng / mL ±10%, the biomarker is MR-proADM and the clinical threshold value is 1 .54 nmol / L ±10% or 0.87 nmol / L ±10%, or the biomarker is copeptin and the clinical threshold value is 10 pmol / L ±10%.

10. The method according to claim 3, wherein the biomarker is PCT or fragment(s) thereof, the clinical threshold value is 2 ng / mL ±10%, and the signal threshold value is 0.3 ±10%, preferably for 2 minutes, the biomarker is MR-proADM, the clinical threshold value is 1 .54 nmol / L±10% and the signal threshold value is 1 ±10%, preferably for 2 minutes, or the biomarker is proADM or fragments(s) thereof, the clinical threshold value is 0.87 nmol / L ±10% and the signal threshold value is 0.93 ±10%, preferably for 2 minutes.11 . The method according to any one of the preceding claims, comprising a diagnosis, prognosis and / or risk stratification of a medical condition in a subject.

12. The method according to the preceding claim, wherein the medical condition is an infectious disease, such as sepsis, severe sepsis or septic shock, and / or an acute medical condition, such as an acute cardiac condition.

13. The method according to any one of the preceding claims, wherein the donor label comprises a rare earth cryptate or chelate, preferably comprising a lanthanide ion, and the acceptor label comprises a fluorescent or chemiluminescent dye.

14. The method according to any one of the preceding claims, wherein the donor label comprises a terbium cryptate or chelate and the acceptor label comprises a fluorescent dye with excitation and emission spectra compatible with the terbium excitation and emission spectra (in a time-resolved fluorescence resonance energy transfer (TR-FRET) reaction and / or a time-resolved amplified cryptate emission (TRACE) reaction), preferably wherein the donor label comprises a terbium cryptate or chelate with emission spectra at 485-495, 540-550, 585-595 and / or 615-625 nm, and the acceptor labelcomprises a fluorescent dye with a excitation spectrum corresponding to the donor label emission spectrum, and an emission spectrum distinct from the donor label emission spectra, preferably in a wavelength range of 500-570, preferably 510-560, or 515-555 nm (green), or 580-770, preferably 600-780, or 670-690 nm (red).

15. A system for assessing whether a biomarker concentration in a sample is above a clinical threshold value, wherein said system is configured for: initiating an automated homogeneous immunoassay in a liquid mixture in said sample and a calibrator comprising a known concentration of said biomarker, said immunoassay preferably comprising time-resolved fluorescence resonance energy transfer (TR-FRET), with non-radiative energy transfer between a donor label and an acceptor label coupled to first and second binding agents, respectively, that bind to said biomarker, wherein proximity of the donor and the acceptor label in complex with said biomarker produces a signal, wherein the system is configured to carry out a signal measurement in said sample and said calibrator within 5 minutes of initiating said immunoassay, and when a ratio of the signal of the sample and the signal of the calibrator (signal ratio sample / calibrator) is above a signal threshold value, the system reports that said signal ratio is above said signal threshold value within 5 minutes of initiating said immunoassay, wherein a signal ratio above said signal threshold value indicates the biomarker concentration is above the clinical threshold value.

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