MR-proADM as marker of extracellular volume status of subject

Abstract

The present invention relates to MR-proADM as a marker of the extracellular volume status of a subject. In particular, the present invention relates to a method for determining an extracellular volume status of a subject. The method comprises determining, in a sample obtained from a subject, the level of a marker, i.e., proadrenomedullin (proADM), or a fragment thereof, preferably MR-proADM. Furthermore, the present invention relates to a method for in vitro diagnosis, risk stratification, therapy control and / or surgical control of a condition or medical condition in a subject wherein the extracellular volume status and salt retention in the subject are determined by the methods provided herein. Furthermore, the present invention relates to a kit and / or diagnostic device for performing the method provided herein.

Description

[0001] The present application is a divisional application of an application with an international application date of November 24, 2016, international application number PCT / EP2016 / 078702, which entered the Chinese national phase on May 28, 2018, application number 201680069503.1, and invention name “MR-proADM as a marker of the extracellular volume state of the subject”. Technical Field

[0002] The present invention relates to the determination of the extracellular volume status of a subject, in particular a patient under health care, most particularly under intensive care. The method comprises determining the level of pro-adrenomedullin (proADM) or fragments thereof, in particular mid-pro-adrenomedullin (MR-proADM), in a sample obtained from the subject. Furthermore, on the basis of the MR-proADM level the fluid balance and / or salt balance can be determined, which in turn is indicative of the extracellular volume status of the subject. Background Art

[0003] The exact blood volume is difficult to assess. Hematocrit can be estimated by determining the hemoglobin concentration (also referred to herein as "Hb") level (Jacob, 2012). Extracellular volume can be estimated based on the subject's weight, e.g., 60% of the body is made up of water, i.e., 42 L for a 70 kg patient, and the extracellular volume is 40% of the body's water, i.e., 17 L for a 70 kg patient; see Figure 1. In clinical practice, in many acute and less acute critical care situations, the "effective" blood volume is estimated several times daily to guide and control the patient's prescription, based on the collection of dynamic information about changes in intravascular pressure and / or cardiac output measurements. This estimate is consistently used as the basis for basic therapeutic decisions involving the amount of volume expanders and the use of catecholamines or blood transfusions. Every day, an estimated 40% of patients in intensive care are offered volume expanders after evaluation (Finfer, 2011). Achieving an appropriate blood volume while avoiding positive fluid balance is a dilemma in the daily care of patients with acute inflammation, such as traumatic stress or sepsis. This challenge is very common in the critical care and anesthesiology literature (Rivers et al., 2001; Chappel et al., 2008; Sakr et al., 2005; Bagshaw et al., 2008; Payen et al., 2008; Murphy et al., 2009; Boyd et al., 2011; Kelm et al., 2015; and Acheampong et al., 2015). Treatment recommendations point to the need for acute control of higher cardiac output to ensure adequate oxygen delivery to the organs. Fluid challenges, consisting primarily of salt and water, are the main tools proposed for volume expansion (Cecconi et al., 2009).

[0004] Increasing blood volume with salt in combination with water causes an increase in extracellular volume, especially when the diuretic effect is reduced by shock. In addition, in the setting of acute inflammation, capillary permeability may be extremely increased, exacerbating the risk of fluid accumulation (Chappell et al., 2009; Jacob et al., 2009; and Ostrowski et al., 2015). It is recognized that excessive volume expansion can provoke organ dysfunction such as acute lung injury, abdominal compartment syndrome, or renal dysfunction (Sakr et al., 2005; Bagshaw et al., 2008; Sakr et al., 2012; and Besen et al., 2015). In addition, studies have reported an increased mortality rate caused by water-sodium overload (Boyd et al., 2011; Kelm et al., 2015; and Acheampong et al., 2015). A cumulative positive fluid balance of 3 to 4 kg of weight gain or 27 to 36 g of salt resulting from a 3 to 4 liter increase in water is considered a threshold for increased mortality and morbidity (Lobo et al., 2002; Brandstrup et al., 2003; and Bjerregaard et al., 2005). Therefore, the accuracy of this assessment is very important. Analytical methods used to prevent or correct these phenomena focus on the “effective” blood volume based on the collection of dynamic information about changes in intravascular pressure and / or cardiac output measurements. Although this strategy has been shown to be effective in the first few hours of shock, it does not prevent excessive plasma expansion (Hilton, 2011).

[0005] Blood transfusion prescriptions are also determined by intravascular volume. Transfusion thresholds are usually considered to be based on hemoglobin (Hb) levels or hematocrit, the latter being by definition the ratio of red blood cell volume to total blood volume. Numerous clinical trials conducted in a variety of critical care settings acknowledge that for Hb concentrations between 7 and 11 g / dL, imprecision makes it difficult to accurately assess the circulating red blood cell volume (Takanishi, 2008; Dorbout Mees, 2011; Jacob, 2012). Indeed, although clinical trials in broad populations of critical care patients have shown that transfusions are ineffective and that a policy of limiting prescriptions using a threshold of 7–8 g / dL is beneficial, other clinical trials conducted in targeted populations have shown that low Hb is detrimental to outcomes (Naidech, 2007; Kellert, 2011). Furthermore, these arbitrary thresholds are controversial due to lack of accuracy and the fact that they do not take into account individual clinical circumstances (Klein, 2015).

[0006] It is possible to obtain a direct and accurate measurement of the total blood volume and to derive the blood cell and plasma volume from it. However, this test is rarely performed because it is costly, time-consuming and labor-intensive. Therefore, this method is only applied to specific diseases (such as polycythemia vera). The most reliable measurement is performed by labeling the patient's red blood cells with chromium-51 (Gore, 2005). Although this test is accepted as the gold standard for measuring intravascular volume, it is impossible to repeat it every day (Gore, 2005). By measuring the albumin distribution volume by labeling albumin with iodine-125, the albumin distribution in the body can be determined. This protein is much more sensitive to capillary permeability damage than red blood cells. As observed for the chromium-51 method, the iodine-125 method cannot be repeated every day, so it is only applied to specific diseases. In addition, this measurement is not suitable when immediate information on the patient's volume status is required, such as in the case of patients in intensive care units.

[0007] In many intensive care units, nurses systematically measure fluid balance by daily weight or daily calculation of fluid input and output. However, in daily practice, nurses cannot devote the necessary time to collect the required information. Moreover, this method is imprecise and never takes into account the assessment of salt balance, a parameter that indicates changes in extracellular volume.

[0008] In health care, especially intensive care, there is a fundamental need for improved methods of assessing the extracellular volume status of a subject in order to improve monitoring of tissue oxygen supply and balance therapy in a less crude manner. Furthermore, improved methods of assessing the extracellular volume status are crucial because a positive daily fluid and salt balance can cause edema, and in critically ill patients with acute kidney injury (Payen, 2008), acute respiratory distress syndrome (Jozwiak, 2013), trauma (Elofson, 2015), subarachnoid hemorrhage (Kissoon, 2015) or sepsis (Acheampong, 2015), the persistence of a positive daily fluid balance over time is associated with a higher mortality rate.

[0009] Therefore, the technical problem to be solved by the present invention is to provide means and methods to provide a rapid and reliable way of assessing the extracellular volume status of a subject.

[0010] The technical problem is solved by providing the embodiments presented below and characterized in the claims. Summary of the invention

[0011] The present invention relates to a method for determining the extracellular volume status of a subject, wherein the method comprises determining the level of pro-Adrenomedullin (proADM) or a fragment thereof, preferably mid-pro-Adrenomedullin (MR-proADM), in a sample obtained from the subject.

[0012] Furthermore, the present invention relates to a method for determining the fluid balance, salt balance and / or hematocrit status of a subject, wherein the method comprises determining the level of pro-Adrenomedullin (proADM) or a fragment thereof, preferably mid-pro-Adrenomedullin (MR-proADM), in a sample obtained from the subject.

[0013] The present invention solves the technical problems identified above, since, as documented hereinafter and in the accompanying examples, it has unexpectedly been found that there is a surprisingly strong statistical relationship between the level of pro-adrenomedullin (proADM) or a fragment thereof, preferably mid-pro-adrenomedullin (MR-proADM), and the extracellular volume status of a subject.

[0014] The results of the clinical study are documented in the accompanying Examples. This clinical study demonstrated that among all biomarkers tested, including Cortisol, catecholamines, renin, angiotensin II, the aldosterone system (RAAS), vasopressin as reflected by CT-pro-AVP (also referred to herein as "copeptin"), endothelin as reflected by proendothelin and natriuretic peptides as reflected by proatrial natriuretic peptide (MR-ProANP), erythropoietin (EPO) and proadrenomedullin as reflected by, for example, MR-pro-ADM, MR-proADM had an unexpectedly strong statistical relationship with the extracellular volume status of the subject (see, e.g., FIG2 , Table 6). This article documents that this relationship is independent of the type of clinical condition the patient has on days 2, 5, and 7 after admission, for example, patients with aneurysms (e.g., aneurysmal subarachnoid hemorrhage (SAH)), multiple trauma (e.g., severe trauma without head trauma (PT)), brain injury or head injury (e.g., severe brain trauma (SBT)), or postoperative patients such as patients with postoperative peritonitis with shock (P); see, e.g., Figure 3.

[0015] Thus, it is shown herein that proADM or fragments thereof, preferably MR-proADM, are good surrogate for the extracellular volume status of a subject.

[0016] The accompanying examples show that high or increased levels of MR-proADM are strongly associated with an increase in salt and / or water in the extracellular volume during the first week after admission to hospital in critically ill patients (see, e.g., FIG. 2 and Example 1), and that almost all subjects had a positive fluid balance and / or salt balance, i.e., an increase in extracellular volume. The increase in extracellular volume is reported as a change in salt balance and a change in water balance in the subject. It is further shown in the accompanying examples that the positive fluid balance and / or salt balance is not associated, e.g., with total blood volume or plasma volume; see, e.g., Example 1.

[0017] To increase plasma volume, a physician may administer a fluid infusion (e.g., crystalloid fluid) to a patient. Indiscriminate fluid manipulation (e.g., by aggressive fluid therapy) may increase fluid migration to the extracellular volume, e.g., into the interstitial space, which in turn may cause, e.g., interstitial edema. The accompanying examples demonstrate that the effective volume is the arterial blood volume that perfuses the tissue.

[0018] As shown herein, MR-proADM has a significant relationship with fluid balance and / or salt balance (e.g., Examples 1 to 4, Figure 2). Specifically, it is demonstrated herein that high levels of MR-proADM are indicative of volume overload. For example, high levels of MR-proADM, e.g., at least 1 to at least 1.5 nmol / l MR-proADM, are indicative of fluid overload. In addition, high levels of MR-proADM and, e.g., at least 27 g to at least 36 g Na+ are indicative of a fluid overload. + and / or an increase of at least 3 L to at least 4 L of water is an alarm signal for the physician to take immediate appropriate action. Excessive salt and / or fluid balance is considered a risk factor for morbidity and mortality in critically ill patients (Acheampong et al., 2015). Therefore, the method of the present invention, comprising the measurement of MR-proADM levels (or the level of proADM or another fragment of proADM), has a high medical potential for quickly, conveniently and reliably determining a subject's fluid balance, salt balance, hematocrit status and extracellular volume, and for determining whether a subject has a critical health status.

[0019] Furthermore, the accompanying examples document that the evaluation of other covariates, such as additional markers and parameters, improves the discrimination power of the single marker proADM (or its fragments), preferably MR-proADM. For example, MR-proADM alone has a good discrimination power for fluid balance with an AUC ("area under the curve") of 0.82 and a variance of 35%, and for salt balance with an AUC of 0.79 and a variance of 42%; see, e.g., Example 2. The inclusion of other markers and other parameters, such as sex, age, serum total protein, BMI, weight and Hb, in addition to MR-proADM, further improves the prediction of fluid balance and sodium balance, e.g. with a ROC curve of 92% (see Tables 13 and 14 and Example 4).

[0020] In many intensive care units, nurses systematically measure fluid balance by daily body weight or daily calculation of fluid input and output. These methods are imprecise. For example, a weak relationship between body weight and fluid balance was found in the accompanying examples (r 2 = 0.33, see the accompanying Example 1). As shown below, conventional markers of extracellular volume described in the prior art, such as plasma proteins or hemoglobin, have a weak relationship with salt and fluid balance (e.g., for plasma proteins and ∆Na + For example 2 = 0.44, or for plasma proteins and ∆H2O r 2 =0.35; for Hb and ∆Na + For example 2 = 0.15, or for Hb and ∆H2O r 2= 0.24; see the accompanying Example 1). In the accompanying Examples, the measurement of salt and fluid balance requires many biological samples and this analysis is always performed and controlled by two physicians, making this procedure very time-consuming and labor-intensive. It is recorded herein that proADM or a fragment thereof, preferably MR-proADM, used in the method of the invention, provides a faster and more accurate measurement of salt balance and / or fluid balance and thus of the extracellular volume state. Therefore, proADM or a fragment thereof, preferably MR-proADM, can be used as an emergency surrogate. In various emergency situations, such as in the first few days after shock, timeliness is crucial. Delayed detection of overload after organ damage such as acute lung injury, abdominal compartment syndrome and renal insufficiency can have serious and potentially fatal consequences. In the accompanying Examples, it was also surprisingly shown that there is a significant relationship between the Sequential Organ Failure Assessment (SOFA score) (Vincent et al., 1996) and salt and fluid balance. This predictive model also documents that proADM or fragments thereof, preferably MR-proADM, are advantageous surrogate and bedside tool.

[0021] Thus, the present invention relates to a method for in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy management / control and / or surgical control of a disorder or medical condition in a subject, wherein the extracellular volume status of the subject is determined by measuring the level of proADM or a fragment thereof, preferably MR-proADM, in whole blood, plasma, serum or urine. The extracellular volume status of the subject may also reflect the sodium retention of the subject.

[0022] Compared with conventional methods, the present invention has the following advantages: the method of the present invention is rapid, easy to perform and accurate for determining the extracellular volume status of a subject, and provides a reliable prediction of the subject's extracellular volume status and positive fluid balance and / or positive salt balance.

[0023] Another advantage of the method of the present invention is that fluid balance and salt balance are related to SOFA score. Therefore, the method provided herein provides a reliable and convenient way to identify critically ill subjects with the risk of organ dysfunction or organ failure caused by edema caused by positive fluid and / or salt balance. In addition, the method of the present invention allows the determination of hematocrit status. Example 3 and in particular Table 9 record the improved predicted value of the method of the present invention compared to the predicted value of hematocrit based on Hb alone.

[0024] In addition, the methods of the invention provided herein can stratify patients with positive salt balance and, therefore, patients with sodium retention, which may be a risk factor for hypertension, renal or heart failure, and pulmonary edema. These patients may require different treatments that direct salt mobilization from the interstitium to the intravascular system.

[0025] In the accompanying examples it is surprisingly shown that MR-proADM has a statistically significant relationship with the extracellular volume status of a subject. Therefore, the present invention relates to a method for determining the extracellular volume status of a subject, wherein said method comprises determining the level of the marker proADM or a fragment thereof, preferably MR-proADM, in a sample obtained from said subject.

[0026] In certain cases, the present invention relates to the use of the marker mid-regional pro-adrenomedullin (MR-proADM) for determining the extracellular volume status of a subject.

[0027] Adrenomedullin (ADM) is a peptide discovered as a hypotensive peptide comprising 52 amino acids that has been isolated from human pheochromocytoma (Kitamura et al., 1993). Adrenomedullin (ADM) is encoded as a precursor peptide ("pre-pro-adrenomedullin" or "pre-proADM") comprising 185 amino acids, which is given herein in SEQ ID NO: 1. ADM comprises positions 95-146 of the pre-proADM amino acid sequence and is a splice product thereof.

[0028] "Pro-Adrenomedullin" ("Pro-ADM") refers to pre-proADM without the signal sequence (amino acids 1 to 21), i.e., amino acid residues 22 to 285 of pre-proADM. "Mid-pro-Adrenomedullin" ("MR-proADM") refers to amino acids 42 to 95 of pre-proADM. The amino acid sequence of MR-proADM is given in SEQ ID NO: 2. It is also contemplated herein that peptides of pre-proADM or MR-proADM and fragments thereof can be used in the methods described herein, such as prediction of the extracellular volume status of a subject. For example, the peptides and fragments thereof can comprise amino acids 22 to 41 of pre-proADM (PAMP peptides) or amino acids 95 to 146 of pre-proADM (mature adrenomedullin). The C-terminal fragment of proADM (amino acids 153 to 185 of preproADM) is known as adrenopressin. Peptides of proADM or fragments of MR-proADM contain, for example, 5 or more amino acids. Thus, the fragment of proADM may for example be selected from the group consisting of MR-proADM, PAMP, vasopressin and mature adrenomedullin, preferably in this context the fragment is MR-proADM.

[0029] It is also envisaged herein to determine the level of MR-proADM polypeptides having at least 75% sequence identity, such as 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence shown in SEQ ID NO: 2, wherein higher sequence identity values ​​are preferred. According to the present invention, in the context of two or more amino acid sequences, the terms "sequence identity", "homology" or "percent homology" or "identical" or "percent identity" or "percent identity" refer to the percentage of two or more sequences or subsequences that are identical or have a specified percentage of identical amino acids over a comparison window (preferably over the entire length) or over a specified region when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm known in the art or by manual alignment and visual inspection. Sequences having, for example, 70% to 90% or more sequence identity can be considered to be substantially identical. This definition also applies to the complementary sequences of the test sequences. Preferably, described identity is present in a length of at least about 15 to about 20 amino acid residues, more preferably a length of at least about 25 to about 45 amino acid residues, most preferably over the full length. Those skilled in the art know how to use algorithms as known in the art, for example based on the CLUSTALW computer program (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci. 6 (1990), 237-245) algorithm to determine the percent identity between the sequences.

[0030] When used herein, the term "level of the marker ProAdrenomedullin (MR-proADM) or fragments thereof" refers to the amount of the molecular entity of the marker ProAdrenomedullin or fragments thereof in a sample obtained from a subject. In other words, the concentration of said marker is determined in said sample. Thus, the term "level of the marker MidProAdrenomedullin (MR-proADM)" refers to the amount of the molecular entity of the marker MidProAdrenomedullin (MR-proADM) in a sample obtained from a subject. In other words, the concentration of said marker is determined in said sample. As mentioned above, it is also envisaged herein that fragments of ProAdrenomedullin (proADM), preferably MR-proADM, can be detected and quantified. Furthermore, fragments of MR-proADM can also be detected and quantified. Methods suitable for determining the level of proADM or fragments thereof, preferably R-proADM, are described in detail below. Immunoassays in various formats can be used, such as sandwich, enzyme-linked immunosorbent assay, luminescent immunoassay, rapid test formats, assays suitable for point-of-care testing, and homogeneous assays such as the Kryptor system (BRAHMS / Thermo Fisher Scientific). In addition, mass spectrometry methods can be used to detect and quantify proADM or a fragment thereof, preferably MR-proADM or a fragment thereof. The skilled person is aware of assays suitable for determining / quantifying the markers described herein.

[0031] The present invention relates to a method for determining the status of a subject's extracellular volume. As used herein, the extracellular volume is a portion of a subject's body water. A subject's body water comprises up to about 55-75% of body weight. A subject's body water is essentially composed of the subject's "extracellular volume" and "intracellular volume"; see Figure 1. When used in this article, "intracellular volume" refers to cytosol or intracellular fluid (ICF) or cytoplasmic matrix, which is a liquid present inside the cell. Typically, the intracellular volume is about 60% of body water. According to Guyton (Guyton Arthur C., (1991), p. 275), a subject whose body contains 40 L of body fluid has an intracellular volume of 25 L. When used in this article, "extracellular volume" is essentially composed of "total blood volume" and "interstitial volume". Typically, extracellular volume is about 40% of body water. Therefore, a subject containing about 40 L of body fluid has an extracellular volume of about 15 L (Guyton Arthur C., (1991), p. 275). When used in this article, "interstitial volume", "interstitial fluid" or "tissue fluid" is a solution that soaks and surrounds the tissue cells of multicellular animals. Typically, interstitial volume is about 28% of body water or about 70% of extracellular volume. As used herein, "total blood volume" or "intravascular volume" is essentially composed of "plasma volume" and "erythrocyte volume". Typically, total blood volume is about 12% of body water and contains about 50% plasma (about 15% of extracellular volume or 6% of body water) and contains about 50% hematocrit (about 15% of extracellular volume). When used herein, "erythrocyte volume" is also called "hematocrit". When used herein, "plasma volume" refers to the volume of "plasma", which is the pale yellow liquid component of blood that usually keeps blood cells suspended in whole blood; this makes plasma the extracellular matrix of blood cells. It accounts for about 55% of the total blood volume of the body. It is the intravascular fluid portion of the extracellular fluid (all body fluids outside the cells). It is mainly water (up to 95% by volume) and contains dissolved proteins (6-8%) (i.e. serum albumin, globulin and fibrinogen), glucose, clotting factors, electrolytes (Na + , Ca 2+ Mg 2+ 、HCO3 − , Cl − Plasma is the main medium for the transport of excretory products. Plasma also serves as a protein reservoir for the human body. It plays a key role in the osmotic pressure effect within the blood vessels to keep electrolytes in a balanced form and protects the body from infection and other blood disorders. When used herein, "red blood cell volume" is also called mean hematocrit or mean cell volume (MCV), which is a measure of the average volume of red blood cells (or erythrocytes).

[0032] The calculation of salt balance and / or fluid balance to estimate changes in extracellular volume is documented daily in the accompanying Examples; see accompanying Example 1. As demonstrated in the accompanying Examples, a complete input-output assessment of salt and water (content) is performed daily on the previous day in order to determine the subject's fluid balance and salt balance. It is shown that, if necessary, the subject's sodium and / or water losses can be measured by determining, for example, diuresis, ileostomy, and ventricular drainage. Sodium (Na + ) losses can be measured from fluids and can be inferred from the salt contribution; however, measuring salt balance is particularly difficult. The difference between water input (e.g., the total amount of enteral nutrition or crystalloid or colloid infusions for the day) and water losses is also calculated. Insensible losses are estimated as a function of body temperature. In the accompanying examples, "Sodium" or "Na + ” (also referred to herein as “∆Na + ”, “ΔSodium” or “Sodium balance”) and water or H2O gain or loss (also referred to herein as “∆H2O” or “Fluid balance”) are added to the previous day’s results as the cumulative “Fluid balance” or “Salt balance”, respectively.

[0033] In the accompanying examples, it was surprisingly demonstrated that the level of MR-proADM in a sample, such as a plasma sample, of a subject has a statistical relationship with said fluid balance and / or salt balance ( FIG. 2 ). Thus, the present invention relates to a method for determining the fluid balance, salt balance and extracellular volume status of a subject, wherein said method comprises determining the level of proADM or fragments thereof, preferably MR-proADM, in a sample obtained from said subject, wherein said fluid balance and / or salt balance is determined on the basis of said level of proADM or fragments thereof, preferably MR-proADM, and wherein said fluid balance and / or salt balance determines / identifies / reflects the extracellular volume status of the subject. It has been found that high MR-proADM levels are associated with high fluid balance (significant increase in body fluid) or high salt balance (significant increase in salt); see e.g. FIG. 2 . Thus, the level of proADM or fragments thereof, preferably MR-proADM, of said subject can be used to predict the salt balance and fluid balance of said subject. In other words, proADM or fragments thereof, preferably MR-proADM can be used as a direct surrogate for said fluid balance and / or salt balance. Hence, the term "on the basis of the level of (MR-)proADM" means that the level of (MR-)proADM identifies / predicts / determines the fluid balance and / or salt balance of the subject.

[0034] In the accompanying examples, the salt balance and / or fluid balance is calculated to estimate the change or fluctuation of the extracellular volume. It is known that the fluid balance and / or salt balance are related to the extracellular volume (Charra et al., 2004). Therefore, the fluid balance and / or salt balance determines the change of the extracellular volume state. The extracellular volume state refers to the fluid in the extracellular volume ( Figure 1). The extracellular volume of a subject is approximately 40% of the body water of the subject. The extracellular volume status of a subject is related to the fluid balance and / or salt balance of the subject. Therefore, changes in the fluid balance or salt balance represent changes in the extracellular volume. Therefore, changes in the fluid balance and / or salt balance of the subject allow for the estimation / determination of the extracellular volume status of the subject. In other words, proADM or a fragment thereof, preferably MR-proADM, is a direct surrogate for the fluid balance and / or salt balance of the subject and it is therefore indicative of the extracellular volume status of the subject. As used herein, "fluid balance" refers to a "water change", "water variation", "Δwater" or "∆H2O" of a subject. In other words, the "fluid balance" is the difference between the input and output of "fluid" or "water" of a subject. In a preferred embodiment of the present invention, the fluid balance is the difference between the input and output of fluid / water of a subject. In an even more preferred embodiment, the fluid balance is a cumulative fluid balance reflecting the difference between the input and output of fluid / water during the hospitalization of the subject. When used in this article, the term "hospitalization" or "each hospitalization" means the time period during which the patient is in a critical health situation. Therefore, when used in this article, the hospitalization of the object can mean the time period during which the object enters the ICU until the critical situation and / or symptom relief. Alternatively, the term refers to the time period during which the patient has accumulated a positive fluid balance of, for example, 4 L or a positive salt balance of, for example, 36 g. In other words, the time period means, for this purpose, that the object has accumulated an increase of, for example, 4 L of body fluid or 36 g of salt. In the accompanying embodiments, the fluid balance and salt balance are calculated every day. Therefore, the fluid balance is the difference between the input and output of the object's body fluid / water within the first day (every day). In a preferred case, the fluid balance is a cumulative fluid balance, which is the difference between the input and output of the object's body fluid / water within the first two days, even more preferably within the first five days, and most preferably within the first week, i.e., the difference between the input and output of the object's body fluid / water after 7 days. It is understood herein that a gain / increase in water of a subject means that the subject has more water compared to an earlier time point (e.g. one day ago) due to the output of body fluids / water being less than the gain of water. It is understood herein that a loss / reduction in water of a subject means that the subject has less water compared to an earlier time point (e.g. one day ago). It is understood herein that no change or no significant change in water of a subject means that the subject has a consistent or similar water content compared to an earlier water content (e.g. one day ago). Preferably, MR-proADM is determined at several time points, e.g. day 0 ("D0"), day 2 ("D2"), day 5 ("D5") and / or day 7 ("D7") after admission to a health care unit, in particular admission to an intensive care unit.It is understood herein that the levels of the markers and / or parameters may be determined at any time and at any time interval, for example hourly or daily (e.g. at admission D0, then on day 1 (D1), day 2 (D2), day 3 (D3), day 4 (D4), day 5 (D5), day 6 (D6) and / or day 7 (D7) after the subject enters the ICU, etc.) or a combination thereof.

[0035] In the accompanying examples it is shown that the level of MR-proADM is correlated with the fluid balance of a subject; see Figure 2. It is understood herein that a high level of proADM or a fragment thereof, preferably MR-proADM, is indicative of a gain / increase in fluid in a subject. As used herein, a subject with a "positive fluid balance" is one in which the gain of fluid is higher than the loss of fluid. Thus, the subject has an imbalance of fluid / water input and output. Thus, a subject with a positive fluid balance accumulates water / fluid in the body. Thus, the subject gains weight. In other words, a subject with an increase in water content or a gain of water has a positive fluid balance. For example, a subject treated with an infusion may have a shift of fluid / water to extravascular fluid. This fluid may shift to the extracellular volume of the subject, such as the interstitial volume. For example, it has been shown that an extracellular volume overload of more than 10 L is trapped in the body after 3 days in resuscitated patients and requires 3 weeks to excrete (Chappell et al., "A rational approach to perioperative fluid management," Anesthesiology; 2008, 109:723-40). Anatomical losses are considered to be physiological phenomena at pathological amounts, i.e., pathological accumulation of fluid within the interstitial space (Chappell, supra). Physiological fluid that migrates from the blood vessels to the interstitial space in the presence of an intact vascular barrier can be considered to contain only small amounts of protein and primarily small molecules. When this migration is quantitatively managed by the lymphatic system, the physiological migration does not cause edema such as interstitial edema. However, overwhelming the lymphatic system, such as by excessive administration of infusion fluids such as crystalloids, can cause edema. There is also a non-anatomical third space loss, which represents a fluid compartment that is functionally and anatomically separated from the interstitial volume. Losses to this third space may be accumulation of body fluids caused by, for example, surgical procedures or trauma in a space that normally contains no or little body fluid. For example, third space losses may be toward the peritoneal cavity, intestines, and traumatic tissue.

[0036] In certain cases of the present invention, a positive fluid balance of 3 to 4 kg of weight gain (during hospitalization, for example, within the first day, preferably within the first two days, even more preferably within the first five days, and most preferably within the first week) is considered to be a threshold for increased mortality and morbidity. Therefore, in certain cases of the present invention, an increase of at least 3L, preferably at least 4L, in body fluid is considered critical. An increase of at least 3L, preferably at least 4L, in the extracellular volume is considered critical. The amount of fluid increase that is considered critical also depends on the patient's characteristics such as the sex, age or weight of the object. For example, the body water of an adult female is 5 to 10% lower than that of an adult male. Therefore, it is understood herein that patients (e.g., women) with lower body weight can react more sensitively to an increase in body fluid and / or salt. In addition, the distribution of body fluid in the body fluid compartment depends on the age of the object, for example, it decreases from 75% in a newborn to 55% in an adult. Therefore, a lower increase in body fluid, such as 3L or less of body fluid, may already have serious consequences in female or elderly subjects. On the other hand, subjects with higher body weight (e.g., males) may not be as sensitive to increases in body fluid and / or salt as the lighter subjects. Thus, mortality rates may be reduced in these subjects.

[0037] In another case, a positive fluid balance of at least 4 L is considered critical. In other words, a positive fluid balance of at least 4 L indicates that the subject has an extracellular volume status that is considered critical. Specifically, it is noted in the accompanying examples that a high increase in water, such as at least 4 L of water, is an alarm signal and indicates a critical extracellular volume status and therefore a critically ill subject. Endothelial damage and / or salt retention can cause an increase in fluid balance.

[0038] As used herein, a subject with a "negative fluid balance" refers to a subject whose fluid loss is greater than fluid gain. Thus, the subject loses water or fluid and therefore loses weight. In other words, a subject with a decreased water content or water loss has a negative fluid balance. As used herein, a subject with a consistent or similar water content is referred to as a subject with a "consistent or similar fluid balance." Such a subject has balanced fluid management, and thus the fluid balance is in a balanced or normal state. In other words, the fluid / water input is consistent or similar to the fluid / water output. In other words, the subject has a normal fluid balance.

[0039] In the context of the present invention, the fluid / water balance of a subject may be increased, for example, by intravenous therapy including, for example, a volume expander. In a particularly preferred case, an increase in water in a subject means that the subject has more water / fluid than the water / fluid determined at an earlier time point, wherein said subject having more water / fluid is a subject with a positive fluid balance. In the accompanying examples it is shown that a high MR-proADM level, for example of at least 1 nmol / l, is indicative of an increase in water, i.e. a positive fluid balance and thus an increased extracellular volume status, wherein said extracellular volume status is considered critical. In other words, a positive fluid balance of at least 4 L indicates that said subject has an extracellular volume status that is considered critical.

[0040] The present invention relates to the methods provided herein, wherein the salt balance is determined on the basis of the level of proADM or a fragment thereof, preferably MR-proADM, and wherein the salt balance determines / identifies the extracellular volume state. As used herein, the "salt balance" or "sodium balance" refers to "variation in sodium", "change in sodium", "Δsodium" or "∆Na + The "salt balance" is the difference between the input and output of "salt" or "sodium" of a subject. In a preferred embodiment of the invention, the salt balance is the difference between the input and output of body fluids / water of a subject. In an even more preferred embodiment, the salt balance is a cumulative salt balance reflecting the difference between the input and output of salt / sodium of the subject during the hospital stay. In some cases, the salt balance is the difference between the input and output of salt / sodium of the subject per day within the first day. In a preferred embodiment, the salt balance is the difference between the input and output of salt / sodium of the subject within the first two days, even more preferably within the first five days, most preferably within the first week, i.e. the difference after 7 days. In the accompanying Examples, it was surprisingly demonstrated that the salt balance has a statistical correlation with the level of MR-proADM in a sample, e.g. a serum sample, of a subject ( Figure 2B). It is further shown in the accompanying examples that the fluid balance of the subject is statistically correlated with the salt balance; see Example 1. It is understood herein that the gain / increase of salt, such as sodium, of a subject means that the subject has a higher amount / content of salt, such as sodium. When used herein, a subject with a higher amount / content of salt refers to a subject with a positive salt balance. Therefore, a subject with a "positive salt balance" means herein that the subject has a higher salt gain than the salt loss of the subject. Therefore, the subject has an imbalance in the input and output of salt / sodium. Therefore, the subject with a positive salt balance accumulates salt / sodium in the body, such as salt retention. In other words, a subject with an increased amount / content of salt / sodium or a gain of sodium / salt has a positive sodium balance. When used herein, "sodium retention" or "salt retention" can indicate renal or heart failure. Salt retention can cause fluid / water retention as well as increased blood volume, increased blood pressure and inflammation. Without being limited by theory, inflammation can cause salt retention. Since MR-proADM has a strong statistical relationship with salt and / or fluid balance, MR-proADM (or proADM or another fragment thereof) may be used as a prognostic marker for inflammation (or vascular damage and permeability caused by inflammation).

[0041] As used herein, a subject with a "negative salt balance" refers to a subject in which the loss of salt, e.g., sodium, is greater than the gain of salt. Thus, the subject loses salt or fluid and therefore loses weight. In other words, a subject with a decrease in salt content or salt loss has a negative salt balance. As used herein, a subject with a consistent or similar salt content is referred to as a subject with a "consistent or similar salt balance." Such a subject has balanced salt management, and therefore the salt balance is in a balanced or normal condition. In other words, the input of salt / sodium is consistent or similar to the output of salt / sodium. In other words, the subject has a normal salt balance.

[0042] In one case, a positive salt balance means that the subject has a higher salt content and thus an increased extracellular volume state compared to an earlier time point. In the accompanying examples it is shown that a high MR-proADM level, e.g. above 1 nmol / l, indicates a salt gain, a positive salt balance and thus an increased extracellular volume state, wherein said increased extracellular volume state is considered to be critical. In particular, it is documented in the accompanying examples that a high salt, e.g. sodium gain of at least 27 g, preferably at least 36 g, is an alarm sign and indicates a critically ill patient. Thus, in certain cases of the present invention, a salt gain of at least about 27 to at least about 36 g is considered to be critical. In preferred cases of the present invention, a positive salt balance of at least 36 g is considered to be critical. In other words, a positive salt balance of at least 27 g or preferably at least 36 g indicates that the subject has an extracellular volume state that is considered to be critical.

[0043] In certain instances of the invention, the methods provided herein determine the hematocrit state of a subject. Specifically, the methods provided herein allow for determining whether a subject's hematocrit is below 20 ml / kg. A hematocrit below 20 ml / kg, or preferably below 15 ml / kg, indicates a critical hematocrit state. In certain instances, a subject's hematocrit below 20 ml / kg indicates a subject with a critical extracellular volume state, wherein the critical extracellular volume state indicates a critical health condition of the subject. Thus, a hematocrit below 20 ml / kg indicates that the subject has a positive fluid balance, wherein the positive fluid balance indicates a critical extracellular volume state.

[0044] The methods provided herein determine the hematocrit status of a subject, wherein the method comprises determining the level of proADM or a fragment thereof, preferably MR-proADM, in the sample, the level of hemoglobin in the sample, the body mass index of the subject, the sex of the subject, the age of the subject, the level of total serum protein in the sample, and optionally the weight of the subject.

[0045] In the accompanying examples, it is documented that the inclusion of further markers or parameters in the statistical analysis improves the predictive power of proADM or a fragment thereof, preferably MR-proADM; see for example Examples 1 to 4. The statistical analysis surprisingly found a consensus model including MR-proADM that has a significant relationship with the extracellular volume status of the subject. As used herein, a consensus model comprises more than one marker and parameter, and on the basis of which the fluid balance and / or salt balance and / or the extracellular volume status of the subject can be determined. In other words, in certain cases, the present invention relates to a method, wherein a set (or sets) of markers and parameters is determined. Thus, in the context of the present invention, further parameters and / or markers can be determined. In other words, the method according to the present invention can be performed in combination with further markers, parameters and / or methods. This means that the measuring method according to the present invention can be performed particularly advantageously as a multiparameter diagnostic. Thereby, at least one further marker, preferably selected from vasodilators, is additionally determined.

[0046] In certain instances of the invention, the methods provided herein comprise determining the level of at least one additional marker selected from the group consisting of hemoglobin, total serum protein, renin, pro-atrial natriuretic peptide (proANP), C-terminal arginine-pro-vasopressin (CT-proAVP) protein, erythropoietin, angiotensin II, aldosterone, cortisol, adrenaline, epinephrine, catecholamines, and pro-endothelin-1 (pro-ET-1).

[0047] In some cases, the present invention relates to the use of an additional marker selected from hemoglobin, total serum protein, renin, pro-atrial natriuretic peptide (proANP), C-terminal arginine-pro-vasopressin (CT-proAVP) protein, erythropoietin, angiotensin II, aldosterone, cortisol, adrenaline, epinephrine, catecholamines and pro-endothelin-1 (pro-ET-1).

[0048] In certain preferred cases, the methods provided herein also include determining the level of the marker "hemoglobin (hemoglobin or heamoglobin)". "Hemoglobin" or "Hb" is an iron-containing metalloprotein that transports oxygen in the red blood cells of vertebrates. Hb concentration can be measured in the context of routine blood tests, usually as part of a complete blood count. Normal Hb concentrations are: for men: 13.8 to 18.0 g / dL (138 to 180 g / L, or 8.56 to 11.17 mmol / L); for women: 12.1 to 15.1 g / dL (121 to 151 g / L, or 7.51 to 9.37 mmol / L); for children: 11 to 16 g / dL (111 to 160 g / L, or 6.83 to 9.93 mmol / L); or for pregnant women: 11 to 14 g / dL 9.5 to 15 (usual values ​​during pregnancy) (110 to 140 g / L, or 6.83 to 8.69 mmol / L). In the context of the present invention, low hemoglobin means that the individual's hemoglobin level is below the lower limit of normal values ​​for their age and sex (see normal range of values ​​above). For example, a 19-year-old male has a low hemoglobin level if the detected blood value is below 13.6 g / dl. In the context of the present invention, a high hemoglobin level means that the measured hemoglobin level is above the upper limit of normal values ​​for the age and gender of the individual (see normal values ​​above). For example, a 19-year-old male with a detected hemoglobin level above 18.2 g / dl has a high hemoglobin level.

[0049] When used in this article, "serum total protein" refers to the total amount of protein in the blood. In a preferred case, the serum total protein refers to the total amount of protein in serum or plasma. The "serum total protein" is measured in routine tests and used in ICU and other medical services. The two main protein components in serum or plasma are albumin and globulin. Globulin is composed of different proteins called α, β and γ types. The test report for serum total protein reports the separate values ​​of total protein, albumin and globulin. Serum total protein can be determined, for example, by biuret reagent or by refractometry. Hypoproteinemia is caused by insufficient synthesis caused by liver failure, malnutrition or by renal loss. There are two main reasons for the increase in serum protein concentration: dehydration, in which less water is present in the body, and reduced blood volume. The most commonly overproduced protein is immunoglobulin, and its level can increase in infection and in hematological tumors. The normal range of serum total protein is about 60 to about 80 g / l.

[0050] As used herein, "renin" or "angiotensinogenase" is an enzyme that participates in the body's renin-angiotensin aldosterone system (RAAS), which mediates extracellular volume (i.e., plasma, lymphatic and interstitial fluid volume) and arterial vasoconstriction. Thus, it regulates the body's mean arterial blood pressure. The level of renin is preferably measured in the plasma or serum of a subject.

[0051] As used herein, "pro-atrial natriuretic peptide" or "proANP" refers to a prohormone comprising 128 amino acids. As used herein, the peptide comprising 28 amino acids (99-126) of the C-terminal segment of the 128 amino acid prohormone (proANP) is referred to as the true hormone ANP. After the release of ANP from its prohormone proANP, an equimolar amount of the remaining larger peptide of proANP consisting of 98 amino acids, namely the N-terminal proANP (NT-proANP; proANP (1-98)), is released into the circulation. Due to the significantly higher half-life and stability of NT-proANP, NT-proANP can be used as a laboratory parameter for diagnosis, follow-up and therapy control; see, for example, Lothar Thomas, ed., Labor und Diagnose, 5th expanded edition, Chapter 2, Section 2.14, Kardiale Diagnostik, pages 116-118 and WO 2008 / 135571. The level of proANP is preferably measured in the plasma or serum of a subject.

[0052] As used herein, endothelin-1 is derived from a larger precursor molecule known as proendothelin-1. Proendothelin-1 can be proteolytically processed into various fragments as described (EP 2 108 958 A1; "Proteolytic processing pattern of the endothelin-1 precursor in vivo. Peptides", 2005 Dec; 26(12): 2482-6). These fragments undergo proteolytic degradation in the blood circulation, which can occur quickly or slowly depending on the type of fragment and the type and concentration / activity of proteases present in the circulation. Therefore, according to the present invention, the level of any one of these fragments of at least 12 amino acids, preferably at least 20 amino acids, more preferably at least 30 amino acids can be measured. Preferably, C-terminal pro-ET-1 (CT-proET-1) or a fragment thereof can be measured. The level of endothelin-1 is preferably measured in the plasma or serum of the subject.

[0053] As used herein, "vasopressin" refers to "AVP". Vasopressin is a potent vasoconstrictor. Its prohormone measurement has been examined as a prognostic and diagnostic factor in cases of diabetes insipidus. Vasopressin or antidiuretic hormone (ADH) is one of the key regulators of body water and water balance. Its secretion is partly related to stress, causing arterial pressure to rise and water to be absorbed, bringing the risk of hyponatremia. However, ADH is unstable. In addition, its concentration depends on its binding to platelets and is therefore unstable. The C-terminal part of the prohormone, "CT-proAVP", is a more stable precursor of ADH, and its plasma concentration reflects ADH secretion (Struck, 2005; Morgenthaler, 2007). When used herein, the C-terminal part of the prohormone is referred to as "CT-proAVP" or "copeptin". Increased plasma levels after septic shock or hemorrhage are associated with blood osmolality and mortality (Morgenthaler, 2007). The level of CT-proAVP is preferably measured in the plasma or serum of a subject.

[0054] Angiotensin I is converted to angiotensin II by removal of two C-terminal residues by angiotensin converting enzyme (ACE), primarily in the lungs (but also in endothelial and renal epithelial cells). Angiotensin II functions as an endocrine, autocrine / paracrine, and intracrine hormone. The level of angiotensin II is preferably measured in the plasma or serum of a subject.

[0055] In some cases, the methods provided herein further include determining at least one parameter selected from the following of the subject: body mass index, weight, age, sex, IGS II, lactate, sodium intake, fluid intake, and patient group. In certain preferred cases, the methods provided herein further include determining at least one parameter selected from body mass index, weight, age, and sex of the subject.

[0056] As used herein, body mass index (BMI) is a value derived from the mass (weight) and height of the subject. BMI is defined as the subject's body mass, i.e., weight, divided by the square of the subject's height, and is commonly expressed in kg / m 2 BMI is expressed in units, derived from weight in kilograms and height in meters. BMI can also be determined using a table or graph (reference value), which uses contour lines or colors for different BMI categories to show the change of BMI with mass and height, and two different units of measurement can be used. BMI is an attempt to quantify the amount of tissue material (muscle, fat and bone) in an individual, and then classify the individual as underweight, normal weight, overweight or obese based on this value. The generally accepted BMI range is underweight: less than 18.5, normal weight: 18.5 to 25, overweight: 25 to 30, and obesity: higher than 30. In some cases of the present invention, the BMI is determined on day 0, such as when the patient is admitted to the hospital.

[0057] As used herein, "weight" refers to the mass of a subject in kg (see BMI). In certain instances of the present invention, the weight is determined on day 0, e.g., when the patient is admitted to a hospital. In the context of the present invention, normal weight can theoretically be calculated according to the Devin formula or the Hamwi method. According to the Hamwi method, the ideal weight for men is 48 kg plus 2.7 kg for every 2.54 cm over 1.5 m. For women, it is 45 kg plus 2.3 kg for every 2.54 cm over 1.5 m. Values ​​below or above these normal values ​​increase the risk of becoming a critical subject.

[0058] As used herein, "age" refers to the length of time in years that an individual has lived. Preferably, the parameter is calculated in the method by adding the square and cube of age, i.e., age 2 and age 3 To add weight.

[0059] As used herein, "IGS II" (which is an abbreviation for "Indice de Gravité Simplifié") or "SAPS II" (which is an abbreviation for "Simplified Acute Physiology Score II") refers to a system for classifying the severity of a disease or condition (see Le Gall JR et al., "A new Simplified Acute Physiology Score (SAPS II) based on aEuropean / North American multicenter study", JAMA. 1993; 270(24): 2957-63). The "IGS II score" consists of 12 physiological variables and 3 disease-related variables. The point score is calculated from 12 conventional physiological measurements, information about previous health status, and some information obtained upon admission to the ICU. The IGS II can be determined at any time, preferably on the second day. The "worst" measurement is defined as the measurement associated with the highest number of points. The SAPS II score ranges from 0 to 163 points. The classification system includes the following parameters: age, heart rate, systolic blood pressure, temperature, Glasgow Coma Scale, mechanical ventilation or CPAP, PaO2, FiO2, urine output, blood urea nitrogen, sodium, potassium, bicarbonate, bilirubin, leukocytes, chronic diseases and type of admission. There is an inverse relationship between mortality and total SAPS II score. The mortality rate of subjects with a SAPS II score of 29 points is 10%, the mortality rate with a SAPS II score of 40 points is 25%, the mortality rate with a SAPS II score of 52 points is 50%, the mortality rate with a SAPS II score of 64 points is 75%, and the mortality rate with a SAPS II score of 77 points is 90% (Le Gall, supra).

[0060] As used herein, "fluid intake" refers to a subject's fluid intake over a given period of time, such as 24 hours. For example, a patient's or subject's fluid intake may be an infusion or fluid resuscitation. Preferably, the fluid intake is determined on day 0, in other words, upon or after the patient's admission to the hospital.

[0061] As used herein, the "sex" of a subject refers to the biological sex of the subject, wherein the subject is either male or female.

[0062] As used herein, "sodium intake" refers to the total amount of salt, preferably sodium, such as sodium chloride, received by an organism from external sources such as nutrients (food and fluids) or infusions. Preferably, the sodium intake is determined on day 0, in other words, at or after the patient's admission to hospital.

[0063] As used herein, "lactate" or "maximum lactate" refers to the highest lactate concentration measured in the blood. Typically, the lactate concentration is assessed daily or even more frequently. The lactate concentration in the blood can be determined by lactate oxidase spectrophotometry.

[0064] As used herein, “total blood volume,” “TBV,” or “TV” can be expressed using chromium 51 (Cr 51 ) labeled red blood cells. The total blood volume can be measured at any time, in particular between day 1 and day 3, such as on day 3; and / or between day 6 and day 10, such as on day 10. It is contemplated herein that the patient's own blood is labeled with chromium 51 (Cr 51 ) are radiolabeled and the radiolabeled red blood cells are selected. A known amount of radiolabeled red blood cells are then reinfused into the whole blood circulation. For example, two samples can be taken in the arterial catheter at two time points, such as at 10 and 30 minutes. To deduce the total blood volume (TBV) in mL or mL / kg, the radioactivity of the two samples is measured and the patient's weight is determined (Gore et al., 2005). The hematocrit number and the measured total blood volume determine the red blood cell volume (RBCV) in mL or mL / kg and the plasma volume (PV) in mL or mL / kg. Normal values ​​(±20%) are about 72±14 mL / kg for TBV, about 32±6 mL / kg for RBCV, and about 40±8 mL / kg for PV (Gore et al., 2005).

[0065] The "plasma volume" or "PV" can be measured using iodine-125 (PVI 125 ). The plasma volume can be measured at any time, for example on day 7. In the accompanying examples, PVI 125 The patient was injected with a defined amount of iodine 125 (I 125 ) radiolabeled albumin and samples were collected at several time points, such as 10 min, 30 min and 2 h after injection (Fairbanks et al., 1996). 125 The normal value of PV measured is about 45 ± 10 mL / kg (Gore et al., 2005). 125 - Plasma volume measured by albumin is slightly greater than that measured by Cr 51– Plasma volume measured by erythrocytes, due to the greater distribution volume of albumin than that of erythrocytes (Gore et al., 2005).

[0066] When used herein, "patient group" or "group" means that the patients are grouped according to their disease pattern or medical condition. When used in the accompanying examples, the subjects were divided into 4 groups, namely severe brain trauma (SBT), aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head trauma (PT) and postoperative peritonitis with shock (P).

[0067] In some cases, the method determines the extracellular volume status of a subject, wherein the method comprises determining at least one marker and / or parameter selected from proADM or a fragment thereof, preferably MR-proADM, sex, hemoglobin, total serum protein, IGS II score, fluid intake and sodium intake. In the accompanying examples, it is demonstrated that random forest analysis can be used to select the combination of markers and parameters that yields the lowest error. It was surprisingly shown herein that the best model for predicting the extracellular volume status of a subject is by using MR-proADM levels in combination with the subject's body mass index, weight, age (age 2 and age 3 ) and sex, hemoglobin and total serum protein. Thus, in the most preferred aspects of the invention, the methods provided herein comprise determining the level of proADM or a fragment thereof, preferably MR-proADM, in the sample, the body mass index, weight, age, sex of the subject, the level of hemoglobin in the sample and the level of total serum protein in the sample. As demonstrated in the accompanying Example 4, markers such as MR-proADM, total serum protein and hemoglobin have good predictive power, e.g. an AUC of 0.94 for fluid balance; see, e.g., Tables 13 and 14. Adding to the markers parameters of the subject such as body mass index, weight, age and sex, increases the AUC for fluid balance to, e.g., 0.95. The individual parameters of the subject such as body mass index, weight, age and sex have an AUC for fluid balance of, e.g., 0.88. Thus, in certain aspects of the invention, the methods provided herein comprise determining the body mass index, weight, age and sex of the subject. In a preferred aspect of the invention, the methods provided herein comprise determining the level of proADM or fragments thereof, preferably MR-proADM, in said sample, the level of hemoglobin in said sample and the level of total serum protein in said sample. In a most preferred aspect of the invention, the methods provided herein comprise determining the level of proADM or fragments thereof, preferably MR-proADM, in said sample, the body mass index, weight, age, sex, the level of hemoglobin in said sample and the level of total serum protein in said sample of said subject.

[0068] As documented in the accompanying Examples, in particular in Examples 3 and 4, different combinations of markers and parameters can be used to determine the extracellular volume status of a subject. In certain aspects of the invention, the methods provided herein determine the fluid balance of a subject, wherein the methods comprise determining the level of proADM or fragments thereof, preferably MR-proADM, in the sample, the subject's body mass index, weight, age, sex, the level of hemoglobin in the sample, the level of serum total protein in the sample, the IGS II score of the subject, and fluid intake. In certain aspects of the invention, the methods provided herein determine the salt balance of a subject, wherein the methods comprise determining the level of proADM or fragments thereof, preferably MR-proADM, in the sample, the subject's body mass index, weight, age, sex, the level of hemoglobin in the sample, the level of serum total protein in the sample, the level of sodium intake in the sample, the IGS II score of the subject, and fluid intake. As shown in the accompanying Example 3, the absence of the parameters IGS II and fluid intake had a small impact on the statistical analysis, with only a loss of 2 to 3% of the r 2 , and had no effect on AUC; see, e.g., Table 6. Thus, in certain other aspects of the invention, the methods provided herein comprise determining the level of proADM or a fragment thereof, preferably MR-proADM, in the sample, the body mass index, weight, age, sex of the subject, the level of hemoglobin in the sample, and the level of total serum protein in the sample.

[0069] In certain instances of the invention, the method comprises determining at least one other marker and / or parameter of the subject, the other marker and / or parameter selected from the group consisting of a proANP level in the sample, a total blood volume level, a hematocrit level in the sample, a red blood cell volume level, a plasma volume level, a total urine volume level, an angiotensin II level in the sample, a patient group of the subject, a cortisol level in the sample, a number of endothelial stem cells in the blood, a catecholamine level in the sample, a whole blood ionogram of the subject, a urine ionogram of the subject, a blood osmolality of the subject, a urine osmolality of the subject, blood glucose of the subject, a pro-endothelin-1 (pro-ET-1) level in the sample, a CT-proAVP level in the sample, aldosterone level in the sample, a lactate level in the sample, an Acute Physiology and Chronic Health Evaluation II (APACHE II) rating of the subject, a World Federation of Neurosurgical Societies (WFNS) rating of the subject, and a Glasgow Coma Scale (GCS) of the subject.

[0070] In the accompanying examples it is documented that there is a statistically significant relationship between the Sequential Organ Failure Assessment (SOFA score) and fluid and / or salt balance in the subject; see Examples 1 and 3 and Figure 4. MR-proADM is associated with fluid and / or salt balance. Thus, in certain cases, the Sequential Organ Failure Assessment (SOFA score) is determined on the basis of the level of proADM or a fragment thereof, preferably MR-proADM. In other words, proADM or a fragment thereof, preferably MR-proADM, is used as a surrogate marker for the SOFA score.

[0071] In certain other aspects of the invention, the sequential organ failure assessment score (SOFA score) is determined on the basis of fluid balance and / or salt balance. In the accompanying examples, it is shown that the inclusion of other parameters such as age, BMI and gender improves the predictive power of determining the SOFA score; see Figure 5 Thus, in some cases, the methods provided herein determine the SOFA score based on fluid balance and / or salt balance, wherein the methods further comprise determining at least one parameter consisting of age, body mass index and sex.

[0072] Furthermore, the present invention relates to a method, wherein the method comprises: (a) determining the level of proADM or a fragment thereof, preferably MR-proADM, in a sample from a subject, and (b1) comparing said level of proADM or said fragments thereof, preferably MR-proADM, with reference data corresponding to the level of said proADM or said fragments thereof, preferably MR-proADM, of at least one reference subject; or (b2) comparing said level of proADM or said fragments thereof, preferably MR-proADM, with data corresponding to the level of said proADM or said fragments thereof, preferably MR-proADM, obtained from a previous analysis for the same subject; (c) identifying the subject's fluid balance and / or salt balance based on said comparing step (b); and (d) determining the hematocrit status and / or the extracellular volume status based on step (c).

[0073] In other words, the present invention relates to the method provided herein, wherein the method comprises: (a) determining the level of proADM or a fragment thereof, preferably MR-proADM, in a sample from a subject, and (b1) comparing said level of proADM or said fragments thereof, preferably MR-proADM, with a reference level of proADM or said fragments thereof, preferably MR-proADM, in at least one reference subject or a population of reference subjects; or (b2) comparing said level of proADM or said fragment thereof, preferably MR-proADM, with a reference level of proADM or said fragment thereof, preferably MR-proADM, of the same subject obtained from a previous analysis; (c) identifying the subject's fluid balance and / or salt balance based on said comparing step (b); and (d) Identification of hematocrit status and / or extracellular volume status.

[0074] Furthermore, the present invention relates to an in vitro method, wherein the method comprises: (a) determining the level of proADM or a fragment thereof, preferably MR-proADM, and the level of at least one other marker and / or at least one other parameter in the subject in the sample, and (b1) comparing the level of said proADM or said fragment thereof, preferably MR-proADM and the level of at least one other marker and / or parameter with reference data corresponding to the level of said proADM or said fragment thereof, preferably MR-proADM and the level of said at least one other marker and / or parameter of at least one reference subject; or (b2) comparing the level of said proADM or said fragment thereof, preferably MR-proADM, and the level of at least one other marker and / or parameter with data corresponding to the level of said proADM or said fragment thereof, preferably MR-proADM, and the level of said at least one other marker and / or parameter for the same subject obtained from a previous analysis; and (c) identifying the fluid balance and / or salt balance of the subject based on the comparing step (b1) or (b2); and (d) determining the hematocrit status and / or the extracellular volume status based on step (c).

[0075] The present invention also relates to an in vitro method, wherein the method comprises: (a) determining the level of proADM or a fragment thereof, preferably MR-proADM, in a sample from a subject, the subject's body mass index, the subject's weight, the subject's age, the subject's sex, the level of hemoglobin in the sample and the level of total serum protein in the sample; and (b1) comparing said level of proADM or said fragment thereof, preferably MR-proADM, said body mass index, said body weight, said age, said sex, said hemoglobin level and said total serum protein level with reference data corresponding to said levels of said markers and said parameters of at least one reference subject; or (b2) comparing said level of proADM or said fragment thereof, preferably MR-proADM, said body mass index, said body weight, said hemoglobin level and said total serum protein level with data corresponding to said levels of said markers and said parameters obtained from a previous analysis for the same subject; (c) identifying the fluid balance and / or salt balance of the subject based on the comparing step (b1) or (b2); and (d) determining the hematocrit status and / or the extracellular volume status based on step (c).

[0076] In certain cases of the present invention, the term "comparing the level of proADM or fragments thereof with reference data" or "comparing the level of proADM or fragments thereof with reference data corresponding to the level of proADM or fragments thereof in at least one reference subject" means that the level of proADM or fragments thereof is determined as described herein and the level of proADM or fragments thereof is compared with the level of proADM or fragments thereof determined in at least one reference subject. Correspondingly, the term "comparing the level of MR-proADM with reference data" or "comparing the level of MR-proADM with reference data corresponding to the level of MR-proADM in at least one reference subject" means that the level of MR-proADM is determined as described herein and the level of MR-proADM is compared with the level of MR-proADM determined in at least one reference subject. In these cases, the reference data correspond to the levels of proADM or fragments thereof, preferably MR-proADM, determined in these reference subjects. In other words, the level of proADM or a fragment thereof, preferably MR-proADM, is compared to a reference level of proADM or a fragment thereof, preferably MR-proADM, of at least one reference subject or a population of reference subjects. Said reference level is generally referred to herein as reference data. Said reference data may contain more levels / values ​​corresponding to, for example, other markers and / or parameters. In a preferred aspect of the present invention, the term "comparing the level of said proADM or said fragments thereof, preferably MR-proADM and the level of at least one other marker and / or parameter with reference data corresponding to the level of said proADM or said fragments thereof, preferably MR-proADM and the level of said at least one other marker and / or parameter of at least one reference subject" means determining the level of proADM or said fragments thereof, preferably MR-proADM and determining at least one level of at least one other marker and / or at least one other parameter and comparing the level of proADM or said fragments thereof, preferably MR-proADM with the corresponding level of proADM or said fragments thereof, preferably MR-proADM of at least one reference subject and comparing the level of at least one other marker and / or at least one other parameter with the corresponding level of said at least one other marker and / or at least one other parameter of at least one reference subject. In some cases, said reference data correspond to the level of proADM or said fragments thereof, preferably MR-proADM and the level of at least one other marker and / or parameter determined in said reference subject.The level of proADM or said fragments thereof, preferably MR-proADM and the level of at least one other marker and / or parameter of said subject to be tested is compared with said reference data of these reference subjects.

[0077] In another aspect of the present invention, said reference data correspond to the levels of proADM or fragments thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin levels and total serum protein levels determined in said reference subjects. Said levels of proADM or fragments thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin levels and total serum protein levels of the subject to be tested are compared with the reference data of these reference subjects.

[0078] In certain aspects of the invention, the reference subject may be a healthy subject, such as a subject with a normal extracellular volume status. In another aspect of the invention, the reference subject may be a subject suffering from a disease or condition. A population of healthy or disease / condition-suffering reference subjects consists essentially of healthy subjects or subjects suffering from a disease or condition, respectively. A population of reference subjects is a population of subjects comprising from 1 to 200 or more reference subjects.

[0079] Specifically, the healthy subjects do not suffer from edema, brain injury, post-aneurysmal rupture, head injury, neurological deficit, multiple traumatic injury, post-surgery, organ failure, lymphatic flow disorder, renal dysfunction, cardiac dysfunction and / or diseases related to fluid balance disorders. Specifically, the healthy subjects do not suffer from aneurysm, multiple trauma, brain injury and / or head injury and are not post-surgery patients.

[0080] In particular, the reference subject or reference subject population suffering from a disease or condition suffers from a disease or condition known to be associated with a critical extracellular volume state and / or a critical hematocrit state, such as edema, brain injury, post-aneurysmal rupture, head injury, neurological deficit, multiple traumatic injury, post-surgery, organ failure, dysregulated lymphatic flow, renal dysfunction, cardiac dysfunction and / or a disease associated with a disordered fluid balance. In particular, the reference subject or reference subject population suffering from a disease or condition suffers from aneurysm, multiple trauma, brain injury and / or head injury, or wherein the reference subject is a post-surgery subject suffering from, for example, peritonitis with shock.

[0081] If the marker and / or parameter profile from the reference subject contains characteristic features of the marker and / or parameter profile from the at least one reference subject, the subject to be tested can be diagnosed accordingly as being healthy, for example having a balanced fluid and / or salt balance, or being at risk of developing or having a positive fluid and / or salt balance, and / or being at risk of or having a critical extracellular volume state and / or a critical hematocrit state.

[0082] In certain aspects of the invention, the method involves determining the fluid balance, salt balance and / or hematocrit status of a subject, wherein the method comprises: determining the level of proADM or a fragment thereof, preferably MR-proADM, in a sample from the subject, and comparing said level of proADM or said fragments thereof, preferably MR-proADM, with a reference level of proADM or said fragments thereof, preferably MR-proADM, of at least one reference subject, wherein each reference subject is healthy; identifying the subject's extracellular volume status, hematocrit status, fluid balance and / or salt balance based on the comparing step; wherein an increase in the level of proADM or said fragment thereof, preferably MR-proADM, in the subject compared to said reference level indicates that the subject has a positive fluid balance, a positive salt balance, a critical hematovolemia state and / or a critical extracellular volume state; a consistent or similar level of proADM or said fragment thereof, preferably MR-proADM, in the subject compared to said reference level indicates that said subject has a consistent or similar fluid balance and / or a consistent or similar salt balance, wherein said consistent fluid balance and / or salt balance indicates that said subject has a normal extracellular volume state and / or a normal hematocrit state; and / or A decrease in the level of proADM or said fragment thereof, preferably MR-proADM, in said subject compared to said reference level indicates that said subject has a negative fluid balance and / or a negative salt balance.

[0083] In these cases of the invention, the reference subject is a healthy subject (see above), for example a subject with a normal extracellular volume status. The healthy subject has a normal fluid balance and / or salt balance. Healthy subjects typically have MR-proADM levels of approximately 0.4 to 1 nmol / L (Angeletti S et al., Procalcitonin and mid-regional pro-adrenomedullin test combination in sepsis diagnosis. Clin Chem Lab Med. 2013 May;51(5):1059-67; Christ-Crain M et al., Mid-regional pro-adrenomedullin as a prognostic marker in sepsis: an observational study. Crit Care. 2005;9(6):R816-24; or Suzuki Y et al., Development and clinical application of an enzyme immunoassay for the determination of mid-regional pro-adrenomedullin. midregionalproadrenomedullin), J Pept Sci. 2013 Jan;19(1):59-63). In one embodiment, the at least one healthy reference subject has a level of proADM or a fragment thereof, preferably a MR-proADM level, of about 0.5 nmol / L. In another embodiment, the at least one healthy reference subject has a level of proADM or a fragment thereof, preferably a MR-proADM level, of about 0.75 nmol / L. In another embodiment, the at least one healthy reference subject has a level of proADM or a fragment thereof, preferably a MR-proADM level, of about 1.0 nmol / L. As demonstrated in the accompanying examples, subjects suffering from, for example, aneurysms, multiple trauma or postoperative conditions show levels of 1.0 nmol / L or more. In other words, subjects suffering from a disease or condition show high levels of proADM or a fragment thereof, preferably MR-proADM. This threshold is also revealed by statistical analysis, such as ROC; see Figure 4 and below.In certain cases of the present invention, the subject to be tested is considered to have a positive fluid balance, a positive salt balance, a critical hematovolemia state and / or a critical extracellular volume state when the level of proADM or fragments thereof, preferably MR-proADM, is increased compared to a reference level in said healthy subject. As used herein, "the level of proADM or fragments thereof, preferably MR-proADM in said subject is increased compared to said reference level" or "higher" level means that the level in said subject is at least 15%, preferably at least 20%, more preferably at least 25%, or even more preferably at least 30% higher than the level of proADM or fragments thereof, preferably MR-proADM in said healthy reference subject or said population of healthy reference subjects. In certain cases, said "elevated" or "higher" level means that the level of proADM or fragments thereof, preferably MR-proADM is at least 0.5 nmol / L, such as at least 0.5 nmol / L, at least 0.75 nmol / L or at least 1.0 nmol / L.

[0084] In certain cases, the level of proADM or fragments thereof, preferably MR-proADM, is compared to a reference level in healthy subjects, wherein the extracellular volume status, hematocrit status, fluid balance and / or salt balance is identified by comparing the level of proADM or fragments thereof, preferably MR-proADM, in the subject to the reference level, wherein an elevated level, e.g. of at least 1 nmol / L, indicates that the subject has a positive fluid balance and / or a positive salt balance, and / or wherein the positive fluid balance and / or positive salt balance indicates that the subject has a critical hematocrit status and / or a critical extracellular volume status.

[0085] In certain aspects of the invention, the methods provided herein comprise comparing the level of MR-proADM with a reference level of a healthy subject, and wherein the level of MR-proADM in the subject is consistent or similar compared to the reference data of a healthy subject, indicating that the subject has consistent or similar fluid balance and / or consistent or similar salt balance, wherein said consistent or similar fluid balance and / or salt balance indicates that the subject has a normal extracellular volume state and / or a normal hematocrit state. As used herein, "the level of proADM or said fragments thereof, preferably MR-proADM in the subject is similar compared to said reference level" means that the level of proADM or said fragments thereof, preferably MR-proADM in the subject is + / - 10%, preferably + / - 5%, more preferably + / - 2% or most preferably the same or consistent as the level of proADM or said fragments thereof, preferably MR-proADM in a healthy reference subject. In an exemplary embodiment, the reference level of proADM or fragments thereof, preferably MR-proADM, is about 0.5 nmol / L to 1.0 nmol / L, wherein the subject has a consistent or similar level of proADM or fragments thereof, preferably MR-proADM, if the subject has a level of proADM or fragments thereof, preferably MR-proADM, of about 0.5 nmol / L to about 1.0 nmol / L. A normal extracellular volume is, for example, about 15 L for a subject having about 40 L of body fluid (Guyton Arthur C., (1991), p. 275). As defined herein, a subject having a normal extracellular volume state may have a consistent or similar fluid and / or salt balance, whereby the input and output of fluids and / or salts of the subject are in balance, i.e. consistent or similar. A normal hematocrit state may be, for example, a hematocrit state above 20 ml / kg.

[0086] As used herein, "the level of proADM or said fragments thereof, preferably MR-proADM in said subject is reduced compared to said reference level" means that said subject's level is 15%, preferably 20%, more preferably 25% or even more preferably 30% lower than the reference level of proADM or said fragments thereof, preferably MR-proADM in healthy reference subjects. In preferred cases, said "reduced" or "lower" level means that said proADM or said fragments thereof, preferably MR-proADM has a level below 1.0 nmol / L, such as below 0.75 nmol / L or below 0.5 nmol / L. In other words, said subject has a reduced level of proADM or said fragments thereof, preferably MR-proADM if said subject has a level below 1.0 nmol / L, such as below 0.75 nmol / L or below about 0.5 nmol / L. In these cases, said subject's level of proADM or said fragments thereof, preferably MR-proADM is reduced compared to said reference data indicating that said subject has a negative fluid balance and / or a negative salt balance.

[0087] The sensitivity and specificity of this method depends not only on the analytical quality of the test, it also depends on the definition of what constitutes an abnormal or normal result. The distribution of the levels of proADM or fragments thereof, preferably MR-proADM, for subjects with and without a disease / condition may overlap. Under these conditions, the test cannot absolutely distinguish normal from disease with 100% accuracy. The skilled person will be aware of the fact that the condition of the subject itself or at least one other marker and / or parameter of the subject may help interpret the data and that such other information allows for a more reliable prognosis in the overlapping areas. Thus, the level of at least one other marker and / or parameter is compared to reference data of at least one healthy subject, wherein the value / level of the at least one other marker and / or parameter is close or consistent compared to the corresponding level of the at least one other marker and / or parameter of the reference data, indicating that the subject has a reduced risk of normal fluid and / or salt balance, and / or wherein the value / level of the at least one other marker and / or parameter is higher or lower than the corresponding level of the at least one other marker and / or parameter of the reference data, indicating an increased risk of having a normal fluid and / or salt balance, and wherein the normal fluid and / or salt balance indicates critical extracellular capacity. In the case where the reference subject is at least one healthy subject, the close or consistent value / level of the at least one other marker and / or parameter is a normal value / level, i.e. the value or level of the marker and parameter is within the normal range. Normal values / levels of markers and parameters are generally known to professionals and technicians. Normal values / levels of certain markers and parameters are described above. In the most preferred aspect of the present invention, said reference data correspond to or contain the levels of proADM or fragments thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin levels and total serum protein levels determined in said reference subjects. The levels of proADM or fragments thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin levels and total serum protein levels of the subject to be tested are compared with the reference data of these reference subjects.

[0088] When used herein, "similar or identical" levels / values ​​mean that the levels / values ​​are + / - 10%, preferably + / - 5%, more preferably + / - 2% or most preferably identical or identical to the corresponding levels / values. When used herein, "lower" or "reduced" or "higher" or "increased" levels / values ​​mean that the levels / values ​​are 15%, preferably 20%, more preferably 25% or most preferably 30% higher or lower, respectively, than the corresponding levels / values.

[0089] In an exemplary embodiment of the invention, the method involves determining the fluid balance, salt balance and / or hematocrit status of a subject, wherein the method comprises: determining the level of proADM or a fragment thereof, preferably MR-proADM, in a sample from the subject, and comparing said level of proADM or said fragment thereof, preferably MR-proADM, with a reference level of proADM or said fragment thereof, preferably MR-proADM, in at least one reference subject or a population of reference subjects, wherein said reference subject is a subject suffering from a disease or condition known to be associated with a critical extracellular volume state and / or a critical hematocrit state, or wherein said reference subject is a post-operative subject suffering from peritonitis with shock; and identifying the subject's extracellular volume status, hematocrit status, fluid balance and / or salt balance based on the comparing step; wherein a similar, identical or increased level of proADM or said fragment thereof, preferably MR-proADM, in said subject compared to said reference level, indicating that said subject has a normal fluid balance, a normal salt balance, a critical hematovolemia state and / or a critical extracellular volume state; and / or A decrease in the level of proADM or said fragment thereof, preferably MR-proADM, in said subject compared to said reference data indicates that said subject has normal fluid balance, normal salt balance, normal extracellular volume status and / or normal hematocrit status.

[0090] In certain preferred aspects of the present invention, the reference subject is a subject suffering from a disease or condition known to be associated with a critical extracellular volume state and / or a critical hematocrit state. These diseases or conditions include, for example, aneurysms, multiple trauma, brain injury and / or head injury or a situation in which the reference subject is a postoperative subject suffering from peritonitis with shock. Thus, it is envisaged that the disease or condition involves a condition in which fluid balance, salt balance, body fluid, extracellular volume and / or intracellular volume are critical. Thus, in an exemplary embodiment, the reference subject suffers from aneurysms, multiple trauma, brain injury and / or head injury and / or is a postoperative subject suffering from a disease or condition such as peritonitis with shock. In another embodiment, the reference subject is a subject suffering from a disease or condition selected from the following: aneurysms (ANE), traumatic brain injury (TC), multiple trauma (POLY), digestive system surgery (CD), severe brain trauma (SBT), aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head trauma (PT) and postoperative peritonitis with shock (P). The MR-proADM level of the subject to be tested is compared with the reference data of these reference subjects. In the accompanying examples it is shown that by plotting ROC curves of proADM or fragments thereof, preferably MR-proADM, in subjects suffering from a disease or condition for predicting fluid and salt balance, threshold values ​​of proADM or fragments thereof, preferably MR-proADM, predicting a critical extracellular volume state can be identified; see Example 1 and Figure 4. It is shown therein that high MR-proADM levels, e.g. of at least about 1.0 to at least about 1.5 nmol / L, are indicative of a gain of water / fluid and / or sodium / salt. Thus, preferably, the determined level of proADM or fragments thereof, preferably MR-proADM, is compared to a reference level of a reference subject suffering from a disease or condition known to be associated with a critical extracellular volume state, such as aneurysm, multiple trauma, brain injury and / or head injury, or wherein the reference subject is a post-operative subject suffering from peritonitis with shock, wherein a high or elevated level, such as of at least 1 nmol / l, determines that the subject has a positive fluid balance and / or salt balance. In other words, a determined level of proADM or fragments thereof, preferably MR-proADM, which is close or identical or even elevated compared to a reference level of a reference subject suffering from said disease or condition, indicates that the subject has a positive fluid balance, a positive salt balance, a critical hematocrit state and / or a critical extracellular volume state.When used herein, "the level of proADM or said fragments thereof, preferably MR-proADM in the subject is similar or identical to the level of proADM or said fragments thereof, preferably MR-proADM in comparison to the reference level", means that the level of proADM or said fragments thereof, preferably MR-proADM in the subject is + / - 10%, preferably + / - 5%, more preferably + / - 2% or even more preferably identical or identical to the level of proADM or said fragments thereof, preferably MR-proADM in at least one reference subject suffering from said disease and / or condition. When used herein, "the level of proADM or said fragments thereof, preferably MR-proADM in the subject is elevated compared to the reference level", means that the level in the subject is at least 15%, preferably at least 20%, more preferably at least 25% or even more preferably at least 30% higher than the level of proADM or said fragments thereof, preferably MR-proADM in the reference subject suffering from said disease or condition. In other words, "the level of proADM or said fragments thereof, preferably MR-proADM in said subject is similar or identical or increased compared to said reference level", means that the level of proADM or said fragments thereof, preferably MR-proADM in said subject is + / - 10%, preferably + / - 5%, more preferably + / - 2% or even more preferably the same or identical as the level of proADM or said fragments thereof, preferably MR-proADM in at least one reference subject suffering from said disease and / or condition; or is at least 15%, preferably at least 20%, more preferably at least 25% or even more preferably at least 30% higher than the level of proADM or said fragments thereof, preferably MR-proADM in said reference subject suffering from said disease and / or condition. In one case, when said reference subject is a subject suffering from said disease or condition, "the level of proADM or said fragments thereof, preferably MR-proADM in said subject is similar or identical or increased", means that said level of proADM or said fragments thereof, preferably MR-proADM is about 0.5 nmol / L or at least 0.5 nmol / L. In another aspect, "the subject has similar or identical levels of proADM or said fragments thereof, preferably MR-proADM or elevated levels" means that said proADM or said fragments thereof, preferably MR-proADM level is about 0.75 nmol / L or at least 0.75 nmol / L.In another aspect, "the subject has similar or identical levels or elevated levels of proADM or said fragments thereof, preferably MR-proADM" means that said level of proADM or said fragments thereof, preferably MR-proADM is about 1 nmol / L or at least 1 nmol / L or even about 1.5 nmol / L or at least 1.5 nmol / L. In other words, the reference level of said proADM or said fragments thereof, preferably MR-proADM is in the range of 0.5 nmol / L to 1.5 nmol / L, such as 0.5 nmol / L, 0.75 nmol / L or 1.0 nmol / L, wherein said subject has similar or identical levels if said level of proADM or said fragments thereof, preferably MR-proADM in said subject is about or at least 0.5 nmol / L to about or at least 1.5 nmol / L.

[0091] When the level of proADM or fragments thereof, preferably MR-proADM determined in said subject is decreased or lower compared to a subject suffering from said disease and / or condition, said subject to be tested does not have a positive fluid and / or salt balance, but a normal fluid and / or salt balance, i.e. a fluid and / or salt balance that is identical or similar. Thus, said fluid and / or salt balance is in balance. Thus, this subject has a normal fluid balance, a normal salt balance, a normal extracellular volume status and / or a normal hematocrit status. Such a decreased or lower level may also indicate that said subject has a negative salt and / or fluid balance. As used herein, the term "the level of proADM or fragments thereof, preferably MR-proADM in said subject is decreased" means that the level of proADM or fragments thereof, preferably MR-proADM determined in said subject to be tested has a level of proADM or fragments thereof, preferably MR-proADM that is at least 15%, preferably at least 20%, more preferably at least 25% or even more preferably at least 30% lower than the level in a reference subject suffering from said disease or condition. Preferably, by "reduced level" or "lower" level of proADM or said fragment thereof, preferably MR-proADM in said subject is intended that said level of proADM or said fragment thereof, preferably MR-proADM is below 1.0 nmol / L, such as below 1.0 nmol / L, below 0.75 nmol / L or below 0.5 nmol / L. In other words, the reference level of proADM or said fragment thereof, preferably MR-proADM is in the range of 0.5 nmol / L to 1.0 nmol / L, wherein said subject has a reduced level of proADM or said fragment thereof, preferably MR-proADM if said subject has said level below 1.0 nmol / L, such as below 1.0 nmol / L, below 0.75 nmol / L or below 0.5 nmol / L.

[0092] In the overlapping area, determining the other condition of the subject can assist in the prognosis. Thus, the level of at least one other marker and / or parameter is compared with reference data of at least one subject suffering from a disease or condition, wherein a similar or identical value / level of the at least one other marker and / or parameter increases the risk of having a normal fluid and / or salt balance, and wherein a higher or lower level / value of the at least one other marker and / or parameter reduces the risk of the subject having a normal fluid and / or salt balance, and wherein the normal fluid and / or salt balance is indicative of a critical extracellular volume.

[0093] As shown in the accompanying examples, the combination of markers and parameters is selected to produce the lowest error. This selection or significance analysis is performed using standard statistical analysis, such as random forest analysis. As shown in the accompanying examples, the markers and parameters proADM or fragments thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin and total serum protein of the subject, yield a very reliable prediction of critical patients suffering from normal fluid and / or salt balance. Therefore, in preferred cases of the present invention, the prediction of patient outcome, i.e. the fluid balance and / or salt balance of the subject, is performed using standard statistical analysis, such as random forest. In these cases, the markers and parameters are implemented in formulas, which can be integrated in a software program. Therefore, in certain embodiments, the present invention relates to a software program suitable for determining the fluid balance, salt balance, extracellular volume and / or hematocrit of a subject using the methods provided herein. Therefore, the level of proADM or fragments thereof, preferably MR-proADM, is determined in a sample of the subject and input into the software. In other embodiments, the subject's level of proADM or a fragment thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin level and serum total protein level are determined and input into the software. In other embodiments, the software automatically calculates / determines a p-critical value based on the level of proADM or a fragment thereof, preferably MR-proADM and / or other parameters and markers and determines whether the subject has a critical fluid balance, a critical salt balance, a critical extracellular volume and / or a critical hematocrit. In other words, the software provides a prognosis of whether the subject is a critical subject. Such software can be used via a graphical user interface. The formulas behind the interface are automatically generated based on patient data using standard statistical methods such as random forest analysis methods implemented in the open scientific software R. Thus, the statistical analysis compares the levels of the markers and parameters with reference data and predicts the subject's fluid balance and / or salt balance and therefore the extracellular volume status. In the ICU, a physician can use the interface to input markers and / or parameters to obtain an estimate of fluid balance and salt balance, which can be used to identify subjects with a positive fluid balance and / or salt balance. In the case where the fluid balance or salt balance exceeds 4L for the fluid balance and exceeds 36g for the salt balance, the patient is in a critical phase (p-critical value> 60%). The predicted result can be illustrated in the graphical user interface, for example, by a traffic light system. For example, the values ​​of the fluid balance and salt balance that exceed 4L or 36g, respectively, are highlighted in red because they indicate a critical patient. If the patient has a fluid balance or salt balance that is lower than 4L or 36g, respectively, the patient or value is highlighted in green (the patient has a p-critical value lower than 30%).In these cases, the methods provided herein can be used: for treatment guidance, e.g., if the p-critical value exceeds 60%, reconsider fluid management; for diagnosis of positive fluid and salt balance to inform the clinician that this patient has fluid overload, even for patients not receiving intravenous fluid resuscitation; or for patient prognosis (in cases where the patient has a p-critical value >90%, the patient has a high SOFA score, a low RBCV, and therefore an even worse prognosis). Figure 6 ). Between 30% and 40%, the patients have intermediate p-critical values, which are highlighted in yellow.

[0094] In certain aspects of the invention, the method involves determining the fluid balance, salt balance and / or hematocrit status of a subject, wherein the method comprises: determining the level of marker proADM or a fragment thereof, preferably the level of MR-proADM, in a sample obtained from said subject; comparing the level of proADM or said fragments thereof, preferably the level of MR-proADM, with the level of proADM or said fragments thereof, preferably the level of MR-proADM, obtained from a previous analysis in the same subject; and The subject's extracellular volume status, hematocrit status, fluid balance and / or salt balance are identified based on the comparison step, wherein A level of at least 1 nmol / L indicates that the subject has a positive fluid balance, a positive salt balance, a critical hematovol state, and / or a critical extracellular volume state.

[0095] In certain contexts of the present invention, the term "comparing the level of proADM or said fragments thereof, preferably the level of MR-proADM, with the level of proADM or said fragments thereof, preferably the level of MR-proADM, obtained from a previous analysis of the same subject" means that the level of proADM or said fragments thereof, preferably the level of MR-proADM is determined as described herein and the level of proADM or said fragments thereof, preferably MR-proADM is compared with one or more levels of proADM or said fragments thereof, preferably MR-proADM, obtained from the same subject at the time of the previous analysis. Preferably, the level of proADM or said fragments thereof, preferably MR-proADM is determined at several time points, i.e. more than one level of proADM or said fragments thereof, preferably MR-proADM, may be obtained from the previous analysis. Using these levels determined at different time points a series may be calculated. This series shows a trend, which may be used to determine, for example, the extracellular volume status and / or the hematocrit status of the subject. In other words, the trend of the MR-proADM level predicts the extracellular volume status. For example, in case a series of measured values ​​of e.g. the level / value of MR-proADM has been determined at several previous time points, the skilled person may calculate a trend which may be used to explain the development of proADM or fragments thereof, preferably MR-proADM and / or said other markers and / or parameters. For example, a positive trend, i.e. an increase in said values ​​or being higher than previously measured levels, may predict that said subject has a positive fluid balance and / or salt balance. In some cases, if the levels of proADM or fragments thereof, preferably MR-proADM, obtained from a previous analysis of the same subject show a positive trend and at least one level of proADM or fragments thereof, preferably MR-proADM, obtained from a previous analysis of the same subject is in the range of at least 0.5 nmol / L to at least 1.5 nmol / L, such as at least 0.5 nmol / L, at least 0.75 nmol / L, at least 1 nmol / L or at least 1.5 nmol / L, it indicates that the subject has a positive fluid balance and / or a positive salt balance, wherein a positive fluid balance and / or salt balance indicates that the subject has a critical extracellular volume state and / or a critical hematocrit state, wherein the subject's health state is deteriorating.Likewise, in certain cases, if the levels of proADM or fragments thereof, preferably MR-proADM, obtained from a previous analysis of the same subject show a negative trend and at least one level of proADM or fragments thereof, preferably MR-proADM, obtained from a previous analysis of the same subject is, for example, at least 0.5 nmol / L, at least 0.75 nmol / L or at least 1 nmol / L, it indicates that the subject has a positive fluid balance and / or a positive salt balance, wherein a positive fluid balance and / or salt balance indicates that the subject has a critical extracellular volume state or a critical hematocrit state, wherein the health state is alleviated. In the accompanying examples, the levels of proADM or fragments thereof, preferably MR-proADM, decrease over time of treatment. Without being limited by theory, the decrease in the concentration of proADM or fragments thereof, preferably MR-proADM, may be caused by a reduction in endothelial damage.

[0096] In some cases, certain fixed thresholds are used to determine the extracellular volume status of the subject. In one embodiment, the patient is determined to have a positive fluid balance and / or salt balance when the level of proADM or fragments thereof, preferably MR-proADM, is above 0.5 nmol / l, wherein the positive fluid balance and / or salt balance indicates that the subject has a critical extracellular volume. In another embodiment, the subject is determined to have a positive fluid balance and / or salt balance when the level of proADM or fragments thereof, preferably MR-proADM, is above 0.75 nmol / l, wherein the positive fluid balance and / or salt balance indicates that the subject has a critical extracellular volume. In another embodiment, the patient is determined to have a positive fluid balance and / or salt balance when the level of proADM or fragments thereof, preferably MR-proADM, is above 1.0 nmol / l, wherein the positive fluid balance and / or salt balance indicates that the subject has a critical extracellular volume. In another embodiment, said patient is determined to have a positive fluid and / or salt balance when said level of proADM or fragments thereof, preferably MR-proADM, is above 1.5 nmol / l, wherein said positive fluid and / or salt balance indicates that said subject has critical extracellular capacity.

[0097] In certain instances of the present invention, the term "comparing the level of said proADM or said fragments thereof, preferably MR-proADM and the level of said at least one other marker and / or parameter with data corresponding to the level of said proADM or said fragments thereof, preferably MR-proADM and the level of said at least one other marker and / or parameter of the same subject obtained from a previous analysis" means determining the level of said proADM or said fragments thereof, preferably MR-proADM and determining at least one other level of at least one other marker and / or at least one other parameter and comparing the level of said proADM or said fragments thereof, preferably MR-proADM with the corresponding level of proADM or said fragments thereof, preferably MR-proADM of the same subject determined at the earlier analysis and comparing the level of said at least one other marker and / or at least one other parameter with the corresponding level of said at least one other marker and / or at least one other parameter of the same subject determined at the earlier analysis. Multiple levels of said at least one other marker and / or parameter obtained from a previous analysis may be compared with themselves in order to predict trends on a multivariate basis. Alternatively, said other marker and / or parameter may be compared with normal data, e.g. data of healthy reference subjects. In case said other markers and / or parameters are above or below said normal levels, the risk of a positive fluid balance and / or salt balance is increased, ie said subject is more susceptible to a critical extracellular volume state.

[0098] In certain aspects of the invention, the level of proADM or fragments thereof, preferably MR-proADM, body mass index, body weight, hemoglobin level and total serum protein level are compared to the corresponding levels determined at an earlier analysis of the same subject. In other words, the levels of the markers and parameters are determined at different time points and the trend of the levels predicts the extracellular volume status.

[0099] As used herein, "preliminary analysis" means that the marker levels are determined at several time points during hospitalization, e.g. day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7, etc. The determination of the markers and / or parameters may also be performed hourly, e.g. a first measurement may be performed when the patient is admitted to the hospital, and the measurements may then be repeated, e.g. every hour, every 2 hours or every 5 hours. The determined level of the marker, e.g. proADM or a fragment thereof, preferably MR-proADM, and optionally the level of the parameter is compared to a level / value of the marker or parameter determined at an earlier time point or to an average of the levels / values ​​calculated from two or more time points. Changes in the marker levels are indicative of the extracellular volume status of the subject.

[0100] As shown in the accompanying Example 1 and Figure 4, by plotting MR-proADM ROC curves for predicting fluid balance and salt balance in intensive care patients, such as patients with aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head trauma (PT), severe brain trauma (SBT) or postoperative peritonitis with shock (P), a threshold value of MR-proADM, e.g. at least 1 nmol / l, for predicting critical patients was identified. It is demonstrated herein that high or increased levels of proADM or fragments thereof, preferably MR-proADM, e.g. above 1 nmol / l, indicate that the subject has fluid overload, i.e. positive fluid balance. Therefore, in a preferred aspect of the present invention, the method provided herein comprises determining the level of proADM or fragments thereof, preferably MR-proADM, in a subject, wherein an increased level of proADM or fragments thereof, preferably MR-proADM, indicates that the subject has a positive fluid balance and / or a positive salt balance.

[0101] In the accompanying examples it is shown that elevated levels of proADM or fragments thereof, preferably MR-proADM, e.g. at least about 1.0 to at least about 1.5 nmol / L, are indicative of a gain of water / fluid and / or sodium, i.e. a positive fluid balance and / or a positive salt balance, for example at least about 3 L to 4 L or about 27 g to 36 g sodium / salt, respectively, in the subject. Thus, in an embodiment of the invention, the elevated or high level of proADM or fragments thereof, preferably MR-proADM, in the subject is at least 0.5 nmol / L or at least 0.6 nmol / L or at least 0.7 nmol / L or at least 0.75 nmol / L or at least 0.8 nmol / L or at least 0.9 nmol / L or at least 1.1 nmol / L or at least 1.2 nmol / L or at least 1.3 nmol / L or at least 1.4 nmol / L or at least 1.5 nmol / L or at least 1.0 nmol / L.

[0102] In the accompanying examples, different patient groups, namely patients with aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head trauma (PT), severe brain trauma (SBT) or patients with postoperative peritonitis with shock (P), all showed elevated or high MR-proADM levels. In particular, patients with postoperative peritonitis with shock (P) showed particularly high MR-proADM values ​​on day 0, day 2 and day 7; see Figure 3. Therefore, it is contemplated herein that the levels of proADM or fragments thereof, preferably MR-proADM, may vary depending on the patient group and that certain conditions, such as postoperatively, may give rise to even higher levels of proADM or fragments thereof, preferably MR-proADM, which are suitable for identifying critical volume status, normal fluid and / or salt balance. Therefore, it is contemplated herein that plotting the levels of proADM or fragments thereof, preferably MR-proADM in a ROC (see below) for the purpose of predicting the fluid and salt balance in patients with specific diseases may result in thresholds higher or lower than 1 nmol / L. For example, a level of proADM or a fragment thereof, preferably MR-proADM, of at least 1.5 nmol / L in a postoperative subject indicates a positive fluid balance and / or a positive salt balance. In general, an elevated value of at least 1.0 nmol / L indicates a subject with a normal fluid and / or salt balance. In a preferred embodiment of the invention, the level of proADM or a fragment thereof, preferably MR-proADM is considered elevated if the concentration of proADM or a fragment thereof, preferably MR-proADM determined in a subject is at least 1 nmol / L (concentration [MR-proADM] ≥ 1.0 nmol / L). In other words, a concentration of proADM or a fragment thereof, preferably MR-proADM in a subject exceeding 1 nmol / L indicates a positive fluid balance (e.g. at least 4 L) or a gain of water. Alternatively, a concentration of proADM or a fragment thereof, preferably MR-proADM in a subject exceeding 1.0 nmol / L indicates a positive salt balance (e.g. at least 36 g) or a gain of salt or a critical extracellular volume state.

[0103] In a preferred aspect of the present invention, the method provided herein determines the extracellular volume of a subject, wherein the method comprises determining the level of said marker proADM or fragments thereof, preferably MR-proADM, in a sample obtained from said subject, wherein on the basis of said level of said proADM or fragments thereof, preferably MR-proADM, the fluid balance is determined, and wherein said fluid balance determines the extracellular volume status, wherein an elevated level of proADM or fragments thereof, preferably MR-proADM, in said subject indicates that said subject has a positive fluid balance, wherein said elevated level of MR-proADM is at least 1 nmol / L, wherein said level indicates that said normal fluid balance is at least about 4 L, and wherein said positive fluid balance indicates that said subject has an extracellular volume status that is considered critical.

[0104] Preferably, the method provided herein determines the extracellular volume of a subject, wherein the method comprises determining the level of the marker proADM or fragments thereof, preferably MR-proADM, in a sample obtained from the subject, wherein on the basis of the level of proADM or fragments thereof, preferably MR-proADM, the salt balance is determined, and wherein the salt balance determines the extracellular volume status, wherein an elevated level of proADM or fragments thereof, preferably MR-proADM, in the subject indicates that the subject has a positive salt balance, wherein the elevated level of proADM or fragments thereof, preferably MR-proADM, is at least 1 nmol / L, wherein the level indicates that the positive salt balance is at least about 36 g, and wherein the positive salt balance indicates that the subject has an extracellular volume status that is considered critical.

[0105] As used herein, a "sample" in the sense of the present invention may be any fluid of the subject, such as plasma, lymph, urine, cerebrospinal fluid, blood, saliva, serum or feces and any biological tissue of the subject.

[0106] Preferably, in the context of the present invention, the sample is a blood sample, more preferably a serum sample, most preferably a plasma sample.

[0107] In a preferred aspect of the invention, the level of proADM or a fragment thereof, preferably MR-proADM, is determined in said sample, wherein said sample is a blood or plasma sample. In the most preferred aspect, said marker is determined in a plasma sample.

[0108] It is contemplated herein that the sample may be a tissue, such as lung tissue, ascites, skin, heart, kidney, digestive tract or lower extremity edema, epithelial tissue, connective tissue such as bone or blood, muscle tissue such as visceral or smooth and skeletal muscle, nervous tissue, bone marrow, cartilage, skin, mucosa or hair. The sample is collected / obtained from the patient or undergoes diagnosis according to the present invention. Where appropriate, as for example in the case of a solid sample, the sample may need to be dissolved, homogenized or extracted with a solvent before being used in the present invention to obtain a liquid sample. In a preferred case, the sample is a liquid sample, such as a solution or suspension. The liquid sample may undergo one or more pretreatments before being used in the present invention. These pretreatments include, but are not limited to, dilution, filtration, centrifugation, concentration, sedimentation, precipitation or dialysis. Pretreatment may also include adding a chemical or biochemical substance to the solution, such as an acid, alkali, buffer, salt, solvent, reactive dye, detergent, emulsifier or chelating agent. In a preferred case, the sample is blood, plasma, serum or urine. In a most preferred case, the sample is plasma.

[0109] In the context of the present invention, "plasma" is the virtually cell-free supernatant of blood containing an anticoagulant obtained after centrifugation. Exemplary anticoagulants include calcium ion binding compounds such as EDTA or citrate and thrombin inhibitors such as heparin salts or hirudin. Cell-free plasma can be obtained by centrifuging anticoagulant-added blood (e.g., citrate, EDTA or heparin-added blood), e.g., at 2000 to 3000 g for at least 15 minutes.

[0110] In the context of the present invention, "serum" is the liquid fraction of whole blood collected after the blood is allowed to clot. When the clotted blood (clotted blood) is centrifuged, serum can be obtained as the supernatant.

[0111] The level of proADM or fragments thereof, preferably MR-proADM, and / or the level of other markers may be determined by an immunoassay. As used herein, an "assay" or diagnostic assay may be of any type used in the field of diagnostics. Preferred detection methods include immunoassays in various formats such as radioimmunoassays, chemiluminescent and fluorescent immunoassays, enzyme-linked immunoassays (ELISA), Luminex-based bead arrays, protein microarray assays, assays suitable for point-of-care testing and rapid assay formats such as immunochromatographic strip tests. Such assays may be based on the binding of the analyte to be detected to one or more capture probes with a certain affinity. As used herein, an immunoassay is a biochemical test that measures the presence or concentration of a macromolecule / polypeptide in a solution by using antibodies or immunoglobulins. According to the invention, the antibodies may be monoclonal as well as polyclonal antibodies. Thus, at least one antibody is a monoclonal or polyclonal antibody. Particularly preferred is a method according to the invention, wherein a partial peptide spanning amino acids 42-95 of pre-proADM or the middle section of the amino acids given in SEQ ID NO: 2 is used to determine MR-proADM or a partial peptide thereof in a sample. In some cases, the level of the marker is determined by high performance liquid chromatography (HPLC). In some cases, the HPLC can be coupled to an immunoassay.

[0112] In certain aspects of the present invention, proADM or fragments thereof, preferably MR-proADM or fragments thereof and / or other markers or fragments thereof are determined using a sandwich immunoassay. In such a sandwich immunoassay, two antibodies are applied, e.g., to one marker, e.g., proADM or fragments thereof, preferably MR-proADM, in a sample. In particular, it is preferred if proADM or fragments thereof, preferably MR-proADM or fragments thereof are determined by using two antibodies, which specifically bind to different partial sequences of proADM or fragments thereof, preferably MR-proADM or fragments thereof.

[0113] In a preferred embodiment of the in vitro method according to the invention, one of the antibodies is labelled and the second is bound or can be selectively bound to a solid phase.

[0114] In a particularly preferred case of the assay, one of the antibodies is labeled and the other is bound to a solid phase or can be selectively bound to a solid phase. In a preferred embodiment, the method is performed as a heterogeneous sandwich immunoassay, wherein one of the antibodies is immobilized on an arbitrarily selected solid phase, such as a coated tube (e.g. a polystyrene tube; coated tube; CT) or the wall of a microtiter plate, for example made of polystyrene, or to particles such as magnetic particles, wherein the other antibody has a group similar to a detectable label or can be selectively attached to a label, and the group is used to detect the sandwich structure formed. It is also possible to use a time-delayed or subsequent immobilization of a suitable solid phase.

[0115] Furthermore, the method according to the invention may be embodied as a homogeneous method, wherein a sandwich complex formed by the one or more antibodies and the marker to be detected, such as proADM or a fragment thereof, preferably MR-proADM or a fragment thereof, is kept suspended in a liquid phase. In this case, preferably, when two antibodies are used, both antibodies are labeled with parts of the detection system, which results in the generation of a signal or a trigger signal when the two antibodies are integrated in a single sandwich. These techniques will be embodied as fluorescence enhancement or fluorescence quenching detection methods. A particularly preferred situation involves the use of detection reagents that should be used in pairs, such as those 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 are possible in which only the reaction products comprising the two labeled components in a single immune complex are directly detected in the reaction mixture. For example, these techniques are marketed under the trade name TRACE ® (Time-Resolved Amplified Cryptate Emission) or KRYPTOR ® The invention provides a method for carrying out the teachings of the above-referenced applications. Therefore, in particularly preferred cases, a diagnostic device is used to carry out the methods provided herein. For example, the level of proADM or a fragment thereof, preferably MR-proADM and / or the level of any other marker of the methods provided herein is determined. In particularly preferred cases, the diagnostic device is a KRYPTOR ®。

[0116] The present invention also relates to the use of a kit for determining the state of extracellular volume in a sample obtained from a test subject, the kit comprising a detection reagent for determining at least one marker selected from the group consisting of proADM or a fragment thereof, preferably MR-proADM, hemoglobin, total serum protein, renin, pro-atrial natriuretic peptide (proANP), C-terminal arginine-pro-vasopressin (CT-proAVP) protein, erythropoietin, angiotensin II, aldosterone, cortisol, adrenaline, epinephrine, catecholamines and pro-endothelin-1 (pro-ET-1) or a fragment thereof, and auxiliary substances for performing the methods provided herein. In certain cases, the present invention relates to the use of a kit for determining the state of extracellular volume in a sample obtained from a test subject, the kit comprising a detection reagent for determining the level of proADM or a fragment thereof, preferably MR-proADM or a fragment thereof, and auxiliary substances for performing the methods provided herein. In a preferred embodiment, the present invention relates to the use of a kit for determining the extracellular volume status in a sample obtained from a test subject, the kit comprising detection reagents for determining the markers proADM or fragments thereof, preferably MR-proADM, hemoglobin and total serum protein, and comprising auxiliary substances for performing the methods provided herein.

[0117] In some cases, the detection reagent for determining the level of proADM or fragments thereof, preferably MR-proADM or fragments thereof, comprises antibodies, wherein one of the antibodies is labeled and the other antibody is bound to a solid phase or can be selectively bound to a solid phase.

[0118] In some cases, the detection reagent for determining the level of at least one marker comprises antibodies, wherein one of the antibodies is labeled and the other antibody is bound to a solid phase or can be selectively bound to a solid phase.

[0119] In principle, all labeling techniques that can be used for assays of the type described can be used, such as labeling with radioisotopes, enzymes, fluorescent, chemiluminescent or bioluminescent labels and directly optically detectable color labels such as gold atoms and dye particles, which are particularly used for point-of-care (POC) or rapid tests. In the case of a heterogeneous sandwich immunoassay, the two antibodies can represent part of a detection system of the type described herein in the context of a homogeneous assay.

[0120] In a preferred case, both the first and second antibodies are dispersed in a liquid reaction medium, wherein a first labeling component as part of a labeling system based on fluorescence or chemiluminescence quenching or enhancement is bound to the first antibody, and wherein a second labeling component of the labeling system is bound to the second antibody, so that after binding of both antibodies to the marker to be detected, such as proADM or a fragment thereof, preferably MR-proADM or a fragment thereof or said other marker or a fragment thereof, a detectable signal is generated, which enables detection of the sandwich complex formed in the measuring solution. One case of this alternative comprises a labeling system such as a rare earth cryptate or chelate in combination with a fluorescent or chemiluminescent dye. In a particularly preferred case, the labeling system comprises a rare earth cryptate in combination with a fluorescent or chemiluminescent dye, in particular of the anthocyanin type. In another preferred case, the detection is performed using a competitive immunoassay. In a preferred case, a radioimmunoassay is used. It is also envisaged herein that the level of the marker can be determined, for example, by mass spectrometry or by a high performance liquid chromatography (HPLC) method or a mass spectrometry-based method that can be coupled to an immunoassay. The skilled artisan will appreciate that any available assay may be used, as long as the level of the marker can be reliably determined.

[0121] It is an object of the present invention to provide an in vitro method for the diagnosis, prognosis, risk assessment, risk stratification, therapy control and / or surgical control of a disorder or medical condition in a subject and / or patient, which in particular provides reliable information for medical practitioners in the Emergency Department (ED) or Intensive Care Unit (ICU).

[0122] Therefore, the present invention relates to a method for in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy control and / or surgical control of a disorder or medical condition in a subject, wherein the extracellular volume status, hematocrit status, fluid balance and / or salt balance of the subject is determined by the methods provided herein.

[0123] In one embodiment, the present invention relates to a method for the in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy control and / or surgical control of a disorder or a medical condition in a subject, wherein the fluid balance, salt balance and / or hematocrit status of the subject is determined on the basis of the level of proADM or fragments thereof, preferably MR-proADM.

[0124] In other embodiments, the present invention relates to a method for in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy control and / or surgical control of a disorder or a medical condition in a subject, wherein the fluid balance and / or salt balance is determined on the basis of the level of proADM or fragments thereof, preferably MR-proADM, and wherein the fluid balance and / or salt balance determines the extracellular volume status.

[0125] In other embodiments, the present invention relates to a method for in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy control and / or surgical control of a disorder or a medical condition in a subject, wherein the fluid balance, salt balance and / or hematocrit status is determined on the basis of the level of proADM or a fragment thereof, preferably MR-proADM, the level of hemoglobin, the level of total serum protein, the weight of the subject, the age of the subject, the sex of the subject.

[0126] As used herein, "diagnosis" in the context of the present invention refers to the identification and (early) detection of a disease or clinical condition in a subject, and may also comprise differential diagnosis. In certain embodiments, the assessment of the severity of a disease or clinical condition may be encompassed by the term "diagnosis".

[0127] As used herein, "prognosis" refers to the prediction of the outcome or specific risk of a subject suffering from a particular disease or clinical condition. This may include an estimate of the subject's chance of recovery or the chance of an adverse outcome.

[0128] In the context of the present invention, the term "therapy control" refers to the monitoring and / or adjustment of the therapeutic treatment of the patient. "Monitoring" refers to keeping track of a diagnosed disease, condition, complication or risk, for example to analyze the progression of the disease or the effect of a particular treatment on the progression of the disease or condition.

[0129] In the present invention, the terms "risk assessment" and "risk stratification" refer to the grouping of subjects into different risk groups according to their further prognosis. Risk assessment also relates to stratification for the use of preventive and / or therapeutic measures. When used herein, the term "surgical control" relates to the pre-operative control, intra-operative control and / or post-operative control of a subject. In particular, it means in this context the control of fluid balance, salt balance, hematocrit status and / or extracellular volume status. Thus, body fluids and / or salts are monitored and controlled in these subjects.

[0130] In some cases, the disorder or medical condition can be water overload, edema, brain injury, post-aneurysm rupture, severe head injury, neurological deficit, severe multiple traumatic injury, post-surgery, cardiac crisis, kidney damage, organ failure, lymphatic flow disorder, renal dysfunction, cardiac dysfunction, and diseases related to fluid balance disorders.

[0131] As shown in the accompanying Examples, a significant statistical relationship was found between MR-proADM and fluid and / or salt balance in a subject. The fluid and / or salt balance is indicative of the extracellular volume of a subject and / or patient. As demonstrated in the accompanying Examples, this strong relationship was found in patients in several clinical situations, such as patients with severe brain trauma (SBT), aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head trauma (PT) and post-operative peritonitis with shock (P) (e.g. Example 1). Thus, in certain cases, the present invention relates to a method for in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy control and / or surgical control of a disorder or medical condition in a subject, wherein the subject has a brain or head injury, a polytraumatic injury or an aneurysm, or is post-operative. In other instances, the present invention relates to a method for in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy control and / or surgical control of a disorder or medical condition in a subject, wherein the subject has severe brain trauma (SBT), aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head trauma (PT), and postoperative peritonitis with shock (P). In other instances, the present invention relates to a method for in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy control and / or surgical control of a disorder or medical condition in a subject, wherein the subject has post-aneurysmal rupture or severe head injury. In some cases, the subject has no neurological deficit. In some cases, the subject has severe multiple traumatic injury or is postoperative.

[0132] The methods provided herein can be used for fluid management of a subject or patient. As used herein, the term "fluid management" means the monitoring and control of the fluid status of a subject or patient and the administration of fluids, for example by intravenous fluid administration. Thus, in some cases, the present invention relates to a method for fluid management of a subject, wherein the extracellular volume status of the subject is determined by the methods provided herein. In some cases, the present invention relates to a method for fluid management of a subject, wherein the extracellular volume status of the subject is determined by the methods provided herein on the basis of the level of proADM or a fragment thereof, preferably MR-proADM.

[0133] In certain cases, the present invention relates to the use of the methods provided herein for fluid management in a subject, wherein therapy of a disorder or medical condition in the subject is controlled based on the level of proADM or fragments thereof, preferably MR-proADM and / or based on the fluid and / or salt balance of the subject.

[0134] In some cases, the present invention relates to predicting a subject's risk of mortality and patient outcome using the methods provided herein, wherein the subject's extracellular volume status is determined by the methods provided herein. In some cases, the present invention relates to a method used as an early warning system for physicians and clinicians to immediately take appropriate therapeutic action, wherein the subject's extracellular volume status is determined by the methods provided herein.

[0135] In certain cases, the present invention relates to using the methods provided herein to predict reduced function or risk of organ failure, dysregulated lymphatic flow, renal dysfunction, cardiac dysfunction in a subject, wherein the extracellular volume status of the subject is determined by the methods provided herein.

[0136] In some cases, the present invention relates to the use of the methods provided herein for the treatment of a subject suffering from a disorder or medical condition, wherein the disorder or medical condition is selected from water overload, edema, brain injury, post-aneurysmal rupture, severe head injury, neurological deficit, severe multiple traumatic injury, post-surgery, cardiac crisis, renal injury, organ failure, dysregulated lymphatic flow, renal dysfunction, cardiac dysfunction, and diseases associated with fluid balance disorders. The terms "treatment", "therapy", and the like are used herein to generally refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease / medical condition / disorder or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease / medical condition / disorder and / or attributing to adverse effects of the disease / medical condition / disorder. As used herein, the term "treatment" covers any treatment of a disease / medical condition / disorder in a subject and includes: (a) preventing and / or alleviating the disease / medical condition / disorder in a subject that may be susceptible to the disease / medical condition / disorder; (b) inhibiting the disease / medical condition / disorder, i.e., stopping its development; or (c) eliminating the disease / medical condition / disorder, i.e., causing regression of the disease / medical condition / disorder. For example, the methods provided herein can be used to control therapy / treatment of resuscitated patients. Thus, for example, the methods provided herein can be used to control fluid management of a subject. The methods provided herein can also be used to control intravenous fluid administration in order to balance fluid balance and / or salt balance in a subject to avoid positive fluid balance associated with increased mortality (Acheampong et al., 2015). In some cases, the methods provided herein can also be used to assess and control fluid management of a subject to avoid pathological amounts of fluid migration to the interstitial space and / or excessive volume expansion. Excessive volume expansion that is considered critical is, for example, more than 4 L within 1 day, 2 days, 3 days, 4 days, 5 days and / or preferably within 7 days.

[0137] In certain aspects of the invention, the methods provided herein include: (a1) comparing the level of said proADM or said fragments thereof, preferably MR-proADM, with reference data corresponding to the level of said proADM or said fragments thereof, preferably MR-proADM, of at least one reference subject; or (a2) comparing said level of proADM or said fragment thereof, preferably MR-proADM, with data corresponding to the level of said proADM or said fragment thereof, preferably MR-proADM, of said same subject obtained from a previous analysis; (b) identifying the subject's fluid balance, salt balance and / or hematocrit status based on the comparing step (a), wherein the subject's fluid balance, salt balance and / or hematocrit status is used to predict the subject's risk of death and patient outcome, and / or is used for the assessment and control of the subject's fluid management.

[0138] In the context of in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy control and / or surgical control of a disorder or medical condition in a subject and / or patient, a subject has a positive fluid balance (elevated fluid balance, i.e. an increase in water content) which is considered to be critical if the level of proADM or a fragment thereof, preferably MR-proADM, is at least 0.5 nmol / L, such as at least 0.5 nmol / L, at least 0.75 nmol / L or at least 1 nmol / L and the subject has an increase in fluid balance of at least 4.0 L (an increase of 4 L of water due to hospitalization). In other words, in a critical subject the positive fluid balance is at least 4 L, i.e. the increase in water in a subject with a critical health condition is at least 4 L.

[0139] Preferably, the method comprises determining the level of proADM or fragments thereof, preferably MR-proADM, in a sample, wherein the level of proADM or fragments thereof, preferably MR-proADM, in the subject is at least 0.5 nmol / L, such as at least 0.5 nmol / L, at least 0.75 nmol / L or at least 1 nmol / L, wherein the level of proADM or fragments thereof, preferably MR-proADM indicates that the subject has a positive fluid balance, wherein the positive fluid balance is at least 4 L, and wherein the positive fluid balance indicates that the subject has a critical health condition. In other words, the positive fluid balance indicates that the subject has a critical extracellular volume status.

[0140] In certain cases, the subject has a positive salt balance that is considered critical if the level of proADM or a fragment thereof, preferably MR-proADM, is at least 0.5 nmol / L, such as at least 0.5 nmol / L, at least 0.75 nmol / L or at least 1 nmol / L and the subject has an increase in salt balance of at least 36 g (an increase of 36 g sodium or salt). In the most preferred case, the increase in sodium is at least 36.0 g, and wherein the change indicates that the subject has a positive fluid balance and / or salt balance that is considered critical. In other words, the positive salt balance and / or fluid balance indicates that the subject has an extracellular volume state that is considered critical. In other words, in a critical subject, the positive salt balance (increase in salt balance, i.e. an increase in the amount of salt) is at least 36 g, i.e. the increase in salt in a critical subject is at least 36 g.

[0141] In other cases, the method comprises determining the level of proADM or fragments thereof, preferably MR-proADM, in a sample, wherein the level of proADM or fragments thereof, preferably MR-proADM, in the subject is at least 1 nmol / L, wherein the level of proADM or fragments thereof, preferably MR-proADM indicates that the subject has a positive salt balance, wherein the positive salt balance is at least 36 g, and wherein the positive salt balance indicates that the subject has a critical health condition.

[0142] It is understood herein that other markers and / or parameters, i.e. in addition to proADM or fragments thereof, preferably MR-proADM, improve the prediction of the fluid and / or salt balance. Thus, in some cases, the method comprises determining the level of proADM or fragments thereof, preferably MR-proADM, in the sample, the body mass index of the subject, the weight of the subject, the age of the subject, the sex of the subject, the level of hemoglobin in the sample and the level of total serum protein in the sample, wherein the fluid balance and / or salt balance is determined on the basis of said markers and said parameters, wherein a salt balance of at least 36 g and / or a fluid balance of 4 L indicates that the subject has a critical health condition. In preferred cases, a salt balance of at least 36 g and a fluid balance of 4 L indicates that the subject has a critical health condition.

[0143] As used herein, "critical condition", "critical health state", "critically ill patient" or "critical subject" means that the subject or patient is in a life-threatening situation because the extracellular volume state is considered critical. As described above, subjects with positive fluid balance and / or salt balance have an increased mortality rate. For example, if a subject has an overload of body fluids or salts, such as caused by excessive intravenous infusion, he can be considered to have a critical health state. Thus, a critical subject has a critical positive fluid balance (e.g., at least 4L), a critical positive salt balance (e.g., at least 36 g) and / or a critical hematocrit state (less than 20 ml / kg). In some cases, if a subject has a low hematocrit state, such as less than about 20 ml / kg or preferably less than about 15 ml / kg, he can be considered to have a critical health state. In some cases, a critical hematocrit state is a hematocrit of less than about 15 ml / kg.

[0144] The level of the marker and / or parameter determined herein is an alarm signal for the physician to take appropriate action immediately. When used herein, "critical extracellular volume state" refers to an elevated or high extracellular volume. In a preferred embodiment of the present invention, the elevated extracellular volume is at least 3 L, preferably at least 4 L, wherein the elevated extracellular volume identifies an object with a critical health state. It is contemplated herein that an increase in body fluid or an increase in salts that increases the mortality rate of the object also depends on the characteristics of the object, such as weight, age or gender, etc. For example, a positive fluid balance of 4 L determined in a male object with a high body weight has a different effect than a positive fluid balance in a child. Therefore, it is contemplated herein that an increase in body fluid and / or an increase in salts indicating a critical object, i.e., 4 L or 36 g, respectively, depends on the characteristics of the object and can be higher or lower than 4 L or 36 g, respectively, depending on the characteristics of the object.

[0145] In the accompanying Examples it is documented that there is a strong statistical relationship between the combined predictor, i.e. combining fluid balance and salt balance, and the identification of a critically ill patient. In certain aspects of the invention, the methods provided herein identify a critically ill patient wherein the increase in water is at least 4 L and the increase in salt is at least 36 g. Thus, in preferred aspects, the method comprises determining the level of proADM or a fragment thereof, preferably MR-proADM, in a sample, wherein the subject has a level of proADM or a fragment thereof, preferably MR-proADM of at least 1 nmol / L, wherein the level of proADM or a fragment thereof, preferably MR-proADM indicates that the subject has a positive salt balance and a positive fluid balance, wherein the positive salt balance is at least 36 g and the positive fluid balance is at least 4 L, and wherein the positive salt balance and the positive fluid balance indicate that the subject has a critical extracellular volume status.

[0146] The accompanying examples demonstrate that there is a strong relationship between the Sequential Organ Failure Assessment score (SOFA score) and salt balance and / or fluid balance; see Examples 1 and 3. Thus, in preferred cases, the Sequential Organ Failure Assessment score (SOFA score) is determined on the basis of fluid balance and / or salt balance. In other preferred cases, the Sequential Organ Failure Assessment score (SOFA score) is determined on the basis of fluid balance and / or salt balance, wherein the fluid balance and / or salt balance is determined on the basis of the level of proADM or a fragment thereof, preferably MR-proADM. Thus, the method provided herein determines the level of proADM or a fragment thereof, preferably MR-proADM, in the sample, wherein the SOFA score is determined on the basis of the level of proADM or a fragment thereof, preferably MR-proADM.

[0147] In some cases, a SOFA score above 14 indicates a very severe health state, indicating a critical health state for the subject. A SOFA score between 0 and 6 indicates a less severe health state, and a SOFA score between 7 and 14 indicates a severe health state. In some cases, an elevated level of proADM or a fragment thereof, preferably MR-proADM, indicates a SOFA score for the subject, wherein a SOFA score above 14 indicates that the subject has a critical health state.

[0148] In the accompanying Examples it is shown that the inclusion of other parameters such as age, BMI and sex improves the predictive power of determining the SOFA score; see Figure 5 Thus, in some cases, the methods provided herein further comprise determining at least one parameter consisting of age, body mass index, and gender.

[0149] It is contemplated herein that the Sequential Organ Failure Assessment score (SOFA score) is at least 15, and wherein the score indicates that the subject has a positive fluid balance and / or salt balance that is considered critical.

[0150] As used herein, the "Sequential Organ Failure Assessment score" or "SOFA score" is a score used to track the status of a patient during a stay in an intensive care unit (ICU). The SOFA score is a scoring system that determines the degree of organ function or failure rate of an individual. The score is based on 6 different scores, each corresponding to the respiratory, cardiovascular, liver, coagulation, kidney and nervous systems. Both the average and the highest SOFA score are predictors of outcome. An increase in the SOFA score during the first 24 to 48 hours in the ICU predicts a mortality rate of at least 50% up to 95%. Scores below 9 give a predicted mortality rate of 33%, while scores above 14 may be close to or above 95%. The following scoring table describes only the conditions under which the points are given (Vincent JL et al., "The SOFA (Sepsis-related Organ Failure Assessment) score to describe organ dysfunction / failure", Intensive Care Med. 1996; 22: 707-710). In case the physiological parameter does not match any row, zero points are given. In case the physiological parameter matches more than one row, the row with the maximum number of points is selected. It helps doctors, nurses and other members of the patient's health care team to estimate the risk of morbidity and mortality caused by sepsis.

[0151]

[0152] In the accompanying Examples 1 and 4, it is shown that the combination of fluid balance and salt balance can efficiently (AUC>0.92) predict whether a subject is facing a critical condition, such as having a positive fluid balance and / or a positive salt balance. In certain aspects of the present invention, the methods provided herein identify the subject with a critical health state based on the subject's fluid balance and sodium balance.

[0153] The accompanying examples also demonstrate that the combination of fluid balance and salt balance can efficiently predict the risk of edema in a subject, i.e. the combined detection of fluid and salt balance can identify critically ill patients at risk of developing edema. In certain instances of the present invention, the methods provided herein identify subjects at critical risk of edema on the basis of the fluid balance and sodium balance. Therefore, in certain instances of the present invention, the methods provided herein are used to control therapy for subjects at critical risk of edema, wherein the risk of edema is determined on the basis of fluid balance and sodium balance. In certain instances of the present invention, the methods provided herein are used to control therapy for subjects at critical risk of edema, wherein the risk of edema is identified on the basis of fluid balance and sodium balance, wherein the fluid balance and / or salt balance is determined on the basis of the level of proADM or a fragment thereof, preferably MR-proADM.

[0154] As mentioned herein in the context of proteins or peptides, the term "fragment" refers to a smaller protein or peptide that can be derived from a larger protein or peptide, which therefore comprises a partial sequence of the larger protein or peptide. The fragment can be derived from the larger protein or peptide by deleting one or more amino acids from the larger protein or peptide.

[0155] When used herein, terms such as "marker", "surrogate", "prognostic marker", "factor" or "biomarker" or "biological marker" are used interchangeably and refer to a measurable and quantifiable biological marker (e.g., a specific enzyme concentration or fragment thereof, a specific hormone concentration or fragment thereof, or the presence of a biological substance or fragment thereof) that serves as an indicator of health and physiological related assessments such as disease / disorder / clinical condition risk. In addition, a biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal physiological processes, pathological processes, or pharmacological responses to therapeutic interventions. Biomarkers can be measured on biological samples (e.g., blood, plasma, urine, or tissue tests).

[0156] When used in this article, parameter is the feature, characteristic or measurable factor that can contribute to define a particular system.Parameter is an important element for the assessment such as disease / illness / clinical condition risk related to health and physiology.In addition, parameter is defined as the feature of the indicator as normal biological process, pathological process or the pharmacological response to therapeutic intervention by objective measurement and assessment.Exemplary parameter can be selected from body mass index, body weight, age, sex, IGS II, fluid intake, acute physiology and chronic health evaluation II (APACHE II), world federation of neurosurgical societies (WFNS) rating, Glasgow Coma Scale (GCS) and sequential organ failure assessment score (SOFA scoring).

[0157] For the purposes of the present invention, a "subject" (or "patient") may be a vertebrate. In the context of the present invention, the term "subject" includes both humans and animals, in particular mammals and other organisms. Thus, the methods provided herein are applicable to both human and animal subjects. Thus, the subject may be an animal such as a mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, bovine species, horse, camel or primate. Preferably, the subject is a mammal. Most preferably, the subject is a human. In the sense of the present invention, any sample collected from cells, tissues, organs, organisms, etc. may be a sample of a patient to be diagnosed. As shown in the accompanying examples, the extracellular volume status of a subject suffering from a variety of different conditions or diseases can be predicted. Thus, the methods provided herein may be used for any subject, whether a healthy subject or a subject suffering from any disease or condition. In a preferred embodiment, the subject suffers from a disease or condition, wherein the disease or condition is selected from edema, brain injury, post-aneurysmal rupture, head injury, neurological deficit, multiple traumatic injury, post-surgery, organ failure, lymphatic flow disorder, renal dysfunction, cardiac dysfunction, and diseases related to fluid balance disorders. In a more preferred embodiment of the present invention, the subject suffers from brain injury, aneurysm, head injury and / or multiple traumatic injury, and / or wherein the subject is post-surgery. In a most preferred embodiment, the subject suffers from severe brain trauma (SBT), aneurysmal subarachnoid hemorrhage (SAH), severe trauma (PT) without head trauma, post-operative peritonitis with shock (P) and / or post-operative gastrointestinal peritonitis.

[0158] As used herein, the terms "polytraumatic injury", "multiple trauma" or "polyinjury" in the context of the present invention encompass a condition with two or more severe injuries in at least two areas of the body, or a condition with multiple injuries, i.e., two or more severe injuries, in one body area. Polyinjuries may be accompanied by traumatic shock and / or hemorrhagic hypotension and seriously endanger one or more vital functions. At least one of the two or more injuries or the sum of all injuries endangers the life of the injured subject with polyinjuries. Trauma is an injury or damage to an organism caused by physical harm from an external source. Severe trauma is an injury that may potentially cause serious long-term consequences such as chronic pain.

[0159] As used herein, brain injury is damage to the brain, such as traumatic brain injury. Brain injury occurs when an external force traumatically injures the brain. Head injury usually refers to brain injury, but is a broader category because it can involve damage to structures outside the brain, such as the scalp and skull. Aneurysm (aneurysm or aneurism) is a localized, blood-filled balloon-like protrusion in the wall of a blood vessel. Aneurysms can occur in any blood vessel, examples include aneurysms of the circle of Willis in the brain, aortic aneurysms affecting the thoracic aorta, and abdominal aortic aneurysms. Aneurysms can also occur in the heart itself.

[0160] When used in this article, the postoperative object is the object undergoing surgery. More preferably, the postoperative object is the object undergoing major surgery. Major surgery can be any surgery in or on the contents of the abdominal cavity, pelvic cavity, cranial cavity or thoracic cavity, or any surgery that poses a risk to the function of life or organs or tissues in a given position, patient condition, difficulty level or time length of execution. Major surgery usually requires general anesthesia, hospital stays of varying lengths (usually one week), and can be performed by a board-certified surgeon in a secondary care hospital or by a surgical specialist in a tertiary care hospital. More preferably, the postoperative object is the object after digestive tract surgery. More preferably, the postoperative object is the object undergoing major surgery and suffering from a life-threatening disease or illness. This disease or illness may be caused by the surgery itself. Most preferably, the postoperative object is the object suffering from peritonitis with shock.

[0161] As used herein, the statistical relationship between the level of a marker, such as proADM or a fragment thereof, preferably MR-proADM and / or parameter, and the extracellular volume status, such as extracellular volume, blood volume or a disorder / disease / clinical condition of a subject is assessed using the statistical methods set out in the Examples appended herein. As demonstrated in the appended Examples, random forest analysis (Breiman, 2001 and 2002; and Boulesteix et al., (2012)), importance analysis, forward selection, linear regression, leave-one-out, "R" analysis, and / or other statistical methods are used to estimate the relationship between the level of a marker, such as proADM or a fragment thereof, preferably MR-proADM and / or parameter, and the extracellular volume status, such as extracellular volume, blood volume or a disorder / disease / clinical condition of a subject. 2 ” or “r 2 ” (coefficient of determination), AUC (area under the curve) and survival analysis. The statistical relationship between the parameters / values ​​can be calculated using any corresponding and suitable algorithm and software package available in the prior art.

[0162] When used herein, the terms "comprise" and "include" or their grammatical variations are considered to specify the stated features, integers, steps or components, but do not exclude the addition of one or more further features, integers, steps, components or groups thereof. The term encompasses the terms "consisting of" and "consisting essentially of.

[0163] Hence, the terms “comprising” / “including” / “having” mean that any additional components (or similarly features, integers, steps, etc.) can / may be present.

[0164] The term "consisting of" means that no other components (or likewise features, integers, steps, etc.) are present.

[0165] The term "consisting essentially of" or its grammatical variations when used herein is deemed to specify the stated features, integers, steps or components, but does not exclude the addition of one or more additional features, integers, steps, components or groups thereof, provided that the additional features, integers, steps, components or groups thereof do not materially alter the basic and novel characteristics of the claimed composition, apparatus or method.

[0166] Thus, the term "consisting essentially of" means that certain other components (or similarly features, integers, steps, etc.) may be present, i.e., those that do not materially affect the essential characteristics of the composition, device or method. In other words, the term "consisting essentially of" (which may be used interchangeably with the term "substantially comprising" herein) allows for the presence of other components in addition to the mandatory components (or similarly features, integers, steps, etc.) in the composition, device and method, as long as the essential characteristics of the device or method are not materially affected by the presence of the other components.

[0167] The term "method" refers to ways, means, techniques and procedures for accomplishing a given task, including but not limited to those ways, means, techniques and procedures known to practitioners in the chemical, biological and biophysical technical fields or which can be readily developed from known ways, means, techniques and procedures.

[0168] The term "about" preferably refers to ±10% of the specified numerical value, more preferably to ±5% of the specified numerical value, and especially to the exact numerical value specified.

[0169] When used in this article, the term "about" refers to ±10% of the specified value, especially ±5% of the specified value. Whenever the term "about" is used, a specific reference to the exact value specified is also included. If the term "about" is used in conjunction with a parameter quantified as an integer, such as the number of nucleotides in a given nucleic acid, the number corresponding to ±10% or ±5% of the specified value should be rounded to the nearest integer. For example, the expression "about 25 amino acids" refers to a range of 23 to 28 amino acids, especially a range of 24 to 26 amino acids, and preferably refers to a specific value of 25 amino acids.

[0170] Unless otherwise indicated, established methods of recombinant genetic technology were used as described in Sambrook, Russell, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, NY (2001), which is incorporated herein by reference in its entirety.

[0171] The sensitivity and specificity of a diagnostic and / or prognostic test depends not only on the analytical "quality" of the test, they also depend on the definition of what constitutes an abnormal result. In practice, a receiver operating characteristic curve (ROC curve) is usually calculated by plotting the value of a variable against its relative frequency in a "normal" (i.e., apparently healthy individuals who do not have a prenatal disorder or condition) and a "disease" population. For any particular marker (such as MR-proADM), the distribution of marker levels for subjects with and without the disease / condition may overlap. Under such conditions, the test cannot absolutely distinguish normal from disease with 100% accuracy, and the area of ​​overlap may indicate where the test cannot distinguish normal from disease. A threshold is chosen below which the test is considered abnormal and above which it is considered normal, or below or above which the test indicates a particular condition. The area under the ROC curve is a measure of the probability that the perceived measurement allows the correct identification of a condition. ROC curves can be used even in cases where the test results do not necessarily give an exact number. As long as one can rank the results, one can generate a ROC curve. For example, the test results for "disease" samples may be ranked by degree (e.g., 1 = low, 2 = normal, 3 = high). This ranking can be associated with the results in the "normal" population and a ROC curve can be generated. These methods are well known in the art; see, for example, Hanley et al., 1982.Radiology 143: 29-36. Preferably, the threshold is selected to provide an ROC curve area greater than about 0.5, more preferably greater than about 0.7, even more preferably greater than about 0.8, even more preferably greater than about 0.85, and most preferably greater than about 0.9. In this case, the term "about" refers to + / - 5% of a given measurement value.

[0172] The horizontal axis of the ROC curve represents (1-specificity), which increases with the false positive rate. The vertical axis of the curve represents sensitivity, which increases with the true positive rate. Therefore, for a selected specific cutoff value, the value of (1-specificity) can be determined and the corresponding sensitivity can be obtained. The area under the ROC curve is a measure of the probability that the measured marker level allows the correct identification of a disease or condition. Therefore, the area under the ROC curve can be used to determine the effectiveness of the test.

[0173] In other embodiments, a positive likelihood ratio, a negative likelihood ratio, an opportunity ratio, or a hazard ratio is used as a measure of the ability of a test to predict risk or diagnose a disorder or condition ("diseased group"). In the case of a positive likelihood ratio, a value of 1 indicates that the likelihood of a positive result is equal between subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that the likelihood of a positive result is greater in the diseased group; a value less than 1 indicates that the likelihood of a positive result is greater in the control group. In the case of a negative likelihood ratio, a value of 1 indicates that the likelihood of a negative result is equal between subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that the likelihood of a negative result is greater in the test group; a value less than 1 indicates that the likelihood of a negative result is greater in the control group.

[0174] In the case of the odds ratio, a value of 1 indicates an equal likelihood of a positive result between subjects in both the "diseased" and "control" groups; a value greater than 1 indicates a greater likelihood of a positive result in the diseased group; and a value less than 1 indicates a greater likelihood of a positive result in the control group.

[0175] In the case of the hazard ratio, a value of 1 indicates that the relative risk of an endpoint (e.g., death) is equal in both the "diseased" and "control" groups; values ​​greater than 1 indicate that the risk is greater in the affected group; and values ​​less than 1 indicate that the risk is greater in the control group.

[0176] It will be appreciated by those skilled in the art that associating a diagnostic or prognostic indicator with a diagnostic or prognostic risk of a future clinical outcome is a statistical analysis. For example, a marker level below X may convey a signal that a patient is more likely to suffer an adverse outcome than a patient with a level greater than or equal to X, as determined by a statistical significance level. In addition, changes in marker concentrations compared to baseline levels may reflect patient prognosis, and the degree of change in marker levels may be associated with the severity of the adverse event. Statistical significance is typically determined by comparing two or more populations and determining confidence intervals and / or p-values; see, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals for the present invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p-values ​​are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001 and 0.0001.

[0177] The invention is further described by reference to the following non-limiting figures and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0178] Figure 1 . Distribution of body water, where the body water can be divided into extracellular volume (the part in the ellipse) and intracellular volume (corresponding to about 57%). The extracellular volume itself can be further divided into blood volume, i.e. hematocrit (corresponding to about 6%) and plasma (corresponding to about 6%) and interstitial volume (corresponding to about 27%).

[0179] Figure 2. Deming regression of MR-proADM (logarithmic scale) with fluid balance (ΔH2O) (A) and salt balance (ΔNa) (B).

[0180] Figure 3. Box and whisker plots of MR-proADM concentrations in nmol / L in intensive care unit patients with aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head trauma (PT), severe brain trauma (SBT), or postoperative peritonitis with shock (P) on days 2 (A), 5 (B), and 7 (C). The mean concentrations of MR-proADM in nmol / L on days 2, 5, and 7 are shown (D).

[0181] Figure 4. ROC plots of MR-proADM for predicting fluid balance (ΔH2O) (A) and salt balance (ΔNa) (B) in intensive care unit patients with aneurysmal subarachnoid hemorrhage, severe trauma without head trauma, severe brain trauma, or postoperative peritonitis with shock.

[0182] Figure 5 . Predicted SOFA (leave one out). Patients are grouped by increasing SOFA. The solid black line gives the true SOFA value. On the x-axis is the patient id (patients are grouped starting with increasing SOFA values), predicted SOFA on the y-axis. The solid black line gives the (increasing) true SOFA values ​​for all patients. The blue circles give the predicted SOFA.

[0183] Figure 6 . Predicted ΔH2O, ΔNa, and P-critical values ​​for 201 patients. 126 "normal" patients (patients without edema) are represented by open circles, and 75 "critical" patients (patients with edema) are represented by closed circles.

[0184] Figure 7 . Random Forest Variable Importance for the Hematocrit Reference Model.

[0185] sequence

[0186] SEQ ID NO: 1: Amino acid sequence of pre-pro-ADM: 1MKLVSVALMY LGSLAFLGAD TARLDVASEF RKKWNKWALS RGKRELRMSS 51SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN 101NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR 151RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

[0187] SEQ ID NO: 2: Amino acid sequence of MR-pro-ADM (AS 45-92 of pre-pro-ADM): ELRMSSSYPT GLADVKAGPA QTLIRPQDMK GASRSPEDSS PDAARIRV

[0188] The examples described were performed by detecting MR-proADM. However, as outlined above, the invention may also be performed by detecting proADM or another peptide fragment thereof.

[0189] The following non-limiting examples illustrate the invention.

[0190] Example 1: Positive fluid balance, blood volume and MR pro-ADM in critically ill patients

[0191] method

[0192] Patients and procedures

[0193] This prospective 7-day observational study was conducted in 30 beds at the Anesthesiology and Intensive Care Unit of Bicêtre University Hospital, France, from March 2012 to September 2014. The Academic Review Board of Bicêtre Hospital approved the study in December 2011, and all patients or their relatives provided written informed consent.

[0194] Four types of patients were studied: patients with severe brain trauma (SBT), aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head trauma (PT), and postoperative peritonitis with shock (P). Patients were included if they required permanent mechanical ventilation on D2 (D2). SBT was defined as brain trauma with a Glasgow Coma Score of less than 9 (GCS < 9). SAH was included when the score was 4 or 5 on the WFNS scale (Brisman et al., 2006). PT was included when the ISS score was 25 or higher. P was included when they had signs of shock after abdominal surgery and had hemodynamic complications (hypotension, low cardiac output) or lactate > 4mmol / L and catecholamines were prescribed on admission to the intensive care unit.

[0195] Exclusion criteria were age < 18 years, pregnancy, and chronic cardiac insufficiency (NYHA II or IV).

[0196] General and demographic data were collected: age, sex, weight and height for BMI, IGS II, ISS, date of admission, and Glasgow Outcome Score (GOS) before leaving intensive care. Sequential Organ Failure Assessment (SOFA) scores were measured on arrival (D0), D2, D5, and D7 (Vincent et al., 1996).

[0197] Each day, mean arterial pressure (MAP) and the dose of norepinephrine, if used, were noted for each patient. Biological parameters were measured: hemoglobin concentration [Hb] and plasma proteins, plasma and urine concentrations of Na+, K+, Cl-, urea, creatinine, and osmolarity. The biological urine results obtained in the morning from a total of 24 hours of diuresis were used to calculate the sum of the previous day's urinary losses of Na+, K+, urea, and creatinine clearance. Body weight and temperature were measured on days 2 (D2), 5 (D5), and 7 (D7).

[0198] A combination of echographic signs, signs of fluid responsiveness in ventilated patients, and repeated measurements of cardiac filling guide daily fluid administration (Feissel et al., 2004; Feissel et al., 2001; Monnet et al., 2013; and Gore et al., 2005). In addition, the amount of intravenous fluid provided is determined by multiple variables including heart rate, arterial pressure, blood lactate levels, and cardiac output.

[0199] Assessment of extracellular volume

[0200] Each day, calculate salt and fluid balance to estimate changes in the extracellular space. Each morning, perform a complete input-output assessment of the previous day for salt and water. Record the exact salt and water contributions. Measure all losses: diuresis, ileostomy, and ventricular drainage if necessary. Sodium (Na+ ) losses are calculated from the fluid measurements and subtracted from the salt contribution. The difference between water input (the sum of enteral nutrition and crystalloid or colloid infusions for the day) and water lost is also calculated. Insensitive losses are estimated as a function of body temperature. Na is calculated daily. + (∆Na + ) and water (∆H2O) gains or losses were calculated and added to the previous day's result as cumulative fluid balance. Creatinine clearance was also calculated daily. All calculations for each patient were performed by one physician and verified by a second physician (BV, PEL, and HF).

[0201] Blood volume measurement

[0202] Total blood volume with red blood cells labeled with chromium 51 (Cr51) is measured on D2 and D7. For practical reasons, D2 is not always exactly the second day after the patient is admitted to the hospital, but somewhere between day 1 and day 3. Day 7 is somewhere between day 6 and day 10.

[0203] In the laboratory, 10 mL of the patient's own blood is radiolabeled with chromium 51 (Cr51) and the radioactively labeled red blood cells are carefully selected by carefully removing all plasma radioactivity. Then, a known amount of radioactive red blood cells is reinjected into the general blood circulation and two samples are taken in the artery at 10 and 30 minutes. Measurement of the radioactivity of the two samples allows the deduction of the total blood volume (TBV) in mL or in mL / Kg if the patient's weight is used (Gore et al., 2005). The hematocrit number and the measured total blood volume then define the red blood cell volume (RBCV) in mL or mL / Kg and the plasma volume (PV) in mL or mL / Kg. Normal values ​​(±20%) are 72±14 mL / Kg for TBV, 32±6 mL / Kg for RBCV and 40±8 mL / Kg for PV (Gore et al., 2005). On D7, a known small amount of radioactive red blood cells is injected into the patient with iodine 125 (I 125 ) Radiolabeled albumin directly measures plasma volume (PVI) 125 ), and samples were collected at 10, 30 minutes, and 2 hours (Fairbanks et al., 1996). 125 The normal value of PV is 45 ± 10 mL / Kg (Gore et al., 2005). 125 -The plasma volume obtained with albumin is slightly greater than that obtained from measurements using Cr51–erythrocytes because albumin has a greater distribution volume than erythrocytes (Gore et al., 2005).

[0204] When blood volume measurements were performed on D2 and D7, two indicators were used to assess stress volume: the change in the inferior vena cava (∆IVC) and E / E' (Vincent et al., 1996; and Feissel et al., 2004).

[0205] Biomarker analysis

[0206] For each patient, plasma biomarkers were studied on D2, D5 and D7. D2 and D7 were blood volume measurement days. The D5 sample was always obtained exactly 2 days before D7. As biomarkers, pro-adrenomedullin (MR pro-ADM), Pro-ANP, renin, angiotensin II, aldosterone, cortisol, epinephrine, CT-arginine pro-vasopressin (copeptin) and pro-endothelin were measured, which potentially interfere with extracellular or plasma volume and reduce (MR pro-ADM and Pro-ANP) or increase arterial pressure. The ability of erythropoietin (EPO) to vary with RBCV was measured. The criteria and techniques used are presented in Table 3.

[0207]

[0208] Statistical analysis

[0209] Power analysis: The required number of patients was calculated using the published range of values ​​for brain natriuretic peptide (BNP) and erythropoietin (EPO) concentrations in similar patients (Dorhout Mees et al., 2011), taking into account a power of 80% and an expected difference of 50% between the groups.

[0210] The normality of data distribution was examined using quantile comparison (qq) plots and Shapiro's test. Statistical comparisons were performed on transformed data when data were log-normally distributed. Data are reported as mean ± standard deviation (SD) or median (25 th Up to 75 th percentiles) or counts and frequencies (percentiles).

[0211] Depending on the normality of the data, linear or Spearman regression was used to investigate the correlation. In addition, since most data were measured with errors, Deming regression with equal variances was used to calculate the slope of the regression curve.

[0212] Comparisons within and between groups were performed using ANOVA (factorial for repeated measures) or nonparametric (Kruskal-Wallis or Friedman) tests followed by Tukey or Mann-Whitney / Wilcoxon tests, the latter corrected for multiple comparisons with the Bonferroni correction.

[0213] Receiver operating curves (ROC) were constructed to calculate the performance of the biomarkers in predicting fluid and sodium balance. The optimal sensitivity / specificity cutoff was calculated using the unweighted Youden index.

[0214] Finally, concordant analysis using four-quadrant plots with and without a 15% "gray zone" exclusion was used to compare markers and changes in fluid and sodium balance (Perrino et al., 1998).

[0215] Analyses were performed using R (The R Foundation for Statistical Computing, Vienna University of Technology, Vienna Austria, http: / / www.r-project.org / accessed June 20, 2015). Statistical significance was set at P < 0.05.

[0216] Independent associations between fluid balance and blood volume and biomarkers were found. A mathematical prediction tool was established using biomarkers and other significant parameters in order to identify clinically useful predictors of fluid status in critically ill patients using a "rational" covariate selection. The SOFA score, a severity score of the patient's condition as a multi-level ordinal variable, was taken as an additional response variable.

[0217] For fluid balance (∆Na + and ∆H2O), a joint predictor was constructed to identify + >36 g and ∆H2O >4 L. For this purpose, we first considered the combined (∆Na + and ∆H2O), its distribution is found to be Gaussian (centered) with a covariance matrix. Therefore, for each pair (∆Na + and ∆H2O) to obtain the probability of crisis (Pcritical), which is easily calculated using the mvtnorm software package (Genz et al., 2009; and Genz et al., 2014).

[0218] result

[0219] During the first 7 days after ICU admission, 67 patients were studied, distributed between SBT (n=21), HSA (n=20), PT (n=20), and P (n=6). General demographic data and the number of patients studied in each clinical setting are shown in Table 4. SOFA scores at D0, D2, D5, and D7 are presented in Table 5.

[0220]

[0221]

[0222] All patients were studied. For each clinical situation, the increase in extracellular volume was reported as the change in salt between D2 and D7 (∆Na + ) and water changes (∆H2O). Almost all patients had + 64 / 67 for ∆H2O or 63 / 67 for ∆H2O) and most in D7 (for ∆Na + 42 / 67 for ∆H2O or 41 / 67 for ∆H2O) showed a positive fluid balance, i.e. an increase in extracellular volume. A positive balance with sodium bicarbonate was observed at D2 and D7 in all diseases, with a higher increase in salt in PT and P compared to SHT and SAH. For example, on D2, PT and P showed a cumulative increase of 70 ± 32 g and 77 ± 28 g of salt, while STB and SAH showed 43 ± 24 g and 28 ± 24 g. ∆Na + is related to ∆H2O, showing that the retained water is tightly bound to the retained salt (r 2 =0.67; p<.0001). As known indicators of the extracellular space, plasma concentrations of protein and body weight changes were associated with ∆Na + and ∆H2O, but those relationships are weak (for plasma proteins and ∆Na + For example 2 = 0.44, for plasma proteins and ∆H2O r 2 = 0.35, and for body weight and ∆H2O r 2 =0.33). As a plasma protein, [Hb] and ∆Na + (r 2 =0.15) and ∆H20 (r 2 =0.24) has a weak relationship.

[0223] Volumes measured using Cr51 (total blood volume (TBV), red blood cell volume (RBCV), and plasma volume (PV)) were studied in 62 patients at D2 and in 63 patients at D7. A decrease in TBV was observed in most patients. Only 21 patients at D2 and only 25 patients at D7 were within 20% of normal. Hypovolemia, with TBV below 20% of normal, was present in 46 patients (74%) at D2 and in 42 patients (66%) at D7. Low RBCV was found in all patients except 1 patient at D2 and 2 patients at D7. All of these patients with normal RBCV were transfused. Even in non-bleeding conditions (SBT or SAH), the decrease in RBCV can be significant, with 50% or less of normal RBCV missing in 25 patients (40%) at D2 and in 21 patients (33%) at D7. There was no statistical relationship between TBV or RBCV and fluid balance, i.e., ∆Na or ∆H2O.

[0224] The distribution of PV was within the normal range. There was no relationship between PV and fluid balance, ∆Na or ∆H2O, and we also found no relationship between PV changes and ∆H2O changes between D2 or D7. Changes in PV were not associated with changes in plasma concentrations of proteins. Hemoglobin concentration was weakly correlated with RBCV (r 2 =0.33) and was not related to VP (r 2 =0.026).

[0225] On D7, PV (PVI125) was measured in 58 patients by I125. There was a statistically significant relationship between the two PVs (PV Cr51 and PVI125). Deming regression found a slope of 0.852 (CI 95% 0.610-1.08) and an intercept of 780 mL (CI 95% 103-1485 mL) (confidence intervals are referred to herein as CIs) in favor of PVI125, and r 2 =0.752.

[0226] No relationship was found between blood volume and the change in inferior vena cava (∆IVC) or E / E'.

[0227] Biomarkers

[0228] The detailed kinetics of all biomarkers are provided in Appendix I. Most of them increased on D2, followed by a statistical decrease on D5 and D7 (copeptin, angiotensin II, renin); some only statistically decreased on D7 (MRpro-ADM, EPO). Other biomarkers were unchanged during the 3 days considered (cortisol, aldosterone, Pro-ANP, proendothelin). Plasma norepinephrine was not relevant for this study due to the bias caused by external infusions used as treatment in patients.

[0229] Of all the biomarkers tested, we found that only two biomarkers, renin and MR pro-ADM, were significantly correlated with ∆Na + and / or ∆H2O have a statistical relationship (Table 6). + The relationship between ΔH2O and / or ΔH2O appears to be very strong and completely independent of the type of clinical situation.

[0230]

[0231] Then, 4L for ∆H2O and / or 4L for ∆Na + For example, 36 g (9‰ of 4 liters gives 36 g of salt) was used as the threshold for positive fluid balance (Bjerregaard et al., 2005). The ROC curve allowed us to find the optimal threshold for MR-proADM that showed the best compromise of sensitivity / specificity using the non-equilibrium Youden index (Figure 4).

[0232] Since MR-proADM was found to predict salt and fluid balance (∆Na + and ∆H2O), we therefore used MR-proADM and other simple covariates to establish a + and / or ∆H2O prediction scores. + For ∆H2O and / or ∆H2O, the best model for prediction required MR-proADM, BMI at D0, age at D0, sex, [Hb], IGS at D0, and fluid intake at D0, from the strongest parameter to the weakest parameter. Removal of MR-proADM from the model reduced the predictive power. Both models independently explained approximately 70% of the variance and had good discrimination, with AUCs for ∆Na and ∆H2O of 1.34 ± 1.13, with AUCs for ∆Na and ∆H2O of 1.37 ± 1.13 ...7 ± 1.13, with AUCs for ∆Na and ∆H2O of + 88% for ∆H2O and 92% for ∆H2O. Furthermore, the absence of IGS and fluid intake at D0 had very limited consequences in both models, with only a 2-3% loss of r2 and no effect on AUC.

[0233] If a combined predictor is constructed as described above in the statistics section to identify+ The performance was significantly improved for critically ill patients with both ∆H2O >36 g and ∆H2O >4 L. The discrimination of this combined predictor was high, with AUC = 0.9987 (95% CI [0.9964-1]). For example, using a threshold of 0.4 on the P-criticality value, a sensitivity of 0.988 and a specificity of 0.949 were obtained. In addition, this fluid balance predictor was significantly associated with the patients' SOFA score.

[0234] In addition, biomarkers were tested and blood volume was measured. Interestingly, among all biomarkers, the same two biomarkers, renin and MR pro-ADM, had a statistical relationship with blood volume, as was the case with fluid balance. However, if blood volume was analyzed again on the logarithm of the biomarker values ​​by regression for mixed purposes, the predictive properties of the markers became weaker, and the ROC curves had low AUCs (for renin: AUC TBV = 0.5798 and AUC PV = 0.6159, for MR pro-ADM: AUC TBV = 0.5967 and AUC PV = 0.6277). In addition, and despite the high probability (p = 0.0002), it was not possible to establish a ROC curve for RBCV and measure the AUC, because the values ​​were too non-characteristic and low. In those situations, there were no experimental thresholds and prediction models.

[0235] discuss

[0236] This study showed that the simple biomarker MR-proADM, known as an indicator of endothelial permeability (Christ-Crain et al., 2005; and Koyama et al., 2013), is a good surrogate for the increase in salt and water in the extracellular volume during the first week after admission to the hospital in critically ill patients. Furthermore, no relationship was found between the increase in salt balance or fluid balance and direct blood volume measurements on D2 and D7.

[0237] It is now clear that excessive salt or fluid balance can be considered a risk factor for morbidity and mortality in critically ill patients (Boyd et al., 2011; Kelm et al., 2015; Acheampong et al., 2015; and Malarian et al., 2014). This situation is frequent because the goal of making cardiac output efficient recommends fluid expansion to achieve therapeutic effects (Cecconi et al., 2011; and Vincent et al., 2011). In the context of hyperinflammatory conditions, usually in the first days after the insult (in sepsis, trauma, or cerebral aneurysm), many patients are at high risk of accumulating salt and water in the extracellular volume due to excessive capillary permeability, increased fluid intake, decreased urine intake, and difficulty maintaining salt in the plasma volume. Patients respond differently to fluid resuscitation. Therefore, clear and rapid surrogate markers are needed to better stratify patients and identify those with a positive salt balance in the interstitium in order to personalize treatment: the choice of infusing more fluid or adjusting with catecholamines or diuretics. Such a marker should also be independent of the type of ICU admission eligibility.

[0238] Given that MR-proADM appears to be a particularly good indicator of salt and fluid balance, it cannot simply be an indicator of capillary permeability. + Balance is not only controlled by the kidneys (Titze et al., 2014). + Interstitial clearance of Na+ appears to be mediated by immune cells, particularly macrophages, which control Na+ production via the interstitial lymphocytic capillary system. + Elimination (Titze et al., 2014). In inflammatory states, macrophages lose control of homeostatic immune cells, which involves their clearance of Na + The failure of the ability to + Interestingly, the adrenomedullin peptide is known to play a role in lymphatic channel tissues, especially in organogenesis (Kahn et al., 2008).

[0239] The excess in the interstitium is not only caused by inflammation and Na + It may be exacerbated by physiological reasons, such as high hydrostatic pressure caused by high mean arterial pressure (MAP) or high infusion rates (Bark et al., 2013) or increased blood stretch caused by excessive cardiac output. High MR-proADM (more than 1 nmol / l) indicates sodium overload, and for example, more than 36 g Na + An increase of 1 L to 4 L of water can serve as an alarm signal for the physician to take immediate appropriate action.

[0240] In many intensive care units, nurses systematically measure fluid balance by measuring body weight daily or calculating daily fluid input and output. These methods are not precise. For example, in our observational study, a weak relationship was found between body weight and fluid balance (r 2 =0.33). Common markers of extracellular volume such as plasma proteins or [Hb] have a weak relationship with salt and fluid balance (r 2 = 0.35, and r 2 =0.24). In this study, the measurement of salt and fluid balance required many biological samples and was always performed and controlled by two physicians. In daily practice, nurses cannot devote the time necessary to collect the required information. MR-proADM appears to provide a more accurate measure of salt balance and extracellular status and can also be used as an emergency surrogate after an emergency situation. In certain emergency situations, in the first few days after shock, timeliness may be crucial. In real life, the presence of overload is often discovered too late, after organ damage (acute lung injury, abdominal compartment syndrome, renal insufficiency). The relationship between the SOFA score and the prediction model of salt and fluid balance strengthens the idea that MR-proADM could be an interesting bedside tool.

[0241] No markers were found for estimating total blood volume (TBV), plasma volume (PV), or red blood cell volume (RBCV). Renin and MR-proADM were found to be significant but insufficient to establish a useful prediction model. Even ∆Na + Neither ∆H2O nor ∆H2O are predictors of blood volume.

[0242] Another interesting result of this study was the confirmation of the absence of a relationship between PV and fluid balance. This was particularly unexpected, since volume expansion is the main reason for fluid perfusion. However, PV was the only volume that was within normal range in most patients at D2 and D7. The absence of a correlation between fluid balance and PV reinforces the hypothesis that Na and water are trapped in the interstitial volume. In daily practice, better control of this plasma volume would be useful, but we did not detect a link with biomarkers or proteins or [Hb] or signs related to stressed volume measured using echography. Additional studies using a combination of biomarkers or other elements must be performed to investigate this issue.

[0243] The correlation found between the two measures of PV (Cr51 and I125) at D7 reinforces the results identified in this paper. Albumin may have a larger distribution volume than erythrocytes, especially if capillary permeability is increased in pathological situations. At D7, the difference was quite strong (780 mL), indicating that capillary permeability was not completely repaired. It may be interesting to discuss this comparison also at D2. Unfortunately, measurements using I125 could not be performed at D2 due to interactions with certain methods used to measure biomarkers (Table 1).

[0244] Low RBCV, anemia is quite common and explains the low TBV results. RBCV is difficult to assess and requires the CR51 method and radioactive device administration. Its assessment is only performed in rare cases such as Waquez disease. In daily practice, [Hb] threshold tests (<7-8 g / dl) are used to determine the need for transfusion. Since [Hb] is the ratio between PV and RBCV, [Hb] is a poor substitute for RBCV (Takanishi et al., 2008). A patient may have an [Hb] of 11 g / dL and only 50% of a normal RBCV. The lack of 50% of normal red blood cells can have important consequences for patient health. Some studies have shown that the total amount of RBCs can be a prognostic marker for cognitive rehabilitation (Naidech et al., 2007). However, RBC transfusion is more than just a replacement of lost cells. In the current study, EPO was independent of RBCV levels, indicating that EPO is not a good substitute for RBCV and that EPO stimulation is explained not only by the number of RBCs but also by the quality of RBCs.

[0245] The clinical situation described in this study reflects the daily practice at our institution. Severe trauma, brain injury, and neurologic conditions are common, and septic shock is less common. However, group as a covariate did not intervene in the prediction of fluid balance or blood volume, indicating that volume disorders are fairly independent of the disease. Additional studies are needed to confirm the current results.

[0246] Overload after shock is an important and underappreciated factor for survival. Positive salt balance is in fact documented as an important prognostic marker. Blood volume does not automatically correlate with volume expansion. MR-proADM is an interesting alternative to assess salt and fluid balance in critically ill patients in the first week after an acute inflammatory situation. It is recommended to withhold frequent excessive volume expansion.

[0247] Example 2: Prediction of fluid balance and / or salt balance by MR-proADM

[0248] 1. Introduction

[0249] The purpose of this study is to answer the following questions: •Can biomarkers and / or other covariates be used to predict changes in Na and H2O? •Can biomarkers and / or other covariates be used to predict volume responsiveness?

[0250] For this purpose, the predictive performance of the provided biomarkers and covariates was evaluated using data mining techniques and model selection.

[0251] 2. Materials and Methods

[0252] Please note that the following Materials and Methods section describes the materials and methods used in Examples 2 to 4 which serve only as exemplary embodiments.

[0253] 2. Materials and Methods 2.1 Data

[0254] Below is a description of the variables and datasets used in this report. A missing data rate of 3.5% was observed (54% for Max Lactate), which is low. As a result, missing data is not actually a significant issue in this study. Therefore, we decided to impute all missing data using the corresponding column mean.

[0255] Response variable A total of 2 target response variables were considered: •ΔH2O (D2, D5, D7): Change in water (H2O) expressed in liters (L). A change ≥ 4.0 L is considered critical. •ΔNa (D2, D5, D7): Change in sodium (Na), expressed in grams (g). A change ≥ 36.0 g is considered critical. •VT (D2, D7): Total volume, expressed in mL / kg. A total volume ≤ 60 mL / kg is considered critical (the classic threshold of 72 has been lowered to increase the number of controls). •VP (D2, D7): plasma volume expressed in mL / kg. A plasma volume of ≤ 40 mL / kg is considered critical. •VG (D2, D7): hematocrit, expressed in mL / kg. A hematocrit of ≤ 15 mL / kg is considered critical (the classic threshold of 32 has been lowered to increase the number of controls). • SOFA (D2, D5, D7): Sequential Organ Failure Assessment score. Multi-level ordinal response: the score increases with the severity of the patient's condition. Note that 4 SOFA scores above 15 were set to 15 to avoid SOFA scores with only one observation. NB: For D7, the second capacity measurement using iodine instead of CR51 was discarded.

[0256] Covariates • Patient covariates (8): age, sex, weight.D0, Bmi.D0 (body mass index), IGS.II (IGS II score), GOS (Glasgow Outcome Scale), fluid.D0 (fluid intake at D0), Na.D0 (sodium intake at D0) • Daily covariates (5): max.temp (maximum body temperature), max.lactate (lactate), min.PAM (minimum median arterial pressure), FC (heart rate), max.cathe (catecholamines) • Biomarker covariates (11): Hb, Prot.D0 (serum total protein on day 0), Prot (serum total protein), Angio (angiotensin II), Renin, Aldo (aldosterone), Pro.ANP, Adre (epinephrine), Pro.Endo (proendothelin-1), CT.proAVP, MR.proADM, Cortisol, Nor (norepinephrine), EPO (all on logarithmic scale, log1p transformed).

[0257] NB: BMI is calculated from height and weight using the following formula: The covariate height.D0 has been dropped to avoid redundancy with weight.D0 and bmi.D0.

[0258] 2.2 Statistical methods

[0259] All statistical calculations were performed using R software (Rmanual), version 3.0.2.

[0260] Random Forest

[0261] Random forests (Breiman, 2001; Breiman, 2002) are used to predict the response variable using covariates. The method consists of repeatedly building decision trees from bootstrapped data. A total of 50,000 trees are built for each run (500 for the leave-one-out procedure). This is a powerful data mining method known to be able to capture even nonlinear effects. For a good introduction to random forests in a biomedical context, see Boulesteix, 2012.

[0262] importance

[0263] A sensitivity analysis was performed by ranking the covariates in decreasing order of importance. For each covariate, random forest predictions were performed with or without the studied covariate and the consequence of its absence in terms of prediction quality was measured.

[0264] Positive selection and negative selection

[0265] Due to the high correlation structure between the covariates, selecting the best model by simply using the k most important variables will not necessarily produce the most accurate predictions. To overcome this problem, the classic approach (Diaz, 2006; Nguyen, 2013) is to use a backward selection procedure using random forests.

[0266] In the case of forward selection, the idea is to perform a sensitivity analysis starting with a null model (one RF per variable), add the variable that provides the lowest improvement in the target criterion (here R2 is used, see description below), and restart from the augmented model. Due to the highly random nature of random forests, the results from the forward selection procedure can vary from one replication to another. Therefore, a total of 5 replications were systematically performed using a high number of trees (trees = 50,000), and a consensus model that appeared stable between replications was selected.

[0267] In the case of backward selection, the idea is to perform a sensitivity analysis starting with the full model (one RF per variable), removing the variable that provides the lowest improvement in the target criterion (here using R2 (also known as r 2 ), see description below), and restart from the reduced model. Due to the highly random nature of random forests, the results from the reverse selection procedure can vary from one replication to another. Therefore, a total of 5 replications were systematically performed using a high number of trees (trees = 50,000), and the consensus model that appeared stable between replications was selected.

[0268] Linear Regression

[0269] Classical linear regression was also performed on the selected model. Studying the linear coefficients also provides a simple way to understand the individual effects of the covariates on the response variable.

[0270] Leave-One-Out

[0271] Since the data should not be split into training and testing datasets, a classic cross-validation technique called "leave one out" is used to avoid overfitting. Using this method, we repeatedly leave out one piece of data, train our model (linear regression or random forest) using the reduced dataset, and then use the resulting model to predict the value of the left-out entry.

[0272] R2

[0273] According to the squared correlation R2 (or r 2 ) measures the correlation between the response variable and the predicted response. In the case of a linear model, it corresponds exactly to the proportion of variance explained. R2 is always between 0 and 100%, the higher the better.

[0274] AUC

[0275] The classification performance is measured by the area under the ROC curve (AUC). This classical criterion is usually preferred because it takes into account all possible thresholds at the same time and does not even require the control of the H0 error rate. The AUC is always between 0 and 100%. An AUC of around 50% corresponds to pure noise, an AUC below 70% is considered weak, an AUC between 70% and 80% is considered correct, between 80% and 90% is good, and above 90% is excellent. Here, AUC estimation is performed using the pROC R package (robin2011proc).

[0276] Survival analysis

[0277] Standard survival analyses were used: Cox models (Andersen and Gill, 1982, Therneau, 2000), Kaplan-Meier nonparametric hazard estimates (Kaplan and Meier, 1958), and the log-rank difference test for significance between survival curves (Harrington and Fleming, 1982). For random forests, the package randomForestSRC (Ishwaran and Kogalur, 2007; Ishwaran et al., 2008; Ishwaran and Kogalur, 2015) was used.

[0278] 3. Results and Discussion

[0279] 3.1 Fluid balance

[0280] MR.proADM showed good performance for predicting ΔH2O. MR.proADM itself achieved good classification ability (AUC≃0.82) ( Figure 2A ), the variance of the response variable is 35%.

[0281] The selection procedure for ΔH2O was performed. The importance analysis pointed to the importance of the following patient and daily covariates: bmi.D0, weight.D0, age, sex, BMI, total protein, Hb, fluid intake at D0 (fluid.D0), patient group (group). The biomarker MR.proADM obtained the highest importance. Other biomarkers seemed to play a role: Pro.Endo, CT.proAVP, EPO, serum total protein and Hb.

[0282] 3.2 Salt balance

[0283] As observed for ΔH2O, MR.proADM had good classification ability (AUC ≃ 0.79), r 2 is 0.42 ( Figure 2B ).

[0284] The significance analysis performed pointed to the same markers / parameters as observed for ΔH2O, with the difference that sodium intake at D0 (Na.D0) replaced fluid intake at D0 (Fluid.D0).

[0285] 4. Conclusion

[0286] The key role of MR-proADM for the prediction of ΔH2O and ΔNa was clearly demonstrated. However, the combination of MR-proADM with other markers and / or parameters might improve the prediction of fluid and / or salt balance.

[0287] Example 3: Improving predictions by including other parameters

[0288] The aim of this study was to use a selection of covariates (other markers or parameters) (see Table 7) to establish clinically exploitable predictors.

[0289]

[0290] 1.1 Fluid and sodium balance

[0291] Reference model for predicting ΔH2O: Of these, both IGS.II and Liquid.DO are optional due to practical difficulties in obtaining them in a clinical setting.

[0292]

[0293] As can be seen in Table 8 (response “H2O”), this model explains about 70% of the variance and has very good discrimination, with an AUC of 92% (the lower boundary of the 95% CI is about 89%). The absence of IGS.II and / or Liquid.D0 has very limited consequences, with only a loss of 2-3% of r 2 , and has no effect on AUC at all.

[0294] This model was also compared to a predictor built from patient weight and Prot. Since weight was not available at D5, this time point was removed entirely, resulting in a smaller data set. The results are presented in Table 8 (response “H2O (-D5)”). The predictor built from weight and Prot only achieved an AUC of 80%. Note that this is a significant drop from our reference model (AUC of 90%). Furthermore, this loss was even more dramatic when only weight (AUC = 71%) or Prot (AUC = 63%) were considered, further confirming the limited benefit of these two covariates. One should also note that when using these models, r 2 Very limited, especially when only linear regression is used.

[0295] Sodium balance

[0296] Reference model for predicting ΔNa: As observed for ΔH2O, both IGS.II and Liquid.D0 are optional.

[0297] As can be seen in Table 8 (response "Na"), this model explains about 70% of the variance and has good discrimination, with an AUC of 88% (the lower boundary of the 95% CI is about 84%). As observed for ΔH2O, the absence of IGS.II and / or Fluid.D0 has very limited consequences. Likewise, as observed for ΔH2O, body weight and Prot have limited interest in predicting the response variable, both with low R 2 (∼ 20%) and AUC (∼ 63-75%).

[0298] Building joint predictors

[0299] In this section, predictors were developed for "critical" patients, i.e. patients with both ΔH2O > 4 L and ΔNa > 36 g. For this purpose, the leave-one-out residuals of (ΔH2O, ΔNa) were considered jointly and their distribution was found to be Gaussian (centered) with the following covariance matrix:

[0300] Therefore, the probability of each pair (x, y) of predicted values ​​(ΔH2O, ΔNa) becoming critical can be calculated as: This probability is easily calculated using the mvtnorm R package (Genz and Bretz, 2009; Genz et al., 2014).

[0301] A graphical representation of the two-dimensional p-critical function allows differentiation between non-critical and critical patients using only the ΔH2O and ΔNa predictive values. Note that the discrimination power of this combined predictor is high, AUC = 0.92 (see also Example 4).

[0302] 2.2 Volume responsiveness

[0303] Total volume and plasma volume

[0304] Surprisingly, ΔH2O and ΔNa were found to be suboptimal predictors for VT and VP (Table 9). In fact, they explained at most 7% of the variance and only marginally outperformed random classifiers in terms of discrimination (best AUC = 64%). This clearly confirms the clinical idea that "priming patients" with saline does not elicit the expected plasma response.

[0305]

[0306] Hematocrit

[0307] For hematocrit (VG), a correlation with ΔH2O and ΔNa was observed. A predictive (reference) model can be established:

[0308] As can be seen in Table 9, this model explained 45% of the variance (which is low), yet provided an AUC of 82% (95% CI lower boundary is 75%). This is a significant improvement over the simple model using Hb exclusively, which achieved an r of only 30%. 2 (using a linear model instead of a random forest) and an AUC of 70% (95% CI lower boundary is 60%). This result is interesting because it highlights the limitations of Hb as a single biomarker for hematocrit responsiveness, the current clinical standard.

[0309] As can be seen in Table 9, the "reference" model without MR-proADM achieved similar performance, r 2 The MR-proADM model identified patients with low (<20 or 15) VG or RBCV.

[0310] When considering the leave-one-out residuals of the best VG model, we clearly observe that these residuals are distributed according to a Gaussian centered distribution with a standard deviation of 3.2-3.3. Despite the fact that most of the observed variance cannot be explained by this model, one can already use them to detect critical patients: those with VG < 20 mL / kg. In fact, if x is the predicted VG, we obtain:

[0311] Figure 7 The P-criticality function is shown. Such a decision function may provide an effective alternative to Hb for detecting critical patients in a reliable manner.

[0312] 2.3 SOFA

[0313] exist Figure 5In Table 10, the SOFA values ​​predicted using the model selected by the experts and the leave-one-out procedure are shown. The correlation is very high, although the prediction accuracy is not great. In fact, in Table 10, the accurate prediction (difference = 0) has only 19% accuracy, but this number increases dramatically when a larger difference is allowed between the prediction and the accurate SOFA. For a difference of 4, a SOFA of 82% is obtained.

[0314]

[0315] Note that we also tried to regroup SOFA into three classes, but there was no significant improvement (33% error rate).

[0316] 2.4 Duration of edema

[0317] We can see the available data in Table 11.

[0318]

[0319]

[0320] Cox model

[0321] The analysis was started by fitting a proportional hazards model on these data (Table 12). Of the covariates tested, only age and SOFA were significant. In particular, MR.proADM did not appear to play a key role. We established an adjusted duration of edema by fitting a Cox model with age and SOFA only, with references of 41.5 (median) for age and 9 (median) for SOFA.

[0322] Kaplan-Meier Estimate

[0323] Kaplan-Meier estimates of the survival curves were analyzed using stratification at D2 with low (MR.proADM < 1.5) and high (MR.proADM > 1.5) values. The difference between the two curves was apparently not significant, confirming the results of Table 12. Note that when the unadjusted duration of edema was used, a significant difference between the two curves was observed (p = 0.01) (data not shown), but this result disappeared when adjustment was made for age and SOFA.

[0324] Random Forest

[0325] Random forest analysis was performed on the same (unadjusted) data. The procedure obtained an overall error rate of 41%, which is high (the exact nature of this error rate is unclear). Based on the variable importance, the key effect of age was confirmed, but both SOFA and MR.proADM seemed to have a weak impact on the results in this non-linear framework. Using the leave-one-out technique, a survival curve was predicted for each patient using random forest on the basis of the covariates (age, SOFA and MR.proADM). The difference between the two data sets was mainly caused by the fact that most of the patients with high MR-proADM at D2 were not cured at the end of the study. However, once the effects of age and SOFA were included, there was no clear trend towards cure in patients with lower MR-proADM at D2.

[0326] 3. Conclusion

[0327] For hematocrit, MR.proADM has a reasonable association. Furthermore, our study points out the limitations of current biomarkers (Hb) and proposes a new model that may be useful for clinicians to monitor patients with critical VG in the future.

[0328] A more interesting achievement of this study is the predictor of edema, which combines ΔH2O and ΔNa predictions to detect patients at critical risk of edema very efficiently (AUC>0.99), using only easily available clinical covariates (bmi.D0, weight.D0, age, sex and optionally IGS.II and fluid.D0) and three biomarkers: MR.proADM, Hb and Prot.

[0329] Example 4: Further improving predictions

[0330] Model

[0331] To simplify the model presented in this paper, IGS.II, Liquid.J0, and Na.J0 were removed from the original model to obtain a single simple model (referred to as "Model 2" below) for predicting both ΔH2O and ΔNa. Age2 = Age was included in the model 2 and age3 = age 3 The prediction power was further improved. This was the only added transformed covariate that appeared to have a significant effect. The model presented in Example 3 for predicting fluid and salt balance, i.e. including the parameters IGS.II, Fluid.J0 and Na.J0, is referred to as "Model 1". • “Model 2: ∼ bmi.J0+weight.J0+age+age2+age3+sex+MR.proADM+Hb+Prot • “Without biomarkers”: ∼ bmi.J0+weight.J0+age+age2+age3+sex • “Biomarker only”: ∼ MR.proADM+Hb+Prot NB: Please note that MR.proADM, Hb and Prot refer to the log1p transformation of the original measurements.

[0332] For each of these models, we can either use the leave-one-out method or use the full data set for prediction. As expected, performance is always higher in the latter case compared to the former. For robust and potentially replicable results, one should prefer the leave-one-out estimation, and for a more optimistic view and for comparison with very crude methods (such as using MR.proADM directly to discriminate between normal and critical patients), one should use the full data set estimation.

[0333] AUC Results

[0334]

[0335]

[0336] Compared to Model 1 presented in Example 3, Model 2 provided herein using only simple covariates and selected biomarkers achieves similar or even better performance. In all cases, P-criticality appears to efficiently combine ΔH2O and ΔNa predictions, slightly improving over the best of the two approaches. When considering a model without any biomarkers, there is a significant drop in performance. However, one should note that this model still indicates patients at high risk for edema. When considering only biomarkers, performance is comparable to the best model, but it is still somewhat inferior.

[0337] For comparison purposes, the performance of MR.proADM alone to discriminate between normal and critical patients achieved AUC = 0.845 [0.791-0.898], which for consistency has to be compared to the AUC of Table 14 (0.990 for the best model). Therefore, the combination of other markers and / or parameters provides even further improvements in predictive power.

[0338] Details about the best model

[0339] The ROCs obtained using three different approaches were compared, namely 1) using only the predicted ΔH2O; 2) using only the predicted ΔNa; and 3) combining both predictions into Pcritical. If ΔH2O is much less effective than the other two (which is consistent with Tables 13 and 14), the ROCs for ΔNa and Pcritical are very similar. However, due to high specificity (e.g., specificity > 0.95), Pcritical is superior to ΔNa. This can be emphasized by considering the (adjusted) partial AUC for specificity ∈ [1.00, 0.95]. Values ​​of 0.781 for ΔH2O, 0.882 for ΔNa, and 0.948 for Pcritical were obtained. These results suggest that Pcritical is even more reliable than ΔNa (and ΔH2O) when high specificity is required.

[0340]

[0341] The predictive performance using P-critical with various thresholds is summarized in Table 15. Depending on the cost of false positives and false negatives, this will allow the threshold to be chosen to obtain the best compromise between the two simultaneous risks.

[0342] The predicted ΔH2O and predicted ΔNa ( Figure 6 ). Both predictions are highly correlated (cor≃0.9), which is consistent with the observed ΔH2O and ΔNa (cor≃0.8). The performance of Pcritical confirms that the high-risk zone is almost exclusively represented by “critical” patients, and the low-risk zone is almost exclusively represented by “normal” patients.

[0343] Correlation between P-crisis, SOFA and VG

[0344] In this section, we compared the new P-crisis score with SOFA and VG (hematocrit).

[0345] In Tables 16 and 17, we can see that there is a good correlation between SOFA and P-crisis.

[0346] From now on, we focused on two specific target groups: a “low-risk” group, which collected a total of 86 patients (out of 201) with Pcriticality < 0:1, and a “high-risk” group, which collected 55 patients with Pcriticality > 0:9.

[0347]

[0348]

[0349] It was confirmed that SOFA was significantly higher in the "high risk" group than in the "low risk" group. In contrast, VG was significantly lower in the "high risk" group.

[0350] Finally, P critical was used to distinguish patients with VG < 15 (91 cases) from patients with VG ≥ 15 (43 controls). An AUROC of 0:76 was obtained (95% CI [0.67 – 0.85]). Note that the CI is quite large since we only have 134 VG measurements in the dataset.

[0351] in conclusion

[0352] The model presented in this paper using only biomarkers (MR.proADM, Hb, Prot) and simple covariates (bmi, weight, age, sex) achieved a maximum AUC of 0.926 in a leave-one-out framework and an AUC of 0.990 when all data were used. This performance is excellent. In terms of clinical application, Figure 6 The figures provided in can provide useful information for clinicians.

[0353] All references cited herein are fully incorporated herein by reference. Having now fully described the invention, it will be appreciated by those skilled in the art that the invention can be practiced within a wide and equivalent range of conditions, parameters, etc. without affecting the spirit or scope of the invention or any embodiment thereof.

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Claims

1. A method for determining the extracellular volume status of a subject, wherein the method comprises determining the level of mid-region pro-adrenomedullin (MR-proADM) in a sample obtained from the subject, wherein the sample is blood, plasma or serum, wherein the method comprises: (a1) comparing the level of MR-proADM in the subject with a reference level of MR-proADM in at least one reference subject or a population of reference subjects; or (a2) comparing the level of MR-proADM in the subject with the level of MR-proADM in the same subject obtained from a previous analysis; and (b) identifying the extracellular volume status of the subject based on the comparison in step (a1) or (a2), respectively; wherein the reference subjects are healthy subjects, and wherein the level of MR-proADM in the subject is elevated compared to the reference level, indicating that the subject has a critical extracellular volume state, wherein a critical extracellular volume state is a positive volume balance of at least 4L.

2. A method for determining the extracellular volume status of a subject, wherein the method comprises determining the level of mid-region pro-adrenomedullin (MR-proADM) in a sample obtained from the subject, wherein the sample is blood, plasma or serum, wherein the method comprises: (a1) comparing the level of MR-proADM in the subject with a reference level of MR-proADM in at least one reference subject or a population of reference subjects; or (a2) comparing the level of MR-proADM in the subject with the level of MR-proADM in the same subject obtained from a previous analysis; and (b) identifying the extracellular volume status of the subject based on the comparison in step (a1) or (a2), respectively; wherein the reference subject is a subject suffering from a disease or condition known to be associated with a critical extracellular volume state, or wherein the reference subject is a post-operative subject suffering from peritonitis with shock, and wherein (i) the subject's MR-proADM level is similar, identical or increased compared to the reference level, indicating that the subject has a critical extracellular volume state, wherein a critical extracellular volume state is a positive volume balance of at least 4 L; and / or (ii) the level of MR-proADM in the subject is reduced compared to the reference level, indicating that the subject has a normal extracellular volume status.

3. The method of claim 1 or 2, wherein the reference level is between 1.0 nmol / L and 1.5 nmol / L.

4. The method of claim 3, wherein the reference level is 1.0 nmol / L.

5. The method of any one of claims 1 to 4, wherein the method further comprises (i) determining the level of hemoglobin and / or the level of total serum protein; (ii) determining at least one parameter of the subject selected from body mass index, weight, age and sex; and / or (iii) determining at least one marker and / or parameter of the subject selected from the group consisting of: a proANP level in the sample, a total blood volume level, a hematocrit level in the sample, a red blood cell volume level, a plasma volume level, a total urine volume level, an angiotensin II level in the sample, a patient group of the subject, a cortisol level in the sample, a number of endothelial stem cells in the blood, a catecholamine level in the sample, a whole blood ionogram of the subject, a urine ionogram of the subject, a blood osmolality of the subject, a urine osmolality of the subject, blood glucose, a pro-endothelin-1 (pro-ET-1) level in the sample, a CT-proAVP level in the sample, aldosterone level in the sample, a lactate level in the sample, an Acute Physiology and Chronic Health Evaluation II (APACHE II) rating of the subject, a World Federation of Neurosurgical Societies (WFNS) rating of the subject, and a Glasgow Coma Scale (GCS) of the subject; or (iv) determining the body mass index of the subject, the weight of the subject, the age of the subject, the sex of the subject, the hemoglobin level in the sample, and the total serum protein level in the sample.

6. The method of any one of claims 1 to 5, wherein the subject suffers from a brain injury, an aneurysm, a head injury, a polytraumatic injury, and / or wherein the subject is post-operative.

7. The method of any one of claims 1 to 6, wherein the level of MR-proADM is determined by an immunoassay, wherein the assay is performed in a homogeneous or heterogeneous phase.

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