RU2778457C2 - MR-proADM AS MARKER FOR EXTRACELLULAR VOLUME STATUS OF SUBJECT - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method includes determination in a subject’s sample of a level of a marker of proadrenomedullin (hereinafter – proADM) or its fragment, preferably MR-proADM. Depending on the level of proADM or its fragment, a status of an extracellular liquid volume, a status of a liquid-salt balance, and a status of a subject’s globular volume are determined. The invention also relates to the use of a kit for the implementation of the specified methods. In addition, the invention relates to in vitro prediction of aggravation of symptoms in a subject.

EFFECT: group of inventions provides for the accuracy of detection of a status of an extracellular liquid volume, a status of a liquid-salt balance, and a status of a subject’s globular volume.

18 cl, 7 dwg, 16 tbl, 4 ex

Description

The present invention relates to determining the status of the extracellular volume of a subject, in particular patients in healthcare facilities, especially in intensive care units. The method includes determining in a sample obtained from the subject, the level of proadrenomedullin (proADM) or its fragments, in particular, the average regional proadrenomedullin (MR-proADM). In addition, based on the level of MR-proADM, you can determine the fluid balance and/or salt balance, which, in turn, show the status of the extracellular volume of the specified object.

BACKGROUND OF THE INVENTION

Exact blood volumes are difficult to estimate. Globular volume can be estimated by determining the concentration of hemoglobin (also referred to in the present description as "Hb") (Jacob, 2012). The extracellular volume can be estimated based on the weight of the subject, for example, the body is 60% water, i.e., 42 L for a 70 kg patient, and the extracellular volume is 40% body water, i.e., 17 L for a 70 kg patient ; see figure 1. In clinical practice, "effective" blood volume, based on the collection of dynamic information about changes in intravascular pressure and/or measurements of cardiac output, is evaluated several times a day in many acute and less acute situations in intensive care to guide and control prescriptions. for patients. This assessment is consistently used as the basis for making fundamental decisions regarding the amount of volume replacement solution and the use of catecholamine or blood transfusion. Every day, approximately 40% of patients in intensive care receive volume replacement after assessment (Finfer, 2011). Obtaining adequate blood volumes while avoiding positive fluid balance is a dilemma in the daily management of patients with acute inflammation, such as the treatment of traumatic stress or sepsis. This issue is very relevant in the resuscitation 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). Therapeutic recommendations focused on the need for emergency control of higher cardiac output to ensure adequate delivery of oxygen to the organs. Fluid administration, mainly composed of salt and water, is the main proposed tool for volume enhancement (Cecconi et al., 2009).

Increasing blood volume with salt dissolved in water leads to an increase in extracellular volume, especially when urine output is reduced by stress. In addition, capillary permeability can be excessively increased in situations of acute inflammation, which increases the risk of fluid accumulation (Chappell et al., 2009; Jacob et al., 2009; Ostrowski et al., 2015). It has been recognized that excessive volume expansion can provoke organ dysfunctions such as acute lung injury, abdominal compression syndrome or kidney dysfunction (Sakr et al., 2005; Bagshaw et al., 2008; Sakr et al., 2012; Besen et al. , 2015). In addition, studies have also reported increased mortality due to hydrosodium overload (Boyd et al. 2011; Kelm et al. 2015; Acheampong et al. 2015). A cumulative positive fluid balance with a weight gain of 3 to 4 kg, or 27 to 36 g of salt, resulting from a gain of 3 to 4 liters of water is considered to be the threshold at which mortality and morbidity increase (Lobo et al., 2002), Brandstrup et al., 2003; Bjerregaard et al., 2005). Therefore, the accuracy of this estimate is very important. The analytical methods used to prevent or correct these phenomena focus on "effective" blood volume based on the collection of dynamic information about changes in intravascular pressure and/or measurements of cardiac output. Although this strategy has proven effective in the early hours of shock, it is unable to prevent excess plasma expansion (Hilton, 2011).

Blood transfusion prescription is also determined by intravascular volume. Transfusion thresholds are usually considered in terms of hemoglobin (Hb) or hematocrit, which, by definition, is the ratio of red blood cell volume to total blood volume. Numerous clinical trials conducted in various intensive care settings suggest that for concentrations between 7 and 11 g/dl Hb, the error is such that it is difficult to accurately estimate the volume of circulating erythrocytes (Takanishi, 2008; Dorbout Mees, 2011, Jacob, 2012). Indeed, although clinical trials of large patient populations in intensive care units indicate that transfusions are ineffective and a policy of limiting prescriptions to a threshold of 7-8 g/dl is preferred, other studies conducted in target populations indicate that low Hb is not beneficial for forecast (Naidech, 2007, Kellert, 2011). Moreover, these arbitrary thresholds have been challenged as not being precise enough, regardless of any individual clinical situation (Klein, 2015).

A direct and accurate measurement of total blood volume can be made and the volume of settled erythrocytes and plasma volume can be derived from it. However, this study is rarely performed because it is expensive, time-consuming and labor intensive. Therefore, this method is used only for specific diseases (eg, polycythemia vera). The most reliable measurement is made by labeling the patient's erythrocytes with chromium-51 (Gore, 2005). Although this study is considered the gold standard for measuring intravascular volumes, it cannot be repeated every day (Gore, 2005). By labeling albumin with iodine-125 to measure the volumetric distribution of albumin, the distribution of albumin in the body can be determined. This protein is much more sensitive than erythrocytes to the deterioration of capillary permeability. As noted for the chromium-51 method, the iodine-125 method also cannot be repeated every day and is therefore only used for specific diseases. In addition, this kind of measurement is not suitable when instantaneous information about the status of the patient's volume is required, for example, in the case of patients in the intensive care unit.

In many intensive care units, nurses systematically measure fluid balance by daily weight measurement or daily calculation of input and output fluids. However, in daily practice, nurses cannot allocate the time needed to collect the necessary information. In addition, this method is not accurate and, in addition, the assessment of salt balance, a parameter indicative of changes in extracellular volume, is never taken into account.

In healthcare, especially in critical care, there is a fundamental need for an improved method for assessing a subject's extracellular volume status in order to better monitor tissue oxygen delivery and provide more balanced and accurate treatment. Moreover, an improved way of assessing extracellular volume status is critical because a positive diurnal fluid and salt balance can cause edema, and the persistence of a positive diurnal fluid balance over time is associated with higher mortality in patients with severe illness such as acute renal failure ( Payen, 2008), acute respiratory distress syndrome (Jozwiak, 2013), trauma (Elofson, 2015), subarachnoid hemorrhage (Kissoon, 2015) or sepsis (Acheampong, 2015).

Thus, the technical problem underlying the present invention is to provide means and methods for providing a fast and reliable method for assessing the extracellular volume status of an object.

The technical problem is solved by providing the embodiments presented below in the present description and in accordance with the appended claims.

Description of the invention

The invention relates to a method for determining the status of a subject's extracellular volume, the method comprising determining, in a sample obtained from said subject, the level of proadrenomedullin (proADM) or a fragment thereof, preferably mid-regional proadrenomedullin (MR-proADM).

In addition, the invention relates to a method for determining the fluid balance, salt balance and/or status of the globular volume of the subject, while the method includes determining in the sample obtained from the specified subject, the level of proadrenomedullin (proADM) or its fragment, preferably, the average regional proadrenomedullin ( MR-proADM).

The present invention solves the above technical problem, because, as described below and in the accompanying examples, it was surprisingly found that there is a surprisingly strong statistical relationship between the level of proadrenomedullin (proADM) or its fragment, preferably mid-regional proadrenomedullin (MR-proADM), and the status extracellular volume of the subject.

The attached examples document the results of a clinical study. This clinical study showed that among all biomarkers tested, including cortisol, catecholamine, renin, angiotensin II, aldosterone system (RAAS), vasopressin, reflected by CT-pro-AVP (also called "copeptin" in the present description), endothelin, reflected by proendothelin , and natriuretic peptides, reflected by proatrial natriuretic peptides (MR-ProANP), erythropoietin (EPO), and proadrenomedullin, reflected by e.g. MR-pro-ADM, MR-proADM has an unexpectedly strong statistical relationship with extracellular volume status (see e. figure 2, table 6). This relationship has been documented to be independent of the type of patient's clinical situation on day 2, day 5, and day 7 after admission, e.g., patients suffering from aneurysm (e.g., aneurysmal subarachnoid hemorrhage (SAH)), multiple trauma ( for example, severe trauma without head injury (PT)), traumatic brain injury or head trauma (eg, severe brain injury (SBT)), or patients after surgery, such as those with post-surgical peritonitis with shock (P); see, for example, figure 3.

Therefore, it is shown herein that proADM or a fragment thereof, preferably MR-proADM, is a good indicator of the extracellular volume status of subjects.

The examples provided show that high or elevated levels of MR-proADM are strongly correlated with an increase in salt and/or water in the extracellular volume during the first week after admission to critically ill patients (see, for example, figure 2 and example 1), and that almost all subjects have a positive fluid balance and/or salt balance, that is, an increase in extracellular volume. Increases in extracellular volume are recorded as changes in the salt balance and changes in the water balance of the subjects. In addition, the accompanying examples show that a positive fluid balance and/or salt balance has no correlation, for example, with total blood volume or plasma volume; see example 1.

To increase plasma volume, physicians may administer liquid infusions (eg, crystalloids) to the patient. Undifferentiated fluid treatment (eg, with aggressive fluid therapy) can increase fluid shear towards the extracellular volume, eg, in the intercellular space, which in turn can cause, for example, interstitial edema. The attached examples demonstrate that the effective volume is the volume of arterial blood flowing through the tissue.

As shown in the present description, MR-proADM has a significant relationship with the fluid balance and/or salt balance (examples 1-4, for example, Fig. 2). In particular, a high level of MR-proADM has been shown to indicate volume overload. For example, a high level of MR-proADM, eg, at least 1 to at least 1.5 nmol/l MR-proADM, is indicative of hypervolemia. Moreover, a high level of MR-proADM and an increase, for example, from at least 27 g to at least 36 g Na ⁺ and/or from at least 3 L to at least 4 liters of water is a warning sign for the doctor to take appropriate action immediately. Increased 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, which includes measuring the level of MR-proADM (or the level of proADM, or another fragment of proADM), has a high medical potential for quickly, conveniently and reliably determining the fluid balance, salt balance, globular volume status and extracellular volume of the subject. , as well as determining if the subject is in critical condition.

In addition, in the attached examples, it is confirmed that the evaluation of additional independent variables, such as additional markers and parameters, improves the discriminating ability of a single proADM marker (or fragment thereof), preferably MR-proADM. For example, only MR-proADM has good discriminating power with an AUC of 0.82 and a variance of 35% for fluid balance, and a discriminating power with an AUC of 0.79 and a variance of 42% for saline. balance; see e.g. example 2. Inclusion of additional markers and additional parameters in MR-proADM such as gender, age, total serum protein, BMI, weight and Hb further improves fluid balance and sodium balance prediction, e.g. with ROC curve 92% (see tables 13 and 14 and example 4).

In many intensive care units, nurses systematically measure fluid balance by daily weight or daily calculation of fluid input and output. These methods are inaccurate. For example, a weak relationship between weight and fluid balance was found in the attached examples (r ² =0.33, see example 1). Common markers of extracellular volume described in the prior art, such as plasma proteins or hemoglobin, have a weak relationship with salt and fluid balance, as shown below (for example, r ² =0.44 for plasma proteins and ΔNa ⁺ , or r ² = 0.35 for plasma proteins and ΔH ₂ O, r ² =0.15 for Hb and ΔNa ⁺ , or r ² =0.24 for Hb and ΔH ₂ O, see example 1). In the accompanying examples, salt and fluid balance measurements required multiple biological samples, and this analysis was always performed and supervised by two physicians, making the procedure time and labor intensive. It has been confirmed that proADM or a fragment thereof, preferably MR-proADM, used in the method of the present invention provides a faster and more accurate measure of salt and/or fluid balance, and thus extracellular volume status. Therefore, proADM or a fragment thereof, preferably MR-proADM, may be used as the critical condition identifier. In various acute situations, for example, in the first days after a shock, time is of the essence. Delay in the detection of overload following organ injury, such as acute lung injury, abdominal compression syndrome, or kidney dysfunction, can have serious and potentially fatal consequences. In the accompanying examples, it was also surprisingly shown that there is a significant relationship between the dynamic organ failure score (SOFA score) (Vincent et al., 1996) and salt and fluid balance. This predictive model also confirms that proADM or a fragment thereof, preferably MR-proADM, is a beneficial indicator and bedside tool.

Thus, the invention relates to a method for in vitro diagnosis, prognosis, risk assessment, risk stratification, management/control of therapy and/or operational monitoring of a disorder or medical condition in a subject, wherein the specified status of the extracellular volume of the specified 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 said subject may also reflect sodium retention in the subject.

The present invention has, among other things, the following advantages over conventional methods: The method of the invention is a fast, easy to perform, and accurate method for determining the extracellular volume status of a subject, providing a reliable prediction of extracellular volume status and positive fluid balance and/or positive salt balance. patient.

Another advantage of the proposed method is that fluid balance and salt balance correlate with the SOFA score. Therefore, the method provided herein provides a reliable and convenient way to identify a critically ill subject who is at risk for organ dysfunction or organ failure due to edema caused by positive fluid and/or salt balance. In addition, the proposed method allows you to determine the status of the globular volume. Example 3, and in particular Table 9, confirms the improved predictive value of the method of the present invention compared to the globular volume predictive value based on Hb alone.

In addition, the method of the present invention can stratify patients with a positive salt balance and thus can stratify patients with sodium retention, which may be a risk factor for hypertension, renal or heart failure, and pulmonary edema. Such patients may require other treatments that direct salt mobilization from the interstitial to the intravascular system.

In the accompanying examples, it was unexpectedly shown that MR-proADM has a significant statistical relationship with the extracellular volume of the patient. Accordingly, the present invention provides a method for determining the extracellular volume status of a subject, the method comprising determining, in a sample obtained from said subject, the level of a proADM marker or a fragment thereof, preferably MR-proADM.

In certain aspects, the present invention relates to the use of the mid-regional proadrenomedulin (MR-proADM) marker to determine the extracellular volume status of a subject.

Peptide adrenomedullin (ADM) was discovered as a 52 amino acid hypotensive peptide that was isolated from human phenochromocytoma (Kitamura et al., 1993). Adrenomedullin (ADM) is encoded as a 185 amino acid precursor peptide ("preproadrenomedullin" or "pre-proADM"), described herein under SEQ ID NO: 1. ADM contains amino acid positions 95-146 of the pre-proADM sequence and is its splicing product.

"Proadrenomedullin" ("Pro-ADM") refers to pre-proADM without a signal sequence (amino acids 1 to 21), that is, amino acid residues 22 to 285 of pre-proADM. "Mid-regional proadrenomedullin" ("MR-proADM") refers to amino acids 42-95 of pre-proADM. The amino acid sequence of MR-proADM is given under SEQ ID NO: 2. It is also contemplated herein that the peptide and its pre-proADM or MR-proADM fragment can be used in the methods described herein, such as predicting a subject's extracellular volume status. For example, the peptide and its fragment may contain pre-proADM amino acids 22-41 (PAMP peptide) or pre-proADM amino acids 95-146 (mature adrenomedullin). The C-terminal fragment of proADM (amino acids 153 to 185 pre-proADM) is called adrenotensin. Peptide fragments of proADM or MR-proADM contain, for example, 5 or more amino acids. Accordingly, the proADM fragment may be selected, for example, from the group consisting of MR-proADM, PAMP, adrenotensin and mature adrenomedullin, preferably, in the present description, the MR-proADM fragment.

It is also provided herein that the level of an MR-proADM polypeptide that has a sequence identity of at least 75%, e.g. 75%, 80%, 85%, 90%, 91%, 92%, 93% is determined. , 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown under SEQ ID NO: 2, with higher sequence identity being preferred. In accordance with the present invention, the terms "sequence identity", "homology" or "percent homology", or "identity", or "percent identity" or "percent identity" in the context of two or more amino acid sequences refers to two or more sequences or subsequences that are the same, or have a certain percentage of amino acids that are the same, when compared and aligned with the best match across the window of comparison (preferably over the entire length) or in a certain region, as measured using a sequence comparison algorithm known in this technical field, or by manual alignment and visual inspection. Sequences having, for example, 70-90% or more sequence identity can be considered to be substantially identical. This definition also refers to a sequence that is complementary to the test. Preferably, the described identity is present in a region that is at least about 15 to about 20 amino acids in length, more preferably in a region that is at least about 25 to about 45 amino acids in length, most preferably the entire length. Those skilled in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on the computer program CLUSTALW (Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp. App. Biosci., 6 (1990), 237-245), as is known in the art.

As used herein, the term "proadrenomedullin marker level (MR-proADM) or fragment thereof" refers to the amount of the proadrenomedullin marker molecular entity or fragments thereof in a sample that is obtained from a subject. In other words, the concentration of the marker is determined in the sample. Therefore, the term “regional mid-proadrenomedullin (MR-proADM) marker level” refers to the amount of the mid-regional pro-adrenomedullin (MR-proADM) marker molecular entity in a sample that is obtained from a subject. In other words, the concentration of the marker is determined in the sample. As described above, it is also contemplated herein that a fragment of proadrenomedullin (proADM), preferably MR-proADM, can be detected and quantified. In addition, MR-proADM fragments can be detected and quantified. Suitable methods for determining the level of proADM or a fragment thereof (preferably MR-proADM) are detailed below. Immunoassays in various formats such as, for example, sandwich assay, enzyme immunosorbent assay, fluorescent immunoassay, rapid test formats, assays suitable for spot treatment testing, and homogeneous assays, such as, for example, the Kryptor system (BRAHMS/Thermo Fisher scientific). In addition, mass spectrometry techniques can be used to detect and quantify proADM or a fragment thereof, preferably MR-proADM or a fragment thereof. Those skilled in the art are aware of assays that are suitable for detecting/quantifying the markers described herein.

The present invention relates to a method for determining the status of the extracellular volume of a subject. As used herein, extracellular volume is the fraction of water in a subject's body. Water in the body of the subject is about 55-75% of body weight. The water in the subject's body consists essentially of the "extracellular volume" and "intracellular volume" of the subject; see fig. 1. As used herein, "intracellular volume" refers to the cytosol or intracellular fluid (ICF), or cytoplasmic matrix, which is the fluid found within a cell. Normally, the intracellular volume is about 60% of the water in the body. According to Guyton (Guyton Arthur C., (1991), p. 275), a subject whose body contains 40 liters of fluid has an intracellular volume of 25 liters. As used herein, "extracellular volume" consists essentially of "total blood volume" and "interstitial volume". Normally, the extracellular volume is about 40% of the water in the body. Accordingly, a subject containing about 40 liters of fluid has about 15 liters of extracellular volume (Guyton Arthur C., (1991), p. 275). As used herein, "interstitial volume", "interstitial fluid" or "tissue fluid" is a solution that bathes and surrounds the tissue cells of metazoans. Typically, the interstitial volume is about 28% of body water, or about 70% of the extracellular volume. As used herein, "total blood volume" or "intravascular volume" consists essentially of "plasma volume" and "erythrocyte volume". Typically, total blood volume is about 12% of body weight and is made up of about 50% plasma (about 15% extracellular volume or 6% body water) and is made up of about 50% globular volume (about 15% extracellular volume). As used herein, "erythrocyte volume" is also referred to as "globular volume". As used herein, "plasma volume" refers to the volume of "blood plasma" or "plasma", which is the pale yellow liquid component of blood that normally holds blood cells in suspension in whole blood; this makes plasma the extracellular matrix of blood cells. It makes up about 55% of the total blood volume. It is the intravascular fluid portion of the extracellular fluid (all body fluid outside of cells). It mainly consists of water (up to 95% by volume) and contains dissolved proteins (6-8%) (for example, serum albumins, globulins and fibrinogen), glucose, blood coagulation factors, electrolytes (Na ⁺ , Ca ²⁺ , Mg ²⁺ , HCO ₃ ^- , Cl ^- etc.), hormones and carbon dioxide (plasma is the main medium for transporting excretory products). Plasma also serves as the protein reserve of the human body. It plays a vital role in the intravascular osmotic effect, which keeps electrolytes in a balanced form and protects the body from infections and other blood disorders. Used in the present description, the term "erythrocyte volume" is also referred to as the average volume of precipitated erythrocytes or the average cell volume (MCV), which is a measure of the average volume of red blood cells (or erythrocytes).

In the accompanying examples, it is documented that salt balance and/or fluid balance is calculated to assess the change in extracellular volume each day; see attached example 1. As shown in the attached examples, a full I/O assessment for the previous day is performed for (content of) salt and water each day to determine the fluid balance and salt balance of the subject. It shows that the loss of sodium and/or water in subjects can be measured by determining, for example, diuresis, ileostomy and ventricular drainage, if necessary. Loss of sodium (Na ⁺ ) can be measured in liquids and can be subtracted from the salt contribution; however, salt balance measurement is particularly difficult. The difference between water intake (eg, enteral nutrition, or total crystalloids, or colloid infusions per day) and water losses is also calculated. Insensitive losses are estimated as a function of body temperature. In the appended examples, the gain or loss of "sodium" or "Na ⁺ " (also referred to herein as "ΔNa ⁺ ", "sodium delta" or "sodium balance"), and the gain or loss of water, or H ₂ O (in also referred to herein as "ΔH ₂ O" or "fluid balance"), were calculated each day and added to the result from the day before as the cumulative "fluid balance" or "salt balance", respectively.

In the accompanying examples, it was surprisingly demonstrated that the level of MR-proADM in a sample, for example a plasma sample of a subject, has a statistical relationship with fluid balance and/or salt balance (figure 2). The invention thus relates to a method for determining the fluid balance, salt balance and extracellular volume status of a subject, the method comprising determining the level of proADM or a fragment thereof, preferably MR-proADM, in a sample obtained from said subject, provided that based on the level of proADM or a fragment thereof, preferably MR-proADM, determine the fluid balance and/or salt balance, and while the specified fluid balance and/or salt balance determines/identifies/reflects the status of the extracellular volume of the subject. High levels of MR-proADM have been found to correlate with high fluid balance (significant fluid gain) or high salt balance (significant salt gain); see, for example, FIG. 2. Therefore, the level of proADM or a fragment thereof, preferably the subject's MR-proADM, can be used to predict the salt balance and fluid balance of the subject. In other words, proADM or a fragment thereof, preferably MR-proADM, can be used as a direct indicator for fluid balance and/or salt balance. Accordingly, the term "based on 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.

In the accompanying examples, salt balance and/or fluid balance is calculated to assess the change or variation in extracellular volume. Fluid balance and/or salt balance are known to correlate with extracellular volume (Charra et al., 2004). Therefore, fluid balance and/or salt balance determine changes in extracellular volume. The status of the extracellular volume refers to the body fluid in the extracellular volume (figure 1). The extracellular volume of the subject is about 40% of the water in the patient's body. The status of the subject's extracellular volume correlates with the fluid balance and/or salt balance of the subject. Therefore, a change in fluid balance or salt balance is a change in extracellular volume. Thus, a change in the fluid balance and/or salt balance of the subject allows you to assess/determine the status of the extracellular volume of the subject. In other words, proADM or a fragment thereof, preferably MR-proADM, is a direct indicator for the fluid balance and/or salt balance of the subject and therefore indicates the status of the subject's extracellular volume. As used herein, "fluid balance" refers to "water variation", "water change", "water delta", or "ΔH ₂ O" of a subject. In other words, "fluid balance" is the difference between the input and output of the subject's "fluid" or "water". In preferred aspects of the invention, fluid balance is the difference between a subject's fluid/water inlet and outlet. In even more preferred aspects, the fluid balance is the cumulative fluid balance reflecting the difference between fluid/water inlet and outlet during the subject's hospitalization. When used in the present description, "during hospitalization" or "in hospitalization" means the period of time during which the patient is in a critical condition. Thus, as used herein, hospitalization of a subject may mean a period of time from the time the subject enters the intensive care unit until the critical situation and/or symptom(s) are alleviated. Alternatively, this term refers to the period of time in which the patient has accumulated a positive fluid balance, for example, 4 L, or a positive salt balance, for example, 36 g. In other words, this aspect refers to the period of time in which the subject has accumulated an increase, for example, per 4 liters of liquid or 36 g of salt. In the attached examples, fluid balance and salt balance were calculated every day. Therefore, the fluid balance is the difference between the fluid/water input and output of the subject during the first day (per day). In preferred aspects, the fluid balance is the cumulative fluid balance, which is the difference between the fluid/water input and output 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 between the inlet and outlet liquid/water flows of the subject after 7 days. As used herein, a subject's water intake/increase refers to a subject that has more water compared to an earlier point in time (e.g., one day before) because the fluid/water output was less than the water intake. As used herein, water loss/decrease in a subject is understood to refer to a subject that has less water compared to an earlier point in time (eg, one day before). As used herein, no change or no significant water change in a subject is understood to refer to a subject that has an identical or similar water content compared to an earlier water content (e.g., one day prior). In preferred aspects, MR-proADM is determined at multiple time points, such as day 0 ("D0"), day 2 ("D2"), day 5 ("D5"), and/or day 7 ("D7") after admission to the health department, in particular, to the intensive care unit. As used herein, it is understood that marker and/or parameter levels may be determined at any time and at any interval, such as hourly or daily (e.g., on admission D0 and 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 admission of the subject to the intensive care unit or the like), or a combination of these.

The accompanying examples show that the level of MR-proADM correlates with the fluid balance of the subject; see Figure 2. As used herein, high levels of proADM or a fragment thereof, preferably MR-proADM, are understood to indicate fluid gain/increase in the subject. As used herein, a subject that has a "positive fluid balance" refers to a subject whose fluid gain exceeds fluid loss. Therefore, the subject has a fluid/water input/output imbalance. Accordingly, a patient with a positive fluid balance accumulates water/fluid in the body. Therefore, the subject gains weight. In other words, a subject that has an increase in water content or a set of water has a positive fluid balance. For example, subjects treated with fluid infusions may have fluid/water fluid shift from the vasculature. This fluid may be dislodged towards an extracellular volume, such as the subject's interstitial volume. For example, an extracellular volume overload greater than 10 L after 3 days for a resuscitation patient has been shown to be retained in the body and take 3 weeks to release (Chappell et al., A rational approach to perioperative fluid management, Anesthesiology; 2008, 109 :723-40). Anatomic loss is considered a physiological phenomenon at the pathological level, that is, the accumulation of pathological fluid within the interstitial space (Chappell, supra). It can be assumed that the physiological fluid transferred from the vessels to the interstitial space with an intact vascular barrier contains only a small amount of protein and, above all, small molecules. When this transfer is quantitatively controlled by the lymphatic system, physiological transfer does not cause edema such as interstitial edema. However, overloading the lymphatic system, such as overuse of fluid infusions such as crystalloids, can cause edema. There are also non-anatomical losses in the third space, representing the fluid compartment, which is functionally and anatomically separated from the interstitial volume. Losses in this third space may be fluid accumulations caused, for example, by surgical procedures or trauma in spaces that are normally empty or low in fluid. For example, losses in the third space may be directed to the abdomen, intestines, and injured tissues.

In certain aspects of the present invention, a positive fluid balance of 3 to 4 kg weight gain (during hospitalization, for example, during the first day, preferably during the first two days, even more preferably during the first five days, most preferably during during the first week) is considered the threshold beyond which mortality and morbidity increase. Therefore, in certain aspects of the present invention, a liquid set of at least 3 liters, preferably at least 4 liters, is considered critical. A fluid set in the extracellular volume of at least 3 liters, preferably at least 4 liters, is considered critical. The amount of fluid that is considered critical also depends on the characteristics of the patient, such as the sex, age, or weight of the subject. For example, the water in the body of an adult woman is 5-10% less than the water in the body of an adult man. Thus, as used herein, it is understood that a lower weight patient (eg, female) may be more responsive to fluid and/or salt intake. In addition, the distribution of fluid in the fluid compartments depends on the age of the subject, for example, it decreases from 75% for a newborn to 55% for an adult. Thus, a lower fluid intake, such as 3 liters of fluid or less, may already have serious consequences for a woman or an elderly subject. On the other hand, a subject that is heavier (eg, a male) may not be as sensitive to fluid and/or salt intake as the lighter subject above. Therefore, the risk of mortality may be reduced for such a subject.

In another aspect, a positive fluid balance of at least 4 liters is considered critical. In other words, a positive fluid balance of at least 4 liters indicates that the subject has an extracellular volume status that is considered critical. In particular, it is documented in the accompanying examples that a high water intake, such as at least 4 liters of water, is a warning sign and indicates the critical status of the extracellular volume and therefore the critical condition of the subject. Endothelial injury and/or salt retention may be responsible for increased fluid balance.

As used herein, a subject that has "negative fluid balance" refers to a subject whose fluid loss is greater than fluid gain. Accordingly, the subject loses water or fluid and therefore loses weight. In other words, a subject that has a decrease in water content or loss of water has a negative fluid balance. As used herein, a subject that has an identical or similar water content refers to a subject that has an "identical or similar fluid balance". Such a subject has balanced fluid management and thus fluid balance is in balance, or is normal. In other words, the fluid/water inlet is identical or similar to the fluid/water outlet. In other words, the subject has a normal fluid balance.

In the context of the present invention, the fluid/water balance of a subject may, for example, be increased by intravenous therapy, including, for example, volume replacement solutions. In particularly preferred aspects, a subject's water intake refers to a subject that has more water/fluid compared to the water/fluid that was determined at an earlier point in time, wherein said subject that has more water/fluid refers to a subject that has a positive fluid balance. The accompanying examples show that a high level of MR-proADM, e.g., at least 1 nmol/l, indicates hydration, i.e., a positive fluid balance and therefore an elevated extracellular volume status, which extracellular volume status is considered critical. In other words, a positive fluid balance of at least 4 liters indicates that the subject has an extracellular volume status that is considered critical.

The invention relates to the method provided herein, in which, based on the level of proADM or a fragment thereof, preferably MR-proADM, salt balance is determined, and in which said salt balance determines/identifies the status of extracellular volume. As used herein, "salt balance" or "sodium balance" refers to "sodium variation", "sodium variation", "sodium delta", or "ΔNa ⁺ ". The "salt balance" is the difference between the input and output of the subject's "salt" or "sodium". In preferred aspects of the present invention, the salt balance is the difference between the fluid/water input and output of the subject. In even more preferred aspects, the salt balance is the cumulative salt balance reflecting the difference between salt/sodium input and output during the subject's hospitalization. In certain aspects, the salt balance is the difference between the salt/sodium input and output of a subject during the first day of the day. In preferred aspects, the salt balance is the difference between the subject's salt/sodium input and output during the first two days, even more preferably during the first five days, most preferably during the first week, i.e., the difference after 7 days. In the accompanying examples, it was unexpectedly demonstrated that the salt balance has a statistical dependence on the level of MR-proADM in the sample, for example, a plasma sample of the subject (figure 2B). In addition, the accompanying examples show that a subject's fluid balance is statistically related to salt balance; see Example 1. As used herein, a gain/increase in salt, eg sodium, in a subject refers to a subject that has a higher amount/content of salt, eg sodium. As used herein, a subject that has a higher amount/content of salt refers to a subject that has a positive salt balance. Therefore, a "salt positive" subject, as used herein, means a subject that has a salt intake in excess of the patient's salt loss. Thus, the subject has a salt/sodium input/output imbalance. Accordingly, a subject with a positive salt balance accumulates salt/sodium in the body, for example, has salt retention. In other words, a subject that has an increase in the amount/content of salt/sodium or a set of sodium/salt has a positive sodium balance. As used herein, "sodium retention" or "salt retention" may be indicative of kidney or heart failure. Salt retention can lead to fluid/water retention and increased blood volume, high blood pressure, and inflammation. Without being limited by theory, inflammation can cause salt retention. Because MR-proADM has a strong statistical relationship with salt and/or fluid balance, MR-proADM (or proADM or another fragment thereof) can be used as a predictive marker for inflammation (or inflammation-induced vascular and permeability damage).

As used herein, a subject that has a "negative salt balance" refers to a subject whose salt loss, eg sodium, is higher than salt gain. Accordingly, the subject loses salt or fluid and therefore loses weight. In other words, a subject that has salt reduction or salt loss has a negative salt balance. As used herein, a subject having an identical or similar salt content refers to a subject that has an "identical or similar salt balance". Such a subject has a balanced salt management, and therefore, the salt balance is in equilibrium, or is normal. In other words, the salt/sodium input is identical or similar to the salt/sodium output. In other words, the subject has a normal salt balance.

In one aspect, a positive salt balance means that the subject has a higher salt content and therefore an increased extracellular volume status compared to an earlier point in time. The accompanying examples show that a high level of MR-proADM, eg greater than 1 nmol/l, indicates salt gain, a positive salt balance, and therefore an elevated extracellular volume status, said elevated extracellular volume status being considered critical. In particular, it is documented in the accompanying examples that a high salt intake, eg of at least 27 g, preferably at least 36 g, of sodium is a warning sign and indicates the critical condition of the patient. Thus, in certain aspects of the present invention, a salt set of at least about 27 grams to at least about 36 grams is considered critical. In preferred aspects of the present invention, a positive salt balance of at least 36 g is considered 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 status that is considered critical.

In certain aspects of the invention, the method provided herein determines the globular volume status of a subject. In particular, the method provided herein determines whether a subject has a globular volume of less than 20 ml/kg. A globular volume of less than 20 ml/kg, or preferably a globular volume of less than 15 ml/kg, indicates a critical globular volume status. In certain aspects, a subject's globular volume of up to 20 ml/kg is predictive of a subject with critical extracellular volume status, said critical extracellular volume status being indicative of the subject's critical health status. Therefore, a globular volume below 20 ml/kg indicates that the subject has a positive fluid balance, with said positive fluid balance indicating critical extracellular volume status.

The method provided herein determines a subject's globular volume status, the method comprising determining the level of proADM or a fragment thereof, preferably MR-proADM, in the sample, the hemoglobin level in the sample, the subject's body mass index, the subject's gender, the subject's age, the level of total whey protein in the sample; and, optionally, the weight of the subject.

The accompanying examples document that the inclusion of additional markers or parameters in the statistical analysis improves the predictive value of proADM or a fragment thereof, preferably MR-proADM; see, for example, Examples 1 to 4. Statistical analysis surprisingly found a consensus model(s) including MR-proADM that has(s) a significant relationship with extracellular volume of the patient. As used herein, a consensus model includes more than one marker and parameter, and based on said consensus model, a subject's fluid balance and/or salt balance and/or extracellular volume status can be determined. In other words, in certain aspects, the invention relates to a method in which a panel (or multiple panels) of marker(s) and parameter(s) is defined. Therefore, other parameters and/or marker may be defined in the context of the invention. In other words, the method according to the present invention may be performed in combination with other markers, parameters and/or methods. This means that the measurement methods of the present invention may be particularly useful for multi-parameter diagnostics. Thus, at least one additional marker is additionally determined, preferably selected from the group of vasodilators.

In certain aspects of the present invention, the method provided herein includes determining the level of at least one additional marker selected from the group consisting of hemoglobin, total serum protein, renin, proatrial natriuretic peptide (proANP), C-terminal proarginine- vasopressin (CT-proAVP), erythropoietin, angiotensin II, aldosterone, cortisol, epinephrine, epinephrine, catecholamines and proendothelin-1 (pro-ET-1).

In certain aspects, the invention relates to the use of another marker selected from the group consisting of hemoglobin, total serum protein, renin, proatrial natriuretic peptide (proANP), C-terminal proarginine-vasopressin protein (CT-proAVP), erythropoietin, angiotensin II, aldosterone, cortisol, adrenaline, epinephrine, catecholamines, and proendothelin-1 (pro-ET-1).

In some preferred aspects, the method provided herein further includes determining the level of the marker "hemoglobin" (herein also referred to as "hemoglobin"). "Hemoglobin", or "Hb", is an iron-containing oxygen-carrying metalloprotein in vertebrate erythrocytes. The Hb concentration can be measured in the context of routine blood tests, usually as a fraction of the total 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); for pregnant women: 11 to 14 g/dl 9.5-15 (usual value 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 a person's hemoglobin level is below the lower normal ranges for their age and sex (see normal ranges 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, high levels of hemoglobin means that the measured levels of hemoglobin are above the upper limits of normal for the age and sex of the person (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.

As used herein, "total serum protein" refers to the total amount of protein in the blood. In preferred aspects, total serum protein refers to the total amount of protein in blood serum or plasma. "Total whey protein" is measured in routine assays and used in intensive care units and other medical services. The two main protein components in serum or plasma are albumins and globulins. Globulin is made up of various proteins called alpha, beta, and gamma types. The total serum protein assay reports separate values for total protein, albumin, and globulins. Total serum protein can, for example, be determined using a biuret reagent or a refractometric method. Hypoproteinemia is the result of insufficient synthesis due to liver failure, malnutrition, or due to loss in the kidneys. An increase in serum protein concentration has two main causes: dehydration, in which there is less water in the body, and blood volume decreases. The proteins most commonly overproduced are immunoglobulins, whose levels can be elevated in infections and hematological neoplasms. The normal range for total whey protein is about 60 to about 80 g/L.

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

As used herein, "proatrial natriuretic peptide" or "proANP" refers to a 128 amino acid prohormone. As used herein, a peptide containing the 28 amino acids (99-126) of the C-terminal portion of the 128 amino acid prohormone (proANP) is referred to as the actual ANP hormone. After ANP is released from its pro-hormone proANP, an equimolar amount of the remaining larger partial peptide of proANP, the 98 amino acid N-terminal proANP (NT-proANP; proANP (1-98)), is released into the circulation. Because NT-proANP has a significantly longer half-life and stability, NT-proANP can be used as a laboratory parameter for diagnosis, post-treatment monitoring, and therapy monitoring; see, for example, Lothar Thomas (editor), Labor und Diagnose, 5th expanded ed., subsection 2.14 of section 2, ^Kardiale Diagnostik, pages 116-118 and WO 2008/135571. The proANP level is preferably measured in the subject's plasma or serum.

As used herein, endothelin-1 is derived from a larger precursor molecule called proendothelin-1. Proendothelin-1 can be proteolytically processed into various fragments as described (EP 2108958 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 circulation, which can occur rapidly or slowly, depending on the type of fragment and the type and concentration/activity of proteases present in the circulation. Thus, according to the present invention, the level of any of these fragments of at least 12 amino acids, preferably fragments of 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.

As used herein, "vasopressin" refers to "AVP". Vasopressin is a strong vasoconstrictor. Analysis of its prohormone has been considered as a prognostic and diagnostic factor for cases of diabetes insipidus. Vasopressin, or antidiuretic hormone (ADH), is one of the keys to the regulation of body water and water balance. Its secretion, which is partly related to stress, causes an increase in blood pressure and water absorption, which can lead to hyponatremia. However, ADH is unstable. Moreover, its concentration depends on its association with platelets and is therefore labile. The C-terminal portion of the prohormone "CT-proAVP" is a more stable ADH precursor and its plasma concentration reflects ADH secretion (Struck, 2005, Morgenthaler, 2007). As used herein, the C-terminal portion of the prohormone is referred to as "CT-proAVP" or "copeptin". Elevated plasma levels after septic shock or hemorrhage correlate with osmolarity and mortality (Morgenthaler, 2007). The level of CT-proAVP is preferably measured in the plasma or serum of the subject.

Angiotensin I is converted to angiotensin II by removal of two C-terminal residues by an angiotensin converting enzyme (ACE), primarily through ACE in the lungs (but also present in endothelial cells and epithelial cells of the kidneys). Angiotensin II acts as an endocrine, autocrine/paracrine and intracranial hormone. The level of angiotensin II is preferably measured in the plasma or serum of the subject.

In certain aspects, the method provided herein further includes determining at least one subject parameter selected from the group consisting of body mass index, weight, age, sex, IGS II, lactate, sodium intake, fluid intake, and group to which the patient belongs. In some preferred aspects, the method provided herein further includes determining at least one subject parameter selected from the group consisting of body mass index, weight, age, and gender.

When used in the present description, the body mass index (BMI) is a value derived from the weight (weight) and height of the subject. BMI is defined as the subject's body weight, that is, weight divided by the square of the subject's height, and is expressed everywhere in units of kg/m ² , with weight in kilograms and height in meters. BMI can also be determined using a table or chart (reference values) that displays BMI as a function of weight and height using contour lines or colors for different BMI categories and can use two different units of measurement. BMI is an attempt to quantify the amount of tissue (muscle, fat, and bone) in an individual and then classify that person as underweight, normal weight, overweight, or obese based on that value. The generally accepted BMI ranges are: underweight: less than 18.5, normal weight: 18.5 to 25, overweight: 25 to 30, obese: more than 30. In certain aspects of the invention, BMI is determined on day 0, for example, at admission patient.

When used in the present description, "weight" refers to the weight of the subject in kg (see BMI). In certain aspects of the invention, weight is determined on day 0, for example, on admission of the patient. In the context of the present invention, normal body weight can be theoretically calculated according to Devin's formula or the Humvee method. According to the Humvee method, the ideal human body weight 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.

As used herein, "age" refers to the length of time an individual has lived, in years. In preferred aspects, the parameter is weighted in the method by adding the square and cube of the age, ie, age ² and age ³ .

As used herein, "IGS II" (i.e., the abbreviation "Indice de Gravité Simplifié") or "SAPS II" (i.e., the abbreviation "Simplified Acute Functional Change Scale II") refers to a classification system for the severity of a disease or disorder. (See Le Gall JR et al., A new Simplified Acute Physiology Score (SAPS II) based on a European/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 score is calculated from 12 routine physiological measurements, previous health information, and some information obtained upon admission to the ICU. IGS II can be determined at any time, preferably on the 2nd day. The "worst" measurement is defined as the measure that correlates with the highest score. 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, nitrogen, urea, sodium, potassium, bicarbonate, bilirubin, leukocytes, chronic diseases and type of admission. There is a sigmoidal relationship between mortality and total SAPS II. Subject mortality is 10% with a SAPSII score of 29, mortality is 25% with a SAPSII score of 40, mortality is 50% with a SAPSII score of 52, mortality is 75% with a SAPSII score of 64, mortality is 90% with a SAPSII score of 77 (Le Gall, see above).

As used herein, "fluid intake" refers to fluid intake by a subject over a specified time period, such as within 24 hours. For example, fluid intake by a patient or subject may be a fluid infusion or fluid resuscitation. Preferably, fluid intake is determined on day 0, in other words, on or after the patient's admission.

As used herein, the "gender" of a subject refers to the biological sex of the subject, whether the subject is male or female.

As used herein, "sodium intake" refers to the total amount of salt, or preferably sodium, such as sodium chloride, that the body receives from external sources such as food (food and fluids) or fluid infusion. Preferably, sodium intake is determined on day 0, in other words, on or after the patient's admission.

As used herein, "lactate" or "max.lactate" refers to the maximum concentration of lactate measured in the blood. Typically, the lactate concentration is measured daily or even more frequently. The concentration of lactate in the blood can be determined using spectrophotometric methods based on lactate oxidase.

As used herein, "total blood volume", "TBV", or "TV" may be measured using chromium 51 (Cr ₅₁ )-labelled red blood cells. The total blood volume can be measured at any time, in particular between the 1st day and the 3rd day, for example on the 3rd day; and/or between the 6th day and the 10th day, for example, on the 10th day. In the present description, it is envisaged that the patient's own blood is labeled with a radioisotope of chromium 51 (Cr ₅₁ ), and radiolabeled red blood cells are selected. A known amount of radiolabeled erythrocytes is then reintroduced into the general circulation. For example, two samples may be taken from an intra-arterial catheter at two time points, such as 10 minutes and 30 minutes. To derive total blood volume (TBV) in ml or ml/kg, the radioactivity of two samples is measured and the patient's weight is determined (Gore et al., 2005). The hematocrit value 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).

"Plasma volume" or "PV" can be measured using iodine-125 (PVI ₁₂₅ ). Plasma volume can be measured at any time, for example on day 7. In the attached examples, PVI ₁₂₅ is measured on day 7. Each patient is injected with a certain amount of radioactively labeled albumin with iodine 125 (I ₁₂₅ ), and samples are collected at several time points, for example, 10 minutes, 30 minutes and 2 hours after injection (Fairbanks et al., 1996). The normal value of PV, measured by I _125, is about 45±10 ml/kg (Gore et al., 2005). Typically, plasma volume measured with I ₁₂₅ -albumin is slightly larger than plasma volume measured with Cr ₅₁ due to a larger volume of distribution in albumin than in erythrocytes (Gore et al., 2005).

As used herein, "patient group" or "group" means that subjects are sorted according to their disease profile or medical picture. As shown in the attached examples, subjects are sorted into 4 groups, namely, severe brain injury (SBT), aneurysmal subarachnoid hemorrhage (SAH), severe injury without head injury (PT), and post-surgical peritonitis with shock (P).

In certain aspects, the method determines a subject's extracellular volume status, the method comprising determining at least one marker and/or parameter selected from the group consisting of proADM or a fragment thereof, preferably MR-proADM, sex, hemoglobin, total whey protein, IGS II score, fluid intake, and sodium intake. The accompanying examples show that random forest analysis can be used to select the combination of markers and parameters that gives the lowest error. Surprisingly, it has been shown that the best model for predicting a subject's extracellular volume status is achieved using the MR-proADM level in combination with body mass index, weight, age (age ² and age ³ ) and the subject's sex, hemoglobin, and total serum protein. Therefore, in most preferred aspects of the invention, the method provided herein includes determining the level of proADM or its fragment, preferably MR-proADM in the sample, the body mass index, weight, age, sex of the subject, the hemoglobin level in the sample and the level of total serum protein in the sample. As shown in the attached Example 4, markers such as MR-proADM, total serum protein and hemoglobin have good predictive value, eg AUC of 0.94 for fluid balance; see, for example, tables 13 and 14. Adding parameters such as body mass index, weight, age, and sex of the subject to the markers improves the AUC, for example, to 0.95 for fluid balance. When using, for example, only parameters such as body mass index, weight, age and sex of the subject, the AUC is 0.88 for fluid balance. Thus, in certain aspects of the invention, the method provided herein includes determining the body mass index, weight, age, sex of the subject. In preferred aspects of the invention, the method provided herein includes determining the level of proADM or a fragment thereof, preferably MR-proADM, in the sample, the hemoglobin level in the sample, and the level of total serum protein in the sample. In the most preferred aspects of the invention, the method provided herein includes 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 hemoglobin level in the sample, and the level of total serum protein in the sample. .

As documented in the accompanying examples, in particular examples 3 and 4, various combinations of markers and parameters can be used to determine the extracellular volume status of a subject. In certain aspects of the invention, the method provided herein determines the fluid balance of a subject, the method including determining the level of proADM or a fragment thereof, preferably MR-proADM in the sample, body mass index, weight, age, sex of the subject, hemoglobin level in the sample , the level of total serum protein in the sample, the IGS II score, and the fluid intake of the subject. In certain aspects of the invention, the method provided herein determines the salt balance of a subject, the method including determining the level of proADM or a fragment thereof, preferably MR-proADM in the sample, body mass index, weight, age, sex of the subject, hemoglobin level in the sample , the level of total whey protein in the sample, the level of sodium intake in the sample, the IGS II score, and the fluid intake of the subject. As shown in the attached example 3, the absence of IGS II parameters and fluid intake has little effect on the statistical analysis, with a loss of only 2 to 3% of r ² , and does not affect AUC; see, for example, Table 6. Thus, in some other aspects of the invention, the method provided herein includes determining the level of proADM or a fragment thereof, preferably MR-proADM in the sample, body mass index, weight, age, sex of the subject, hemoglobin level in the sample; and total serum protein level in the sample.

In certain aspects of the present invention, the method includes determining at least one additional marker and/or subject parameter selected from the group consisting of proANP level in the sample, total blood volume level, hematocrit level in the sample, erythrocyte volume level, plasma volume, total urine volume level , angiotensin II level in the sample, patient group for the subject, cortisol level in the sample, the number of endothelial stem cells in the blood, the level of catecholamines in the sample, the subject's total blood ionogram, the subject's bladder ionogram, the subject's blood osmolarity, subject's urine osmolarity, subject's blood sugar, sample proendothelin-1 (pro-ET-1), sample CT-proAVP, sample aldosterone, sample lactate, Acute and Chronic Functional Change Scale II (APACHE II) of the subject, the subject's World Federation of Neurosurgical Societies (WFNS) score, and the subject's score subject's ki according to the Glasgow Coma Scale (GCS).

The accompanying examples document that there is a significant statistical relationship between the dynamic organ failure score (SOFA score) and fluid balance and/or salt balance; see examples 1 and 3 and figure 4. MR-proADM correlates with fluid balance and/or salt balance. Thus, in certain aspects, a dynamic organ failure score (SOFA score) is determined based on 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 an indicator marker for SOFA evaluation.

In some other aspects of the present invention, a dynamic organ failure score (SOFA score) is determined based on fluid balance and/or salt balance. The accompanying examples show that the inclusion of additional parameters such as age, BMI, and gender improves the predictive value for determining the SOFA score; see fig. 5. Thus, in certain aspects, the method provided herein determines a SOFA score based on fluid balance and/or salt balance, the method further comprising determining at least one parameter consisting of age, body mass index, and gender.

Furthermore, the invention relates to a method, said method comprising:

(a) determining the level of proADM or a fragment thereof, preferably MR-proADM, in the subject's sample, and

(b1) comparing said level of proADM or said fragment, preferably MR-proADM, with reference data corresponding to said level of proADM or said fragment, preferably MR-proADM, of at least one reference subject; or

(b2) comparing said proADM level or said fragment, preferably MR-proADM, with data corresponding to said proADM level or said fragment, preferably MR-proADM, from the same subject from a previous assay;

(c) determining the fluid balance and/or salt balance of the specified subject based on step (b) comparison; and

(d) determining the state of the globular volume and/or the status of the extracellular volume based on step (c).

In other words, the invention relates to the method provided herein, said method including:

(a) determining the level of proADM or a fragment thereof, preferably MR-proADM, in the subject's sample, and

(b1) comparing said level of proADM or said fragment, preferably MR-proADM, with a reference level of proADM or said fragment, preferably MR-proADM, of at least one reference subject or 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, from the same subject obtained from a previous assay;

(c) determining the fluid balance and/or salt balance of the specified subject based on step (b) comparison; and

(d) determining the status of the globular volume and/or the status of the extracellular volume.

The invention further relates to an in vitro method, said method comprising:

(a) determining the level of proADM or a fragment thereof, preferably MR-proADM, in the sample and the level(s) of at least one additional marker and/or at least one additional subject parameter, and

(b1) comparing said proADM level or said fragment thereof, preferably MR-proADM, and the level(s) of at least one additional marker and/or parameter with reference data corresponding to said proADM level or said fragment thereof, preferably, MR-proADM and the specified(s) level(s), at least one additional marker and/or parameter, at least one reference subject; or

(b2) comparing said proADM level or said fragment thereof, preferably MR-proADM, and the level(s) of at least one additional marker and/or parameter with data corresponding to said proADM level or said fragment thereof, preferably MR -proADM, and the specified(s) level(s), at least one additional marker and/or parameter of the same subject obtained from the previous analysis; and

(c) determining the fluid balance and/or salt balance of the specified subject based on step (b1) or (b2) comparison; and

(d) determining the status of the globular volume and/or the status of the extracellular volume based on step (c).

The invention also relates to an in vitro method, said method comprising:

(a) determining the level of proADM or a fragment thereof, preferably MR-proADM, in the subject's sample, the subject's body mass index, the subject's weight, the subject's age, the subject's sex, the hemoglobin level in the sample, and the level of total serum protein in the sample; and

(b1) comparing said proADM level, or said fragment thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin level, and total serum protein level, with reference data corresponding to said levels of said markers and said parameters, at least at least one reference subject; or

(b2) comparing said proADM level or said fragment, preferably MR-proADM, body mass index, weight, hemoglobin level and total serum protein level with data corresponding to said levels of said markers and said parameters of the same subject obtained from a previous analysis;

(c) determining the fluid balance and/or salt balance of the specified subject based on step (b1) or (b2) comparison; and

(d) determining the status of the globular volume and/or the status of the extracellular volume based on step (c).

In certain aspects of the invention, the term "comparison of a specified level of proADM or a fragment thereof with reference data" or "comparison of a specified level of proADM or a fragment thereof with reference data corresponding to a specified level of proADM or a specified fragment of at least one reference subject" means that the level of proADM or its specified fragment is determined as described herein, and the level of proADM or its specified fragment is compared with the level(s) of proADM or its specified fragment determined(s) for at least one reference subject. Accordingly, the term "comparison of the specified level of MR-proADM with reference data" or "comparison of the specified level of MR-proADM with reference data corresponding to the specified level of MR-proADM of at least one reference subject" means that the level of MR-proADM is determined in as described herein, and the level of MR-proADM is compared with the level(s) of MR-proADM determined(s) for at least one reference subject. In these aspects, the reference data correspond to the levels of proADM or a fragment thereof, preferably MR-proADM, determined for these reference subjects. In other words, said 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 population of reference subjects. The reference level is usually referred to in the present description as reference data. The reference data may contain more levels/values corresponding to, for example, an additional marker and/or parameter. In preferred aspects of the invention, the term "comparison of the specified proADM level or specified fragment, preferably MR-proADM and the level(s) of at least one additional marker and/or parameter with reference data corresponding to the specified proADM level or specified fragment, preferably MR-proADM, and the specified level(s) of at least one additional marker and/or parameter of at least one reference subject" means that the level of proADM or its specified fragment is determined, preferably , MR-proADM, and determine at least one level of at least one additional marker and/or at least one additional parameter, and that the level of proADM or its specified fragment, preferably MR-proADM, is compared with corresponding level of proADM or specified fragment thereof, preferably MR-proADM, of at least one reference subject, and that the level(s) of at least one to additional marker and/or at least one additional parameter is compared with the corresponding level(s) of at least one additional marker and/or at least one additional parameter of at least one reference subject. In certain aspects, the reference data correspond to the levels of proADM or a specified fragment thereof, preferably MR-proADM, and the level(s) of at least one additional marker and/or parameter defined for the reference subject(s). (s). The level of proADM or said fragment thereof, preferably MR-proADM, and the level(s) of at least one additional marker and/or parameter of the subject to be tested are compared to the reference data of such reference subjects.

In another aspect of the invention, reference data correspond to levels of proADM or a fragment thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin level and total serum protein level determined for reference subjects. The level of proADM or a fragment thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin level and total serum protein level of the subject is compared with the reference data of such reference subjects.

In certain aspects of the invention, the reference subject may be a healthy subject, for example, a subject having a normal extracellular volume status. In a further aspect of the invention, the reference subject may be a subject suffering from a disease or disorder. The population of healthy reference subjects and reference subjects with diseases/disorders consists essentially of healthy subjects or subjects suffering from diseases or disorders, respectively. A reference subject population is a subject population including 1 to 200 or more reference subjects.

In particular, healthy subject(s) does not suffer from edema, brain injury, rupture after aneurysm, head trauma, neurological disorders, multiple traumatic injuries, postoperative, organ failure, dysregulation of lymphatic activity, dysfunction kidneys, heart dysfunction and / or diseases associated with impaired fluid balance. In particular, the healthy subject(s) does not suffer from aneurysm, multiple trauma, traumatic brain injury and/or head trauma and is not(are) the patient(s) after surgery.

In particular, the reference subject or population of reference subjects suffering from a disease or disorder suffers from a disease or disorder known to be associated with critical extracellular volume status and/or critical globular volume status, such as edema, brain injury, post-aneurysm rupture. , head trauma, neurological disorders, multiple traumatic injuries, postoperative, organ failure, dysregulation of lymphatic activity, kidney dysfunction, cardiac dysfunction and / or a disease associated with impaired fluid balance. In particular, the reference subject or population of reference subjects suffering from a disease or disorder suffering from an aneurysm, multiple trauma, traumatic brain injury and/or head trauma, or the reference subjects are postoperative subjects suffering from, for example, peritonitis with shock.

If the reference subject's marker and/or parameter profile contains characteristic elements of the marker and/or parameter profile for at least one specified reference subject, then the test subject may be diagnosed as healthy, for example, having a balanced fluid and/or salt balance, or as at risk of developing or having a positive fluid balance and/or salt balance, and/or as being at risk or having a critical extracellular volume status and/or a critical globular volume status.

In certain aspects of the invention, the method relates to determining the fluid balance, salt balance and/or globular volume status of a subject, the method including:

determining the level of proADM or a fragment thereof, preferably MR-proADM, in the sample of said subject, and

comparing said level of proADM or said fragment, preferably MR-proADM, with a reference level of proADM or said fragment, preferably MR-proADM, of at least one reference subject, each reference being healthy;

determining an extracellular volume status, a globular volume status, a fluid balance and/or a salt balance of said subject based on the comparison step; wherein

an elevated level of proADM or said fragment thereof, preferably MR-proADM, of the subject compared to said reference level indicates that said subject has positive fluid balance, positive salt balance, critical globular volume status, and/or critical extracellular volume status;

an identical or similar level of proADM or said fragment, preferably MR-proADM, of a subject compared to said reference level indicates that said subject has identical or similar fluid balance and/or identical or similar salt balance, wherein said identical fluid balance and /or salt balance indicates that the subject has a normal extracellular volume status and/or a normal globular volume status; and/or

a reduced level of proADM or said fragment thereof, preferably MR-proADM, of a subject compared to said reference level indicates that said subject has negative fluid balance and/or negative salt balance.

In these aspects of the invention, the reference subject is a healthy subject (see above), for example, a subject having a normal extracellular volume status. A healthy subject has a normal fluid balance and/or salt balance. Healthy subjects typically have an MR-proADM level of about 0.4 to about 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 midregional proadrenomedullin, J Pept Sci. 2013 Jan; 19(1):59-63). In one embodiment, said at least one healthy reference subject has a proADM or fragment thereof level, preferably an MR-proADM level of about 0.5 nmol/L. In another embodiment, said at least one healthy reference subject has a proADM or fragment thereof level, preferably an MR-proADM level of about 0.75 nmol/L. In a further embodiment, said at least one healthy reference subject has a proADM or fragment thereof, preferably MR-proADM, level of about 1.0 nmol/L. As shown in the accompanying examples, subjects suffering from, for example, aneurysm, multiple traumatic or postoperative disorders, showed levels of 1.0 nmol/l or more. In other words, subjects suffering from the disease or disorder exhibit elevated levels of proADM or a fragment thereof, preferably levels of MR-proADM. This threshold has also been identified through statistical analysis such as ROC; see figure 4 and below. In certain aspects of the invention, when the level of proADM or a fragment thereof, preferably MR-proADM, is elevated above the specified reference level of healthy subjects, the subject is considered to have positive fluid balance, positive salt balance, critical globular volume status, and/or critical status extracellular volume. As used herein, "increased level of proADM or a fragment thereof, preferably MR-proADM of the subject compared to the specified reference level" or "higher" level means that the level of 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 levels of proADM or a fragment thereof, preferably MR-proADM of said healthy reference subjects or said populations of said healthy reference subjects. In certain aspects, an "increased" or "higher" level means that the level of proADM, or said fragment thereof, preferably MR-proADM, is at least 0.5 nmol/L, e.g., at least 0.5 nmol /l, at least 0.75 nmol/l or at least 1.0 nmol/l.

In certain aspects, the level of proADM or a fragment thereof, preferably MR-proADM, is compared with the indicated reference level of healthy subjects, while the status of extracellular volume, the status of globular volume, fluid balance and/or salt balance is identified by comparing the level of proADM or its fragment, preferably , MR-proADM of the subject at the indicated reference level, wherein an elevated level, e.g., at least 1 nmol/l, indicates that the subject has a positive fluid balance and/or a positive salt balance, and/or that the specified positive fluid balance and/or positive salt balance indicate that the subjects have critical globular volume status and/or critical extracellular volume status.

In certain aspects of the invention, the method provided herein includes comparing said MR-proADM level to said reference level of healthy subjects, wherein the subject's identical or similar MR-proADM level compared to reference healthy subjects indicates that said subject has identical or a similar fluid balance and/or an identical or similar salt balance, wherein said identical or similar fluid balance and/or salt balance indicates that the subject has a normal extracellular volume status and/or a normal globular volume status. As used herein, "similar level of proADM or a specified fragment thereof, preferably MR-proADM, of a subject compared to a specified reference level" means that the level of proADM or a specified fragment thereof, preferably MR-proADM, of a subject is +/- 10%, preferably +/-5%, more preferably +/-2%, or most preferably equal or identical to the levels of proADM or said fragment, preferably MR-proADM, of healthy reference subjects. In a typical embodiment, said reference level of proADM or said fragment thereof, preferably MR-proADM, is from about 0.5 nmol/l to 1.0 nmol/l, wherein the subject has an identical or similar level of proADM or said fragment thereof, preferably MR-proADM if the level is from about 0.5 nmol/L to about 1.0 nmol/L. The normal extracellular volume is, for example, about 15 liters for a subject who contains about 40 liters of fluid (Guyton Arthur C., (1991), p. 275). As defined herein, a subject with a normal extracellular volume status may have an identical or similar fluid and/or salt balance, hence the subject's fluid and/or salt intake and exit are in balance, i.e., are identical or similar. The normal status of the globular volume may be, for example, the status of the globular volume above 20 ml/kg.

As used herein, "reduced level of proADM or a specified fragment thereof, preferably MR-proADM, of a subject compared to a reference level" means that the subject's level is 15%, preferably 20%, more preferably 25% , or even more preferably, 30% below the reference levels of proADM or the specified fragment, preferably MR-proADM, healthy reference subjects. In preferred aspects, "reduced" or "lower" level means that the level of proADM or said fragment thereof, preferably MR-proADM, is below 1.0 nmol/l, for example below 0.75 nmol/l or below 0, 5 nmol/l. In other words, the subject has a reduced level of proADM or said fragment thereof, preferably MR-proADM, if said level is below 1.0 nmol/L, such as below 0.75 nmol/L or below about 0.5 nmol/L. In these aspects, a reduced level of proADM, or said fragment thereof, preferably MR-proADM, of a subject compared to reference data indicates that said subject has a negative fluid balance and/or a negative salt balance.

The sensitivity and specificity of such a method depend not only on the analytical quality of the test, but also on the determination of what constitutes an abnormal or normal result. Distributions of levels of proADM or a fragment thereof, preferably levels of MR-proADM, for subjects with and without the disease/condition may overlap. Under such conditions, the test does not always distinguish the normal state from the disease with 100% accuracy. One of ordinary skill in the art will appreciate that the state of the subject or at least one other marker and/or parameter of the subject may itself be helpful in interpreting the data, and that this additional information allows for a more reliable prediction in areas of overlap. Accordingly, the level(s) of at least one additional marker and/or parameter is compared with the reference data of at least one healthy subject, with the same or identical values/levels of said at least one additional marker and/ or parameter compared to corresponding levels of said at least one other marker and/or parameter of said reference data indicate a reduced risk of the subject having a positive fluid and/or salt balance, and/or higher or lower levels/values of said at least one additional marker and/or parameter compared to the corresponding levels of said at least one additional marker and/or parameter of said reference data show an increased risk of having a positive fluid and/or salt balance, and at the same time a positive fluid and/or salt balance indicates critical extracellular volume. In the event that the reference object is at least one healthy subject, the indicated same or identical values/levels of the indicated at least one additional marker and/or parameter are normal values/levels, that is, the values or levels of the indicated markers and parameters are in the normal range. Normal values/levels of markers and parameters are generally known to those skilled in the art. The normal values/levels of certain markers and parameters are described above. In the most preferred aspects of the invention, the reference data correspond to or contain levels of proADM or a fragment thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin level and total serum protein level determined for reference subjects. The level of proADM or a fragment thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin level and total serum protein level of the test subject is compared with the reference data of such reference subjects.

As used herein, "same or identical" level/value means that the level/value is +/-10%, preferably +/-5%, more preferably +/-2%, or most preferably the same or identical value compared to the corresponding level/value. As used herein, "lower" or "reduced" or "higher" or "increased" level/value means that the level/value is 15%, preferably 20%, more preferably 25% or, most preferably, 30% higher or lower, respectively, compared to the corresponding level/value.

In an exemplary embodiment of the invention, the method relates to determining the fluid balance, salt balance, and/or globular volume status of a subject, the method comprising:

determining the level of proADM or a fragment thereof, preferably MR-proADM, in the sample of said subject, and

comparison of the specified level of proADM or the specified fragment, preferably MR-proADM, with the reference level of proADM or the specified fragment, preferably MR-proADM, at least one reference subject or population of reference subjects, while the reference subjects are subjects suffering from a disease or disorder known to be associated with critical extracellular volume status and/or critical globular volume status, or wherein the reference subjects are post-operatively suffering peritonitis with shock; and

determining an extracellular volume status, a globular volume status, a fluid balance and/or a salt balance of said subject based on the comparison step; wherein

a similar level, identical level, or elevated level of proADM or a specified fragment thereof, preferably MR-proADM, of the subject compared to the specified reference level indicates that the specified subject has positive fluid balance, positive salt balance, critical globular volume status and/or critical status extracellular volume; and/or

a reduced level of proADM or said fragment thereof, preferably MR-proADM, of the subject compared to said reference indicates that said subject has normal fluid balance, normal salt balance, normal extracellular volume status, and/or normal globular volume status.

In some preferred aspects of the invention, reference subjects are subjects suffering from a disease or disorder known to be associated with critical extracellular volume status and/or critical globular volume status. Such diseases or disorders include, for example, aneurysm, multiple trauma, traumatic brain injury and/or head trauma, or cases where the reference subjects are postoperative subjects suffering from peritonitis with shock. Therefore, the disease or disorder is intended to include conditions 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 an aneurysm, multiple trauma, traumatic brain injury, and/or head injury, and/or is a post-operative subject suffering from a disease or disorder such as peritonitis with shock. In another embodiment, the reference subject is a subject suffering from a disease or disorder selected from Aneurysm (ANE), Traumatic Traumatic Brain Injury (TC), Multiple Trauma (POLY), Digestive Surgery (CD), Severe Brain Injury (SBT ), aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head injury (PT), and postoperative peritonitis with shock (P). The level of MR-proADM of the test subject is compared with the reference data of such reference subjects. The accompanying examples show that a threshold for proADM or a fragment thereof, preferably MR-proADM predicting critical extracellular volume conditions, was identified by plotting proADM or a fragment thereof, preferably MR-proADM ROC curves for subjects with a disease or disorder. , to predict fluid balance and salt balance; see example 1 and figure 4. They show that a high level of MR-proADM, for example, constituting at least about 1.0 to at least about 1.5 nmol/l, shows a set of water / liquid and/or sodium/salt. Thus, in preferred aspects, said level of proADM or a fragment thereof, preferably MR-proADM, is compared to a reference level of reference subjects suffering from a disease or disorder known to be associated with critical extracellular volume status such as aneurysm, multiple trauma, traumatic brain injury and/or head injury, or with reference subjects being postoperative subjects suffering from peritonitis with shock, where a high or elevated level, e.g., at least 1 nmol/l, indicates that the subject has positive fluid balance and/or salt balance. In other words, a similar or identical level, or even an increased level of proADM or a fragment thereof, preferably MR-proADM, determined compared to the reference level of reference subjects suffering from the specified disease or disorder, indicates that the specified subject has a positive fluid balance, a positive salt balance, globular volume critical status and/or extracellular volume critical status. As used herein, "a similar level or identical level of proADM or a specified fragment thereof, preferably MR-proADM, of a subject compared to a specified reference level" means that the level of proADM or a specified fragment thereof, preferably MR-proADM of the subject is + /-10%, preferably +/-5%, more preferably +/-2%, or even more preferably, is the same or identical compared to the level of proADM or a fragment thereof, preferably MR-proADM, of at least one a reference subject suffering from said disease and/or disorder. As used herein, "increased level of proADM, or said fragment thereof, preferably MR-proADM, of a subject compared to said reference level" means that the subject's level is at least 15%, preferably at least 20%, more preferably at least 25% or even more preferably at least 30% above the levels of proADM or said fragment, preferably MR-proADM, of said reference subjects suffering from said disease or disorder. In other words, "a similar level or identical level, or an increased level of proADM or a specified fragment thereof, preferably MR-proADM, of a subject compared to a specified reference level" means that the level of proADM or a specified fragment thereof, preferably MR-proADM, of a subject is +/-10%, preferably +/-5%, more preferably +/-2% or even more preferably, is the same or identical compared to the level of proADM or its specified fragment, preferably MR-proADM, at least at least one reference subject suffering from said disease and/or disorder; or is at least 15%, preferably at least 20%, more preferably at least 25%, or even more preferably at least 30%, above the levels of proADM or its specified a fragment, preferably MR-proADM, of said reference subjects suffering from said disease or disorder. In one aspect, when the reference subjects are subjects suffering from the specified disease or disorder, "a similar level or identical level, or an increased level of proADM or the specified fragment, preferably MR-proADM, of the subject" means that the level of proADM or the specified fragment , preferably MR-proADM, is about 0.5 nmol/l or at least 0.5 nmol/l. In another aspect, "a similar or identical level or elevated level of proADM or a specified fragment thereof, preferably MR-proADM" of a subject" means that the level of proADM or a specified fragment thereof, preferably MR-proADM, is about 0.75 nmol/l or at least 0.75 nmol/l. In yet another aspect, "similar or identical level or elevated level of proADM or a specified fragment thereof, preferably MR-proADM" of the subject" means that the level of proADM or a specified fragment 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, said reference level of proADM or said fragment thereof, preferably MR-proADM, is in the range of 0.5 nmol/L to 1.5 nmol/L, e.g. 0.5 nmol/L, 0.75 nmol /l or 1.0 nmol/l, while the subject has the same or identical level, or an increased level of proADM or its specified fragment, preferably MR-proADM, if the specified level is from about, or not less than, 0.5 nmol/ l up to about, or not less than, 1.5 nmol/l.

When the level of proADM, or a fragment thereof, preferably MR-proADM, as determined in a subject is reduced or lower than in subjects having said disease and/or disorder, the test subject does not have a positive fluid and/or salt balance, but has a normal fluid and/or salt balance, i.e. identical or similar fluid balance and/or salt balance. Therefore, the fluid and/or salt balance is in balance. Thus, this subject has normal fluid balance, normal salt balance, normal extracellular volume status, and/or normal globular volume status. This reduced or lower level may also indicate that the subject has a negative salt balance and/or fluid balance. As used herein, "a reduced level of proADM or a specified fragment thereof, preferably MR-proADM in a subject" means that the level of proADM or a specified fragment thereof, preferably MR-proADM, detected in a test subject is at least 15%, preferably at least 20%, more preferably at least 25% or even more preferably at least 30% lower level of proADM or said fragment thereof, preferably MR-proADM, compared with the levels of reference subjects suffering from the specified disease or disorder. In preferred aspects, "a reduced level of proADM or a specified fragment thereof, preferably MR-proADM in a subject" or "lower" level means that the level of proADM or a specified fragment thereof, preferably MR-proADM, is below 1.0 nmol/L , for example, below 1.0 nmol/l, below 0.75 nmol/l or below 0.5 nmol/l. In other words, said 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 the subject has a reduced level of proADM or said fragment thereof, preferably MR-proADM if the indicated level is below 1.0 nmol/L, for example, below 1.0 nmol/L, below 0.75 nmol/L, or below 0.5 nmol/L.

In the area of overlap, the definition of additional states of the subject can help in prediction. Accordingly, the level(s) of at least one additional marker and/or parameter is compared to the reference data of at least one subject suffering from a disease or disorder, with the same or identical values/levels of said at least one an additional marker and/or parameter increase the risk of having a positive fluid and/or salt balance, while higher or lower levels/values of the specified at least one additional marker and/or parameter reduce the risk of having a positive fluid and/or salt balance in a subject, and a positive fluid and/or salt balance indicates a critical extracellular volume.

As shown in the attached examples, the combination of markers and parameters is chosen in such a way as to obtain the smallest error. This selection or importance analysis is carried out by standard statistical analysis, such as random forest analysis. As shown in the accompanying examples, markers and parameters of proADM or a fragment thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin, and total serum protein of the subject give a very reliable prediction of critically ill patients who suffer from positive fluid and/or salt balance. Therefore, in preferred aspects of the invention, prediction of a patient's disease outcome, ie fluid balance and/or salt balance of a subject, is performed using standard statistical analysis such as random forest analysis. In these aspects, markers and parameters are implemented in a formula that can be integrated into software. Therefore, in some embodiments, the invention relates to software suitable for determining fluid balance, salt balance, extracellular volume, and/or globular volume of a subject using the method provided herein. Accordingly, the level of proADM or a fragment thereof, preferably MR-proADM, is determined in the subject's sample and entered into the software. In other embodiments, the level of proADM or a fragment thereof, preferably MR-proADM, body mass index, weight, age, sex, hemoglobin level, and total serum protein level of the subject is determined and entered into the software. In additional embodiments, the software automatically calculates/determines a P-critical value based on levels of proADM or a fragment thereof, preferably MR-proADM, and/or additional parameters and markers, and determines if the subject has critical fluid balance, critical salt balance, critical extracellular volume and/or critical globular volume. In other words, the software makes a prediction whether the subject is in critical condition or not. Such software may be used with a graphical user interface. The formula underlying the interface is generated automatically using standard statistical methods, such as random forests implemented in the R open science software, and based on patient data. Thus, statistical analysis compares the levels of markers and parameters with reference data and predicts the fluid balance and/or salt balance and, therefore, the status of the extracellular volume of the subject. In the ICU, the clinician may use an interface to enter markers and/or parameters to obtain a fluid balance and salt balance score, which may be used to identify subjects with positive fluid balance and/or salt balance. If the fluid balance or salt balance is more than 4 liters for fluid balance and more than 36 g for salt balance, the patient is in the critical phase (P-critical value > 60%). The results of the prediction can be illustrated in a graphical user interface, for example with a traffic light system. For example, fluid balance and salt balance values that are more than 4 L or 36 g, respectively, are highlighted in red, as they indicate the critical condition of the patient. If the patient has a fluid balance or salt balance below 4 L or 36 g, respectively, then the patient or values are highlighted in green (patients have a P-critical value below 30%). In these aspects, the method proposed in the present description can be applied: for direction of treatment, for example, if the P-critical value is greater than 60%, fluid management is reviewed; to diagnose positive fluid and salt balance in order to inform the clinician that a given patient has fluid overload, even for patients not receiving intravenous fluid resuscitation; or to obtain a prognosis for a patient in case the patient has a P-critical >90%, the patient has a high SOFA score, a low RBCV and hence an even worse prognosis (Figure 6). Between 30% and 40%, the patient has an intermediate P-critical value highlighted in yellow.

In certain aspects of the invention, the method relates to determining the fluid balance, salt balance and/or globular volume status of a subject, the method including:

determination in the sample obtained from the specified subject, the level of the proADM marker or its fragment, preferably the level of MR-proADM;

comparing said proADM level or said fragment, preferably MR-proADM level, with proADM level(s) or said fragment thereof, preferably MR-proADM level(s) of the same subject obtained from a previous assay; and

determining the extracellular volume status, globular volume status, fluid balance and/or salt balance of said subject based on the comparison step, wherein

a level of at least 1 nmol/L indicates that the subject has positive fluid balance, positive salt balance, critical globular volume status, and/or critical extracellular volume status.

In certain aspects of the invention, the term "comparison of a specified proADM level or a specified fragment thereof, preferably an MR-proADM level, with a proADM level(s) or a fragment thereof, preferably an MR-proADM level(s) of the same subject obtained from a previous assay" means that the level of proADM or the specified fragment, preferably the level of MR-proADM, is determined as described herein, and that the level of proADM or the specified fragment, preferably MR-proADM, is compared with the level of proADM or its the specified fragment, preferably MR-proADM, or the levels of proADM or the specified fragment, preferably MR-proADM, which(s) obtained from the same subject in the previous analysis. Preferably, the level of proADM or its specified fragment, preferably MR-proADM, is determined at multiple time points, i.e., more than one level of proADM or its specified fragment, preferably MR-proADM, is available from the previous analysis. A series can be calculated based on these levels determined at different points in time. This series shows a trend that can be used to determine, for example, the extracellular volume status and/or the globular volume status of a subject. In other words, the MR-proADM level trend predicts the state of the extracellular volume. For example, if a set of levels/values measurements, e.g. MR-proADM, has been determined at several previous time points, then one skilled in the art can calculate a trend that can be used to interpret the development of proADM or a fragment thereof, preferably MR-proADM and/or additional markers and/or parameters. For example, a positive trend, that is, when values increase or exceed levels measured previously, may predict that the subject has a positive fluid balance and/or salt balance. In certain aspects, if the levels of proADM or a fragment thereof, preferably MR-proADM obtained from a preliminary analysis of the same subject, show a positive trend and at least one level of proADM or a fragment thereof, preferably MR-proADM obtained from a preliminary analysis of the same subject is in the range from at least 0.5 nmol/l to at least 1.5 nmol/l, for example, is at least 0.5 nmol/l, at least , 0.75 nmol/l, at least 1 nmol/l or less than 1.5 nmol/l, then the subject is considered to have a positive fluid balance and/or a positive salt balance, while a positive fluid balance and/or salt balance is shown that the subject has an extracellular volume critical status and/or a globular volume critical status in which the subject's health is deteriorating. Similarly, in certain aspects, if the levels of proADM or a fragment thereof, preferably MR-proADM, the levels obtained from the preliminary analysis of the same subject show a negative trend and at least one level of proADM or a fragment 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, then the subject is considered to have a positive fluid balance and/or positive salt balance, wherein a positive fluid balance and/or salt balance indicates that the subject has or had an extracellular volume critical status or a critical globular volume status in which the state of health deteriorates. In the attached examples, the level of proADM, or a fragment thereof, preferably MR-proADM, decreases during treatment. Without being bound by theory, a decrease in the concentration of proADM or a fragment thereof, preferably MR-proADM, may be due to a decrease in endothelial damage.

In certain aspects, certain fixed thresholds are used to determine the extracellular volume status of a subject. In one embodiment, when the level of proADM or a fragment thereof, preferably MR-proADM, is greater than 0.5 nmol/L, the patient is determined to be fluid and/or salt positive, fluid positive and/or salt positive. the balance indicates that the subject has a critical extracellular volume. In another embodiment, when the level of proADM or a fragment thereof, preferably MR-proADM, is greater than 0.75 nmol/L, the patient is determined to be fluid and/or salt positive, fluid and/or salt positive. indicates that the subject has a critical extracellular volume. In another embodiment, when the level of proADM or said fragment thereof, preferably MR-proADM, is greater than 1.0 nmol/L, the patient is determined to have a positive fluid balance and/or salt balance, while a positive fluid balance and/or salt the balance indicates that the subject has a critical extracellular volume. In another embodiment, when the level of proADM or a fragment thereof, preferably MR-proADM, is greater than 1.5 nmol/L, the patient is determined to be fluid and/or salt positive, fluid and/or salt positive. indicates that the subject has a critical extracellular volume.

In certain aspects of the invention, the term "comparison of the specified proADM level or a specified fragment, preferably MR-proADM, and the specified level(s) of at least one additional marker and/or parameter with data corresponding to the specified proADM level or its specified fragment, preferably MR-proADM, and the specified level(s) of at least one additional marker and/or parameter of the same subject obtained from a previous analysis" means that determine the level of proADM or its said fragment, preferably MR-proADM, and at least one additional level of at least one additional marker and/or at least one additional parameter, and that the level of proADM or said fragment thereof, preferably MR-proADM, compared with the corresponding level of proADM or a specified fragment thereof, preferably MR-proADM, of the same subject as determined in the previous assay, and that the level(s) at least one other marker and/or at least one additional parameter is compared with the corresponding level(s) of at least one additional marker and/or at least one additional parameter of the same subject, that were identified in the previous analysis. The levels of at least one specified additional marker and/or parameter obtained from the previous analysis can be compared with themselves to predict a trend based on a plurality of parameters. Alternatively, additional markers and/or parameters can be compared with normal data, such as data from healthy reference subjects. If additional markers and/or parameters are above or below normal levels, then the risk of positive fluid balance and/or salt balance increases, ie, the subject is more susceptible to critical extracellular volume status.

In certain aspects of the invention, the level of proADM or a fragment thereof, preferably MR-proADM, body mass index, weight, hemoglobin level and total serum protein level are compared with the corresponding levels of the same subject, which were determined in a previous analysis. In other words, the levels of markers and parameters are determined at different time points, and the level trends predict the state of the extracellular volume.

As used herein, "previous assay" means that the marker level is determined at multiple time points during hospitalization, e.g. day 0, day 1, day 2, day 3, day 4, day 5, day 6, day 7 etc. Determination of markers and/or parameters may also be performed hourly, for example, the first measurement may be performed on admission of the patient, and then the measurement may be repeated, for example, every hour, every two hours, or every five hours. The level of the marker, e.g., proADM or a fragment thereof, preferably MR-proADM, and optionally the level of a certain parameter is compared with one of the levels/values of the marker or parameter determined at a previous point in time, or with an average of the levels/values calculated for two or more previous points in time. The change in marker level indicates the status of the subject's extracellular volume.

As shown in the attached examples 1 and 4, MR-proADM thresholds for predicting critical patients, e.g., at least 1 nmol/l, were determined by plotting MR-proADM ROC curves to predict fluid balance and salt balance of patients. intensive care units; for example, patients suffering from aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head injury (PT), severe brain injury (SBT), or postoperative peritonitis with shock (P). As used herein, a high or elevated level of proADM or a fragment thereof, preferably MR-proADM, eg greater than 1 nmol/L, indicates that the subject has fluid overload, ie, positive fluid balance. Thus, in preferred aspects of the present invention, the method provided herein includes determining the level of proADM or a fragment thereof, preferably MR-proADM in a subject, wherein an elevated level of proADM or a fragment thereof, preferably MR-proADM, indicates that said subject has a positive fluid balance and/or a positive salt balance.

The accompanying examples show that a high level of proADM or a fragment thereof, preferably MR-proADM, e.g. at least 1.0 to at least about 1.5 nmol/L, indicates water/fluid intake and/or sodium, that is, positive fluid balance and/or positive salt balance of the subject, for example, at least 3 L to 4 L, or about 27 g to about 36 g sodium/salt, respectively. Thus, in embodiments of the invention, the elevated or high level of proADM or a fragment thereof, preferably the subject's MR-proADM, 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.

In the accompanying examples, different patient groups, i.e., patients suffering from aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head injury (PT), severe brain injury (SBT), or postoperative peritonitis with shock (P), showed increased or high levels MR proADM. In particular, patients suffering from 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 level of proADM or a fragment thereof, preferably MR-proADM, may vary depending on the patient population, and certain disorders, such as conditions after surgery, may lead to even higher levels. proADM or a fragment thereof, preferably MR-proADM, which are suitable for determining critical volume, positive fluid and/or salt balance. Thus, it is contemplated herein that plotting proADM or a fragment thereof, preferably MR-proADM levels, on an ROC curve (see below) to predict fluid balance and salt balance in patients suffering from a particular disease, may give higher or greater lower thresholds 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 subject after surgery indicates a positive fluid balance and/or a positive salt balance. Typically, an elevated value of at least 1.0 nmol/L indicates a subject with a positive fluid and/or salt balance. In preferred aspects of the invention, the level of proADM or a fragment thereof, preferably MR-proADM, as determined in a subject is considered elevated if the concentration of proADM or a fragment thereof, preferably MR-proADM is at least 1 nmol/l (concentration of [MR- proADM]≥ 1.0 nmol/L). In other words, a concentration above 1 nmol/L of proADM or a fragment thereof, preferably MR-proADM, in the subject shows a positive fluid balance (eg, at least 4 L) or water intake. Alternatively, a concentration greater than 1.0 nmol/L of proADM or a fragment thereof, preferably MR-proADM, in the subject shows a positive salt balance (eg, at least 36 g) or salt intake, or a critical extracellular volume status.

In preferred aspects of the invention, the method provided herein determines the extracellular volume of a subject, the method comprising determining, in a sample obtained from said subject, the level of a proADM marker or a fragment thereof, preferably MR-proADM, while based on the level of proADM or its fragment, preferably MR-proADM, determine fluid balance, and while the specified fluid balance determines the status of the extracellular volume, while an increased level of proADM or a fragment, preferably MR-proADM in the subject, indicates that the specified subject has a positive fluid balance, wherein the elevated level of MR-proADM is at least 1 nmol/L, wherein said level indicates that said positive fluid balance is at least about 4 L, and wherein said positive fluid balance indicates that said subject has the status of extracellular volume, which is considered critical.

In preferred aspects, the method provided herein determines the extracellular volume of a subject, the method comprising determining, in a sample obtained from said subject, the level of a proADM marker or fragment thereof, preferably MR-proADM, based on the level of proADM or a fragment thereof. , preferably MR-proADM, determine the salt balance, and while the specified salt balance determines the status of the extracellular volume, while an increased level of proADM or its specified fragment, preferably MR-proADM of the subject, indicates that the specified subject has a positive salt balance, when the elevated level of proADM, or a fragment thereof, preferably MR-proADM, is at least 1 nmol/L, said level indicating that said positive salt balance is at least about 36 g, and said a positive salt balance indicates that said subject has an extracellular volume status that is considered melt critical.

As used herein, "sample" as used herein can be any fluid from a subject, such as plasma, lymph, urine, cerebrospinal fluid, blood, saliva, serum, or feces, and any biological tissue from a subject.

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

In preferred aspects of the present invention, the level of proADM or a fragment thereof, preferably MR-proADM, is determined in a sample, said sample being a blood or plasma sample. In most preferred aspects, the marker is determined in a plasma sample.

In the present description, it is contemplated that the sample may be tissue such as lung tissue, ascites, skin, heart, kidney, digestive tract or lower lymph node edema, epithelial tissue, connective tissue such as bone or blood, muscle tissue such as visceral or smooth muscle and skeletal muscle, nervous tissue, bone marrow, cartilage, skin, mucous membrane or hair. The sample is collected/obtained from the patient or subjected to diagnostics in accordance with the invention. If necessary, for example in the case of solid samples, the sample may be solubilized, homogenized or solvent extracted before being used in the present invention to obtain a liquid sample. In preferred aspects, the sample is a liquid sample, such as a solution or suspension. Liquid samples may be subjected to one or more pre-treatments prior to use in the present invention. Such pretreatments include, but are not limited to, dilution, filtration, centrifugation, concentration, settling, precipitation, or dialysis. Pre-treatment may also include adding chemicals or biochemicals to the solution, such as acids, bases, buffers, salts, solvents, reactive dyes, detergents, emulsifiers, or chelators. In preferred aspects, said sample is blood, blood plasma, serum or urine. In most preferred aspects, the sample is blood plasma.

"Plasma" in the context of the present invention is a practically cell-free blood supernatant containing an anticoagulant obtained after centrifugation. Typical anticoagulants include calcium-binding compounds such as EDTA or citrate and thrombin inhibitors such as heparins or hirudin. Cell-free plasma can be obtained by centrifuging anticoagulated blood (eg, citrated, EDTA or heparinized blood), for example, for at least 15 minutes at 2000-3000 rpm.

"Serum" in the context of the present invention is the liquid fraction of whole blood, which is collected after the blood has begun to clot. When the coagulated blood (clotted blood) is subjected to centrifugation, serum can be obtained as a supernatant.

The level of proADM or a fragment thereof, preferably MR-proADM, and/or the level of additional markers can be determined by immunoassay. When used in the present description, "analysis" or diagnostic analysis can be of any type used in the field of diagnostics. Preferred detection methods include immunoassays in various formats such as, for example, radioimmunoassays, chemiluminescence and fluorescence immunoassays, enzyme-linked immunosorbent assay (ELISA), Luminex bead-based assays, protein microarray assays, assays suitable for in situ testing therapy and rapid test formats such as immunochromatography tests. Such an assay may be based on the binding of the analyte to be detected to one or more capture probes with a specific affinity. As used herein, an immunoassay is a biochemical test that measures the presence or concentration of a macromolecule/polypeptide in solution using an antibody or immunoglobulin. According to the invention, antibodies can be monoclonal as well as polyclonal antibodies. Thus, at least one antibody is a monoclonal or polyclonal antibody. Particularly preferred is the method of the present invention, in which a regional average partial peptide spanning amino acids 42-95 of pre-proADM or the amino acids indicated in SEQ ID NO: 2 is used to detect MR-proADM or its partial peptides in a sample. In certain aspects, the marker level is determined by high performance liquid chromatography (HPLC). In certain aspects, HPLC may be combined with an immunoassay.

In certain aspects of the present invention, proADM or a fragment thereof, preferably MR-proADM or a fragment thereof, and/or additional markers or fragments thereof are determined using a sandwich immunoassay. In this sandwich immunoassay, two antibodies are added to the sample, for example for one marker such as proADM or a fragment thereof, preferably MR-proADM. In particular, it is preferred if proADM or a fragment thereof, preferably MR-proADM or a fragment thereof, is detected using two antibodies that specifically bind to different partial sequences of proADM or a fragment thereof, preferably MR-proADM or a fragment thereof.

In a preferred aspect of the in vitro method according to the invention, one of the antibodies is labeled and the other is or can be selectively bound to the solid phase.

In a particularly preferred aspect of the assay, one of the antibodies is labeled and the other is either bound to the solid phase or can selectively bind to the solid phase. In a preferred embodiment, the method is carried out in the form of a heterogeneous sandwich immunoassay, in which one of the antibodies is immobilized on a randomly selected solid phase, for example, on the walls of coated tubes (for example, polystyrene assay tubes, coated tubes, CT) or on microtiter plates , for example, consisting of polystyrene, or on particles, such as, for example, magnetic particles, whereby the other antibody has a group similar to a detectable label, or allows selective attachment to the label, and which serves to detect the formed sandwich structures. Time delay immobilization or serial immobilization using suitable solid phases is also possible.

In addition, the method according to the present invention can be carried out as a homogeneous method, in which the sandwich complexes formed by the antibody/antibodies and a marker, for example, proADM or a fragment thereof, preferably MR-proADM or a fragment thereof, to be detected, remain suspended in the liquid phase. In this case, it is preferable that when two antibodies are used, both antibodies are labeled with parts of the detection system, resulting in signal generation or signal triggering if both antibodies are integrated into one sandwich. Such techniques can be implemented, in particular, in methods for detecting fluorescence enhancement or fluorescence quenching. A particularly preferred aspect is the use of detection reagents to be used in pairs, such as, for example, those described in US 4882733A, EP-B1 0180492 or EP-B1 0539477 and the prior art cited therein. Thus, it becomes possible to carry out measurements in which only reaction products are detected, including both labeling components in one immune complex directly in the reaction mixture. For example, such technologies are offered under the trademarks TRACE ^® (time resolved cryptate enhanced emission) or KRYPTOR ^® , embodying the principles of the aforementioned applications. Thus, in particularly preferred aspects, a diagnostic device is used to carry out the method as provided herein. For example, the level of proADM or a fragment thereof, preferably MR-proADM, and/or the level of any additional marker from the method provided herein is determined. In particularly preferred aspects, the diagnostic device is ^KRYPTOR® .

The invention also relates to the use of a kit for determining the status of extracellular volume in a sample obtained from a test subject, containing detection reagents 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, proatrial natriuretic peptide (proANP), C-terminal proarginine-vasopressin (CT-proAVP), protein, erythropoietin, angiotensin II, aldosterone, cortisol, adrenaline, epinephrine, catecholamines and pro-andothelin-1 (pro -ET-1) or a fragment thereof, and containing excipients for the implementation of the method proposed in the present description. In certain aspects, the invention relates to the use of a kit for determining the status of the extracellular volume in a sample obtained from a test subject, containing detection reagents for determining the level of proADM or a fragment thereof, preferably MR-proADM or a fragment thereof, and containing excipients for the implementation provided herein invention of the method. In preferred aspects, the invention relates to the use of a kit for determining the status of extracellular volume in a sample obtained from a test subject, containing detection reagents for detecting proADM markers or a fragment thereof, preferably MR-proADM, hemoglobin and total serum protein, and containing excipients for exercising proposed in the present description of the method.

In certain aspects, these detection reagents for determining the level of proADM or a fragment thereof, preferably MR-proADM or a fragment thereof, contain antibodies, wherein one of the antibodies is labeled and the other antibody is associated with the solid phase or can selectively bind to the solid phase.

In certain aspects, these detection reagents for determining the level of at least one marker contain antibodies, wherein one of the antibodies is labeled and the other antibody is associated with the solid phase or can selectively bind to the solid phase.

In principle, all labeling techniques that can be used in this type of assay 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 used in in particular in on-site diagnostics (POC) or rapid tests. In the case of heterogeneous sandwich immunoassays, both antibodies can expose portions of a detection system of the kind described herein in the context of homogeneous assays.

In a preferred aspect, both the first and second antibodies are dispersed in a liquid reaction medium, whereby the first labeling component, which is part of a labeling system based on quenching or enhancing fluorescence or chemiluminescence, binds to the first antibody, and as a result, the second labeling component of this labeling system binds to a second antibody so that after both antibodies bind to a marker, e.g. proADM or a fragment thereof, preferably MR-proADM or a fragment thereof, or an additional marker or fragment thereof to be detected, a detectable signal is generated which makes it possible to detect sandwich complexes formed in the measuring solution. One aspect of this alternative includes a labeling system such as rare earth cryptates or chelates in combination with a fluorescent or chemiluminescent dye. In a particular preferred aspect, the labeling system comprises a rare earth cryptate in combination with a fluorescent or chemiluminescent dye, in particular of the cyanine type. In yet another preferred aspect, detection is carried out using a competitive immunoassay. In a preferred aspect, radioimmunoassay is used. It is also contemplated herein that the level of the marker may be determined, for example, by mass spectrometry-based methods, or by high performance liquid chromatography (HPLC), which may be combined with an immunoassay or mass spectrometric analysis method. The specialist will understand that any available analysis can be used if it allows you to reliably determine the level of the marker.

It is an object of the present invention to provide an in vitro method for diagnosing, predicting, risk assessment, risk stratification, therapy monitoring and/or operational monitoring of a disorder or medical condition in a subject and/or patient, which provides reliable information, in particular to a healthcare professional in the emergency room. care (ICU) or intensive care unit (ICU).

Thus, the invention relates to a method for in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy control and/or operational control of a disorder or health condition in a subject, in which the status of the extracellular volume, the status of the globular volume, fluid balance and/or salt the balance of the specified subject is determined in accordance with the method presented in the present description.

In one embodiment, the invention relates to a method for in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy control and/or operational control of a disorder or medical condition in a subject, in which, based on the level of proADM or a fragment thereof, preferably MR- proADM determine the fluid balance, salt balance, and/or globular volume status of the subject.

In other embodiments, the invention relates to an in vitro method for diagnosing, predicting, risk assessment, risk stratification, therapy control and/or operational control of a disorder or medical condition in a subject, in which, based on the level of proADM or a fragment thereof, preferably MR-proADM, determine the fluid balance and/or salt balance, while the specified fluid balance and/or salt balance determines the status of the extracellular volume.

In other embodiments, the invention relates to an in vitro method for diagnosing, predicting, risk assessment, risk stratification, therapy control and/or operational control of a disorder or medical condition in a subject, in which, based on the level of proADM or a fragment thereof, preferably MR-proADM, hemoglobin level, total serum protein level, subject weight, subject age, and subject sex determine fluid balance, salt balance, and/or globular volume status.

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

As used herein, "prediction" refers to predicting the outcome of a disease or a particular risk for a subject suffering from a particular disease or clinical condition. It may include an assessment of the likelihood of recovery or the likelihood of a poor outcome for the specified subject.

The term "control therapy" in the context of the present invention refers to the monitoring and/or adjustment of therapeutic treatment of the specified patient. "Monitoring" refers to tracking an already diagnosed disease, disorder, complication, or risk, for example, to analyze the progression of a disease or the effect of a particular treatment on the progression of a disease or disorder.

In the present invention, the terms "risk assessment" and "risk stratification" refer to the grouping of subjects into different risk groups in accordance with their future prognosis. Risk assessment also refers to stratification for the application of preventive and/or therapeutic measures. As used herein, "operative control" refers to preoperative control, intraoperative control, and/or postoperative control of a subject. In particular, as used herein, it means that fluid balance, salt balance, globular volume status, and/or extracellular volume status are monitored. Therefore, fluid and/or salt is monitored and controlled in such subjects.

In certain aspects, the disorder or medical condition may be water overload, edema, brain injury, rupture after aneurysm, severe head trauma, neurological disorder, severe multiple traumatic injury, postoperative disorder, cardiac risk, kidney injury, organ failure, lymphatic dysregulation , kidney dysfunction, cardiac dysfunction, a disease associated with impaired fluid balance.

As shown in the attached examples, a significant statistical relationship was found between MR-proADM and the patient's fluid and/or salt balance. Fluid and/or salt balance indicates the extracellular volume of the subject and/or patient. As demonstrated in the attached examples, this strong relationship was found in several patient clinical situations such as patients with severe brain injury (SBT), aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head injury (PT), and postoperative peritonitis with shock ( P) (for example, example 1). Therefore, in certain aspects, the invention relates to an in vitro method for diagnosing, predicting, risk assessing, risk stratifying, monitoring therapy and/or operational monitoring of a disorder or medical condition in a subject, wherein the subject has a brain or head injury, multiple traumatic injuries or aneurysm, or is the subject after surgery. In further aspects, the invention relates to an in vitro method for diagnosing, predicting, risk assessment, risk stratification, therapy control and/or operational control of a disorder or medical condition in a subject, wherein the subject has a severe brain injury (SBT), aneurysmal subarachnoid hemorrhage (SAH ), severe trauma without head injury (PT), and postoperative peritonitis with shock (P). In the following aspects, the invention relates to an in vitro method for diagnosing, predicting, risk assessment, risk stratification, therapy control and/or operational control of a disorder or medical condition in a subject, wherein said subject has a ruptured post-aneurysm or severe head injury. In certain aspects, the specified subject does not have neurological disorders. In certain aspects, the specified subject has severe multiple traumatic injuries or is a patient after surgery.

The method provided herein can be used to manage the fluid of a subject or patient. As used herein, "fluid management" means monitoring and controlling the fluid status of a subject or patient and administering the fluid, such as by intravenous fluid administration. Thus, in certain aspects, the invention provides a method for use in fluid management of a subject, wherein said subject's extracellular volume status is determined by the method provided herein. In certain aspects, the invention relates to a method for use in fluid management of a subject, in which, based on the level of proADM or a fragment thereof, preferably MR-proADM, the extracellular volume status of the subject is determined by the method provided herein.

In certain aspects, the invention relates to a method provided herein for use in fluid management of a subject, in which, based on the level of proADM or a fragment thereof, preferably MR-proADM, and/or in which therapy is controlled based on the fluid and/or salt balance of the subject. in relation to the disorder or medical condition of the subject.

In certain aspects, the invention relates to a method provided herein for predicting mortality risk and treatment outcome of a subject, wherein the extracellular volume status of said subject is determined by the method provided herein. In certain aspects, the invention relates to a method used as an alert system for physicians and clinicians to immediately take appropriate therapeutic actions, wherein said subject's extracellular volume status is determined by the method provided herein.

In certain aspects, the invention relates to a method provided herein for predicting organ failure, dysregulation of lymphatic activity, kidney dysfunction, reduced function, or risk of cardiac dysfunction in a subject, said subject's extracellular volume status being determined by the method provided herein.

In certain aspects, the invention relates to a method provided herein for use in the treatment of a subject suffering from a disorder or medical condition selected from the group consisting of water overload, edema, brain injury, rupture after an aneurysm, severe head trauma, neurological disorders , severe multiple traumatic injuries, postoperative condition, cardiac risk, kidney injury, organ failure, dysregulation of the activity of lymphatic flows, kidney dysfunction, heart dysfunction, a disease associated with impaired fluid balance. The terms "treatment", "therapy" and the like are used in the present description in a general sense to denote the achievement of the desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of the complete or partial prevention of the disease/medical condition/disorder or its symptoms, and/or may be therapeutic in terms of the partial or complete treatment of the disease/medical condition/disorder and/or side effect associated with the disease/ medical condition/disorder. As used herein, "treatment" encompasses any treatment of a disease/medical condition/disorder in a subject and includes: (a) preventing and/or ameliorating a disease/medical condition/disorder in a subject that may be predisposed to the disease/medical condition/violation; (b) suppressing the disease/medical condition/disorder, that is, stopping its development; or (c) alleviating the disease/medical condition/disorder, that is, causing the disease/medical condition/disorder to regress. For example, the method provided herein may be used to monitor the therapy/treatment of a resuscitation patient. Thus, for example, the method provided herein may be used to control a subject's fluid management. The method described 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, which is associated with increased mortality (Acheampong et al., 2015). In certain aspects, the method provided herein can also be used to assess and monitor a subject's fluid management to avoid fluid overflow into the interstitial space when there is an abnormal amount and/or overload of a volume replacement solution. An overload of a volume-replacing solution that is considered critical is, for example, more than 4 liters in one day, in two days, in three days, in four days, in five days, or preferably in seven days.

In certain aspects of the invention, the method provided herein includes:

(a1) comparing said level of proADM or fragment thereof, preferably MR-proADM, with reference data corresponding to said level of proADM or said fragment, preferably MR-proADM, of at least one reference subject; or

(a2) comparing said level of proADM or fragment thereof, preferably MR-proADM, with data corresponding to said level of proADM or said fragment, preferably MR-proADM, from the same subject from a previous assay;

(b) determining the fluid balance, salt balance and/or globular volume status of said subject based on step (a) of the comparison, wherein the fluid balance, salt balance and/or globular volume of said subject is used to predict mortality risk and disease outcome of the subject and/ or used to evaluate and monitor a subject's fluid management.

In the context of in vitro diagnosis, prognosis, risk assessment, risk stratification, therapy control and/or operational control of a disorder or medical condition in a subject and/or patient, if the level of proADM or a fragment thereof, preferably MR-proADM, is at least , 0.5 nmol/l, for example, 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, by by at least 4.0 liters (a set of 4 liters of water during hospitalization), then the subject has a positive fluid balance (increased fluid balance, that is, a set of water content), which is considered critical. In other words, a critically ill subject has a positive fluid balance of at least 4 L, that is, a water intake of at least 4 L in a critically ill subject.

In preferred aspects, the method includes determining the level of proADM or a fragment thereof, preferably MR-proADM in the sample, wherein the level of proADM or a fragment thereof, preferably MR-proADM, of the subject is at least 0.5 nmol/l , for example, at least 0.5 nmol/l, at least 0.75 nmol/l or at least 1 nmol/l, while the specified level of proADM or its fragment, preferably MR-proADM, indicates that the specified subject has a positive fluid balance, while the specified positive fluid balance is at least 4 liters, while the positive fluid balance indicates that the subject is in a critical state of health. In other words, said positive fluid balance indicates that said subject has a critical extracellular volume status.

In a certain aspect, if the level of proADM or a fragment thereof, preferably MR-proADM, is at least 0.5 nmol/l, for example, 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 (a set of 36 g of sodium or salt), then the subject has a positive salt balance, which is considered critical. In the most preferred aspects, the increase in sodium is at least 36.0 g, and this change indicates that the subject has a positive fluid balance and/or salt balance, which is considered critical. In other words, said positive salt balance and/or fluid balance indicates that said subject has an extracellular volume status that is considered critical. In other words, a critically ill subject has a positive salt balance (increase in salt balance, i.e., salt gain) of at least 36 g, i.e., a salt intake of at least 36 g in a critically ill subject.

In other aspects, the method includes determining the level of proADM or a fragment thereof, preferably MR-proADM in the sample, wherein the level of proADM or a fragment thereof, preferably MR-proADM, of the subject is at least 1 nmol/L, wherein the indicated level of proADM or a fragment thereof, preferably MR-proADM, indicates that the specified subject has a positive salt balance, while the specified positive salt balance is at least 36 g, and the positive salt balance indicates that the subject is in critical health condition.

In the present description, it is understood that additional markers and/or parameters, ie, in addition to proADM or a fragment thereof, preferably MR-proADM, improve the prediction of fluid and/or salt balance. Therefore, in certain aspects, the method includes determining the level of proADM or a fragment 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 hemoglobin level in the sample, and the level of total serum protein in the sample, at the same time, based on the indicated markers and the indicated parameters, the fluid balance and/or salt balance is determined, while the salt balance of at least 36 g and/or the fluid balance of 4 l indicate that the subject is in critical condition health. In preferred aspects, a salt balance of at least 36 g and a fluid balance of 4 L indicate that the subject is in critical health.

As used herein, "critical condition", "critically ill patient", "critically ill patient" or "critical subject" means that the subject or patient is in a life-threatening situation because the status of the extracellular volume is considered critical. As described above, patients with positive fluid balance and/or salt balance have increased mortality. For example, a subject may be considered critically ill if they have fluid or salt overload, such as caused by excessive intravenous infusion. Therefore, a critical subject has a critical positive fluid balance (eg, at least 4 L), a critical positive salt balance (eg, at least 36 g), and/or a critical globular volume status (below 20 ml/kg). In certain aspects, a subject may be considered critically healthy if they have a low globular volume level, such as below about 20 ml/kg, or preferably below about 15 ml/kg. In certain aspects, a globular volume critical status is a globular volume below about 15 ml/kg.

The levels of markers and/or parameters defined herein are a warning sign for the clinician to take appropriate action immediately. As used herein, "critical extracellular volume status" refers to elevated or high extracellular volume. In preferred aspects of the invention, the increased extracellular volume is at least 3 L, preferably at least 4 L, wherein said increased extracellular volume identifies a subject in critical health. It is contemplated herein that the fluid intake or salt intake that increases the mortality of the subject also depends on the characteristics of the subject, such as weight, age or sex, etc. For example, a positive fluid balance of 4 L measured in a heavy male has a different effect than a positive fluid balance in a small child. Therefore, it is envisaged that the fluid set and/or the salt set, i.e., 4 L or 36 g, respectively, which indicates a critical subject, depends on the characteristics of the subject and may be higher or lower than 4 L or 36 g, respectively, depending on characteristics of the subject.

In the accompanying examples, it is documented that there is a strong statistical relationship between the pooled predictor, ie, the combination of fluid balance and salt balance, and the definition of critically ill patients. In certain aspects of the invention, the method provided herein detects critically ill patients in whom the water increase is at least 4 L and the salt increase is at least 36 g. Therefore, in preferred aspects, the method includes determining in sample level of proADM or its fragment, preferably MR-proADM, while the level of proADM or its fragment, preferably MR-proADM of the subject, is at least 1 nmol/l, while the specified level of proADM or its fragment, preferably, MR-proADM shows that said subject has a positive salt balance and a positive fluid balance, wherein said positive salt balance is at least 36 g and said positive fluid balance is at least 4 L, and the indicated positive salt balance and the indicated positive fluid balance indicate that the subject has a critical status ec lethal volume.

The attached examples demonstrate that there is a strong relationship between the dynamic organ failure score (SOFA score) and salt and/or fluid balance; see Examples 1 and 3. Therefore, in preferred aspects, a dynamic organ failure score (SOFA score) is determined based on fluid balance and/or salt balance. In other preferred aspects, a dynamic organ failure score (SOFA score) is determined based on fluid balance and/or salt balance, with fluid balance and/or salt balance determined based on 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 a sample, whereby the SOFA score is determined based on the level of proADM or a fragment thereof, preferably MR-proADM.

In certain aspects, a SOFA score above 14 is indicative of a very severe health condition, indicative of a critical health condition in the subject. A SOFA score of 0 to 6 indicates less severe health, and a SOFA score of 7 to 14 indicates severe health. In certain aspects, an elevated level of proADM or a fragment thereof, preferably MR-proADM, is indicative of a subject's SOFA score, with a SOFA score greater than 14 indicating that the subject is in a critical health condition.

The accompanying examples show that the inclusion of additional parameters such as age, BMI, and gender improves the predictive value for SOFA score determination; see fig. 5. Thus, in certain aspects, the method provided herein further includes determining at least one parameter consisting of age, body mass index, and gender.

It is contemplated herein that the dynamic organ failure score (SOFA score) is at least 15, which score indicates that the subject has a positive fluid balance and/or salt balance that is considered critical.

As used herein, a "dynamic organ failure score" or "SOFA score" is one of the scores used to track a patient's condition while in an intensive care unit (ICU). The SOFA score is a scoring system for determining the degree of functioning of a human organ or failure rate. The score is based on six different measures, one each for the respiratory, cardiovascular, hepatic, coagulation, renal, and neurological systems. Both the average and highest SOFA scores are predictors of outcome. An increase in SOFA during the first 24-48 hours in the ICU predicts a mortality rate of at least 50% to 95%. Values less than 9 give a predicted mortality of 33%, and above 14 - close to or above 95%. The score tables below only describe conditions that add points to the score ((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 cases where physiological parameters do not correspond to any line, indicate zero points. In cases where physiological parameters correspond to more than one line, choose the line with a higher number of points. The assessment helps doctors, nurses and other members of the health care team patient in assessing the risk of complications and mortality from sepsis.

Tables 1 and 2: SOFA scoring table - scoring scheme

SOFA score 0 one 2 3 four respiratory
PaO2/FIO2 >400 ≤400 ≤300 ≤ 200 with artificial ventilation ≤ 100 with artificial ventilation coagulation
platelets >150
10 ^3/ mm ³ ≤150
10 ^3/ mm ³ ≤100
10 ^3/ mm ³ ≤50
10 ^3/ mm ³ ≤20
10 ^3/ mm ³ hepatic
Bilirubin <20 µmol/l 20-32
µmol/l 33-101
µmol/l 102-204
µmol/l > 204
µmol/l cardiovascular _
hypotension absence MAP <70 mmHg Dopa ≤ 5 or dobutrex Dopamine > 5, or epinephrine ≤0.1, or norepinephrine ≤0.1 Dopamine > 15, or epinephrine > 0.1, or norepinephrine > 0.1 Central Nervous System GCS fifteen 13-14 10-12 6-9 <6 renal
creatinine or diuresis <110 µmol/l 110-170 171-299 300-440 or <500 ml/day >440 or <200 ml/day Total =.........

organ 0 one 2 3 four respiratory twenty% 27% 32% 46% 64% cardiovascular 22% 32% 55% 55% 55% coagulation 35% 35% 35% 64% 64% central nervous system 26% 35% 46% 56% 70% hepatic 32% 34% fifty% 53% 56% renal 25% 40% 46% 56% 64%

The accompanying examples 1 and 4 show that the combination of fluid balance and salt balance can effectively predict (AUC>0.92) whether a subject will experience a critical condition, such as having a positive fluid balance and/or a positive salt balance. In certain aspects of the invention, the method provided herein identifies a subject who is in critical health condition based on the patient's fluid balance and sodium balance.

The attached examples also demonstrate that the combination of fluid balance and salt balance can effectively predict the risk of edema in a subject, that is, the combined detection of fluid and salt balance can identify a critically ill patient at risk of developing edema. In certain aspects of the invention, the method provided herein identifies a subject who is at critical risk for edema based on fluid balance and sodium balance. Thus, in certain aspects of the invention, the method provided herein is used to monitor therapy for a subject who is at critical risk for edema, wherein the risk of edema is determined based on fluid balance and sodium balance. In certain aspects of the invention, the method provided herein is used to monitor therapy for a subject who is at critical risk for edema, wherein the risk of edema is identified based on fluid balance and sodium balance, fluid balance and/or salt balance being determined based on proADM level or its fragment, preferably MR-proADM.

As mentioned herein in the context of proteins or peptides, the term "fragment" refers to smaller proteins or peptides that are derivatives of larger proteins or peptides, which therefore contain a partial sequence of a larger protein or peptide. These fragments can be obtained from larger proteins or peptides by removing one or more amino acids from the larger protein or peptide.

As used herein, terms such as "marker", "indicator", "prognostic marker", "factor" or "biomarker", or "biological marker" are used interchangeably and refer to measurable and quantitative biological markers (e.g., the concentration of a particular enzyme or fragment thereof, the concentration of a particular hormone or fragment thereof, or the presence of biological substances or fragments thereof) that serve as indicators for health and physiological assessments such as the risk of a disease/disorder/clinical condition. In addition, a biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. A biomarker can be measured in a biological sample (such as blood, plasma, urine or tissue).

As used herein, a parameter is a characteristic, property, or measurable factor that can help define a particular system. The parameter is an important element for health and physiological assessments such as the risk of a disease/disorder/clinical condition. In addition, a parameter is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. The sample parameter may be selected from the group consisting of body mass index, weight, age, sex, IGS II, fluid intake, Acute and Chronic Functional Change Scale II (APACHE II), World Federation of Neurosurgical Societies (WFNS) score , Glasgow Coma Scale (GCS) scores, and dynamic organ failure scores (SOFA scores).

For 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 humans and animals. Accordingly, said subject may be an animal such as a mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, bull, horse, camel, or primate. Preferably, the subject is a mammal. Most preferably, the subject is a human. Within the meaning of the invention, any sample collected from cells, tissues, organs, organisms, or the like may be a patient sample to be diagnosed. As shown in the attached examples, the extracellular volume status of patients suffering from various disorders or diseases can be predicted. Thus, the method provided herein can be applied to any subject who is a healthy subject or a subject who suffers from an arbitrary disease or disorder. In preferred aspects, the subject suffers from a disease or disorder, wherein the disease or disorder is selected from the group consisting of edema, brain injury, post-aneurysm rupture, head injury, neurological disorder, multiple traumatic injuries, postoperative disorders, organ failure, dysregulation of the activity of lymphatic flows, kidney dysfunction, heart dysfunction, diseases associated with impaired fluid balance. In more preferred aspects of the invention, the subject suffers from a traumatic brain injury, aneurysm, head trauma and/or multiple traumatic injuries, and/or said subject is a subject after surgery. In most preferred aspects, the subject suffers from severe brain injury (SBT), aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head injury (PT), postoperative peritonitis with shock (P), and/or is a postoperative subject for digestive peritonitis.

As used herein, "multiple traumatic injuries", "multitrauma", "polytrauma" or "multitrauma" in the context of the invention covers a condition with two or more severe injuries in at least two areas of the body, or a condition with multiple trauma, that is, two or more severe injuries in the same area of the body. Polytrauma may be accompanied by traumatic shock and/or hemorrhagic hypotension and a serious threat to one or more vital functions. At least one of the two or more injuries, or the sum total of all injuries, is life-threatening for the affected subject with polytrauma. Trauma is a bruise or damage to a biological organism caused by physical damage from an external source. A major injury is an injury that has the potential to lead to serious long-term consequences such as chronic pain.

As used herein, a brain injury is an injury to the brain, such as a traumatic brain injury. Brain damage occurs when an external force causes traumatic injury to the brain. Head injury is usually referred to as a head injury, but is a broader category because it can result in damage to structures other than the brain, such as the scalp and skull. Aneurysms, or vasodilatations, are localized, blood-filled, spherical bulges 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 aneurysms of the abdominal aorta. Aneurysms can also form in the heart itself.

As used herein, a postoperative subject is a subject who has undergone surgery. More preferably, the postoperative subject is one who has undergone major surgery. Major surgery can be any surgery within or on the contents of the abdominal, pelvic, cranial, or thoracic cavity; or an intervention that, given the location, the condition of the patient, the level of complexity or duration of execution, poses a threat to the life or function of an organ or tissue. Major surgery usually requires general anesthesia, a hospital stay of varying lengths (often a week), and may be performed by a board certified general surgeon in a specialized care hospital or a subspecialty surgeon in a tertiary care hospital. More preferably, the subject after surgery is a subject who has undergone surgery on the digestive system. More preferably, the postoperative subject is one who has undergone major surgery and is suffering from a life-threatening disease or disorder. This disease or disorder may be caused by the surgery itself. Most preferably, the subject suffers from postoperative peritonitis with shock.

As used herein, the statistical relationship between the level of the marker(s), e.g. proADM or a fragment thereof, preferably MR-proADM and/or parameter(s), and the status of extracellular volume, e.g. extracellular volume, blood volume, or disorder (s)/disease(s)/clinical(s) condition(s) of the subject were evaluated using statistical methods, as shown in the attached examples. As shown in the accompanying examples, random forest analysis can be applied (Breiman, 2001 and 2002; ^Boulesteix et al. (2012); importance analysis; inclusion selection; linear regressions; one-by-one exclusion; "R2" or " ^r2 " (coefficient of certainty), AUC (area under the curve), and survival analysis Any appropriate and suitable algorithm and software package available in the prior art can be used to calculate and analyze the statistical relationship between parameters/values.

As used herein, the terms "comprising" and "comprising" or their grammatical variants are to be construed as indicating given elements, integers, steps, or components, but do not preclude the addition of one or more additional elements, integers, steps, components, or groups thereof. This term encompasses the terms "consisting of" and "consisting essentially of".

Thus, the terms "comprising"/"comprising"/"having" mean that any other component (or similar elements, integers, steps, and the like) may or may not be present.

The term "consisting of" means the absence of any other components (or similar elements, integers, steps, and the like).

The term "consisting essentially of" or its grammatical variants when used herein should be considered as indicating the given elements, integers, steps, or components, but it does not exclude the addition of one or more additional elements, integers, steps, components, or groups of them. , but only if the additional elements, integers, steps, components, or groups thereof do not significantly change the main and new characteristics of the claimed composition, device, or method.

Thus, the term "consisting essentially of" means that specific additional components (or similar elements, integers, steps, and the like) may be present, namely those that do not significantly affect the essential characteristics of the composition, device, or method. In other words, the term "consisting essentially of" (which may be used interchangeably herein with the term "comprising essentially") allows for the presence of other components in a composition, device, or method in addition to the required components (or similar elements, integers , steps, etc.), provided that the desired characteristics of the device or method are not significantly affected by the presence of other components.

The term "method" refers to mechanisms, tools, techniques and procedures for performing a given task, including, but not limited to, those mechanisms, tools, techniques and procedures that are known, or can be easily developed from known mechanisms, tools, techniques and procedures by practitioners in the field of chemistry, biology and biophysics.

The term "about" preferably refers to ±10% of the specified numerical value, more preferably ±5% of the specified numerical value, and in particular to the exact specified numerical value.

When used in the present description, "about" refers to ±10% of the specified numerical value and, in particular, to ±5% of the specified numerical value. Whenever the term "about" is used, specific reference is also made to an exact numerical value. When the term "about" is used in connection with a parameter that is quantified in whole numbers, such as the number of nucleotides in a given nucleic acid, then numbers corresponding to ±10% or ±5% of the stated numerical value should be rounded to the nearest whole number. For example, the expression "about 25 amino acids" refers to the range of 23 to 28 amino acids, in particular 24 to 26 amino acids, and preferably refers to the specific value of 25 amino acids.

Unless otherwise indicated, established methods from recombinant gene technology have been used as described, for example, in Sambrook, Russell "Molecular Cloning, A Laboratory Manual", Cold Cold Harbor Laboratory, NY (2001)), which is incorporated herein by links in their entirety.

The sensitivity and specificity of a diagnostic and/or prognostic test depend not only on the analytical "quality" of the test, but also on the definition of what constitutes an abnormal result. In practice, true/false detection ratio curves (ROC curves) are usually computed by plotting the value of a variable against its relative frequency in "normal" (i.e., apparently healthy individuals with no prenatal disorder or condition) and "diseased" populations. . For any particular marker (eg, MR-proADM), the distribution of marker levels for subjects with and without the disease/condition is likely to overlap. Under such conditions, the test does not always distinguish normal from disease with 100% accuracy, and the area of overlap may indicate where the test cannot distinguish normal from disease. A threshold value is selected below which the test is considered pathological and above which the test 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 likelihood that a given measurement will correctly identify a condition. ROC curves may be used even if test results do not necessarily give an accurate number. As long as you can rank the results, you can create an ROC curve. For example, test results for "diseased" samples may be ranked according to grade (eg, 1=low, 2=normal, and 3=high). This ranking can be correlated with the results of a "normal" population and an ROC curve can be plotted. These methods are well known in the art; see, for example, Hanley et al. 1982. Radiology 143: 29-36. Preferably, the threshold is chosen to provide an area under the ROC curve greater than about 0.5, more preferably greater than 0.7, even more preferably greater than 0.8, even more preferably greater than 0.85, and most preferably , more than 0.9. The term "about" in this context refers to +/-5% of a given measurement.

The horizontal axis of the ROC curve is (1-specificity), which increases with the false positive rate. The vertical axis of the curve represents the sensitivity, which increases with the true positive rate. Thus, for a certain selected cutoff, a value (1-specificity) can be determined and an appropriate sensitivity can be obtained. The area under the ROC curve is a measure of the likelihood that a measured marker level will correctly identify a disease or condition. Thus, the area under the ROC curve can be used to determine the effectiveness of the test.

In other embodiments, a positive likelihood ratio, a negative likelihood ratio, an odds ratio, or a hazard ratio are used as a measure of a test's ability to predict risk or diagnose a disorder or condition ("disease group"). In the case of a positive likelihood ratio, a value of 1 indicates that a positive result is equally likely among both "diseased" and "control" subjects; a value greater than 1 indicates that a positive result is more likely in the group with the disease; and a value less than 1 indicates that a positive result is more likely in the control group. In the case of a negative likelihood ratio, a value of 1 indicates that a negative result is equally likely among subjects in both the "diseased" and "control" groups; a value greater than 1 indicates that a negative result is more likely in the test group; and a value less than 1 indicates that a negative result is more likely in the control group.

In the case of an odds ratio, a value of 1 indicates that a positive result is equally likely among both "diseased" and "control" subjects; a value greater than 1 indicates that a positive result is more likely in the diseased group; and a value less than 1 indicates that a positive result is more likely in the control group.

In the case of a hazard ratio, a value of 1 indicates that the relative risk of the endpoint (eg, death) is the same in both "diseased" and "control" groups; a value greater than 1 indicates that the risk is higher in the group with the disease; and a value less than 1 indicates that the risk is higher in the control group.

One skilled in the art will appreciate that comparing a diagnostic or prognostic indicator with a diagnosis or predicted risk of a future clinical outcome is a statistical analysis. For example, a marker level below X may signal that a patient is more likely to suffer a poor outcome than patients with a level greater than or equal to X, as determined by the level of statistical significance. In addition, changes in marker concentrations relative to baseline levels may be a reflection of the patient's prognosis, and the degree of marker level change may be related to the severity of side effects. Statistical significance is often determined by comparing two or more populations and determining a confidence interval and/or p-value; see, for example, Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals for the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5% , 99.9% and 99.99%, and the preferred p-values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

The present invention is further described with reference to the following non-limiting figures and examples.

Description of figures

Figure 1. The distribution of water in the body, while the water in the body 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, ie, globular volume (corresponding to about 6%) and plasma (corresponding to about 6%), and interstitial volume (corresponding to about 27%).

Figure 2. Deming regression for MR-proADM (logarithmic scale) and fluid balance (delta H ₂ O) (A) and salt balance (delta Na) (B).

Figure 3. Box plot of MR-proADM concentration in nmol/L for ICU patients suffering from aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head injury (PT), severe brain injury (SBT), or postoperative peritonitis with shock (P ) on day 2 (A), day 5 (B), and day 7 (C). Mean MR-proADM concentration in nmol/l is shown on day 2, day 5 and day 7 (D).

Figure 4.Schedule ROC for MR-proADM to predict fluid balance (delta H₂O) (A) and salt balance (Na delta) (B) in ICU patients suffering from aneurysmal subarachnoid hemorrhage, severe trauma without head injury, severe brain injury, or postoperative peritonitis with shock.

Figure 5. Predicted SOFA score (eliminate one at a time). Patients are sorted in ascending SOFA. The solid line shows the true SOFA values. The patient ID is shown on the x-axis (patients are sorted by ascending SOFA value), the predicted SOFA values are shown on the y-axis. The solid black line shows the (increasing) true SOFA values for all patients. The blue circles show the predicted SOFA score.

Figure 6. Predicted delta.H ₂ O, delta.Na and P-critical value for 201 patients. The 126 "normal" patients (patients without edema) are represented by empty circles, and the 75 "critical" patients (patients with edema) are represented by filled circles.

Figure 7. Random forest variable value for globular volume reference model.

Sequences

SEQ ID NO: 1: pre-pro-ADM amino acid sequence:

1 MKLVSVALMY LGSLAFLGAD TARLDVASEF RKKWNKWALS RGKRELRMSS

51 SYPTGLADVK AGPAQTLIRP QDMKGASRSP EDSSPDAARI RVKRYRQSMN

101 NFQGLRSFGC RFGTCTVQKL AHQIYQFTDK DKDNVAPRSK ISPQGYGRRR

151 RRSLPEAGPG RTLVSSKPQA HGAPAPPSGS APHFL

SEQ ID NO: 2: Amino acid sequence of MR-pro-ADM (pre-pro-ADM amino acids 45-92):

ELRMSSSYPT GLADVKAGPA QTLIRPQDMK GASRSPEDSS PDAARIRV

Examples were performed by detecting MR-proADM. However, as stated above, the invention can also be carried out by detecting proADM or another peptide fragment thereof.

The following non-limiting examples illustrate the invention.

Example 1 Positive Fluid Balance, Blood Volumes, and MR pro-ADM in Critically Ill Patients

Methods Patients and Procedures

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

Four types of patients were studied: patients with severe brain injury (SBT), aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head injury (PT), and postoperative peritonitis with shock (P). Patients were included if they required mechanical ventilation in D2 (D2) on an ongoing basis. SBT was defined as brain injury with Glasgow coma scores less than 9 (GCS<9). SAHs were included when the WFNS score was 4 or 5 (Brisman et al., 2006)]. PTs were included when the ISS score was 25 or more. P were included after abdominal surgery with signs of shock with hemodynamic complications (hypotension, low cardiac output) or lactate >4 mmol/l and catecholamine administration on admission to the intensive care unit.

Exclusion criteria: age <18 years, pregnancy, and congestive heart failure (NYHA II or IV).

General and demographic data were collected: age, sex, weight and height for BMI, IGS II, ISS, date of admission, Glasgow Outcome Scale (GOS) score before discharge from the intensive care unit. Dynamic Organ Failure Score (SOFA) was measured at admission (D0), at D2, D5 and D7 (Vincent et al., 1996).

Mean arterial pressure (MAP) and norepinephrine dose, if used, were recorded every day for each patient. Biological parameters were measured: the concentration of hemoglobin [Hb] and plasma proteins, plasma and urine concentrations of Na ⁺ , K ⁺ , Cl ^- , urea, creatinine and osmolarity. Urinary biological results obtained in the morning from the 24-hour total urine output were used to calculate the sum of the previous day's urinary loss of Na ⁺ , K ⁺ , urea and creatinine excretion. Weight and temperature were measured on day 2 (D2), day 5 (D5) and day 7 (D7).

A combination of echographic evidence, evidence of fluid response in ventilated patients, and repeated measurements of cardiac filling guided fluid administration throughout the day Feissel et al., 2004; Feissel et al., 2001; Monnet et al., 2013; Gore et al., 2005). In addition, the amount of intravenous fluid administered was also determined by a number of variables, including heart rate, blood pressure, blood lactate levels, and cardiac output.

Estimation of extracellular volume

Every day, salt and fluid balance was calculated to assess the change in the extracellular space. Each morning, a full input-output assessment for the previous day was performed for salt and water. Accurate data on salt and water were noted. All losses were measured: diuresis, ileostomy and ventricular drainage, if necessary. Loss of sodium (Na ⁺ ) was measured in liquids and subtracted from salt data. The difference between input consumption (enteral nutrition and total infusion of crystalloids or colloids per day) and water losses was also calculated. Insensitive losses were evaluated as a function of body temperature. The gain or loss of Na ⁺ (ΔNa ⁺ ) and water (ΔH ₂ O) was calculated each day and added to the result the day before as the total fluid balance. Creatinine clearance was also calculated every day. All calculations for each patient were made by a physician and validated by a second physician (BV, PEL and HF).

Blood volume measurements

Total blood volume with chromium 51 (Cr51) labeled erythrocytes was measured in D2 and D7. For practical reasons, D2 was not always carried out on the second day after the admission of the patient, but at some time from 1 day to 3 days. Day 7 measurements were taken from day 6 to day 10.

In the laboratory, 10 ml of the patient's own blood was labeled with the radioactive isotope chromium 51 (Cr51), and the radiolabeled erythrocytes were carefully selected by careful removal of all plasma radioactivity. Then, a known amount of radioactive erythrocytes was re-introduced into the general circulation and two samples were obtained in the arterial line after 10 and 30 minutes. Measurement of the radioactivity of two samples allowed derivation of total blood volume (TBV) in ml or ml/kg using the patient's weight (Gore et al., 2005). The erythrocyte volume (RBCV) (ml or ml/kg) and plasma volume (PV) in ml or ml/kg were then determined from the hematocrit value and the measured total blood volume. 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). In D7, plasma volume (PVI ₁₂₅ ) was directly measured with a known small amount of radioactively labeled 125 (I ₁₂₅ )albumin administered to the patient and samples were collected at 10, 30 minutes and 2 hours (Fairbanks et al., 1996). The normal PV value measured with I ₁₂₅ is 45±10 ml/kg (Gore et al., 2005). Typically, the plasma volume obtained with I ₁₂₅ -albumin is slightly larger than the plasma volume obtained from measurements with Cr51-erythrocytes due to the larger volume of distribution of albumin than that of erythrocytes (Gore et al., 2005).

When blood volume measurements were taken at D2 and D7, transthoracic echographic measurements were used to estimate stress volume with two measures: inferior vena cava change (ΔIVC) and E/E (Vincent et al., 1996 and Feissel et al., 2004).

Biomarker analysis

For each patient, plasma biomarkers in D2, D5 and D7 were studied. In D2 and D7, blood volume measurements were performed. D5 samples were always taken exactly 2 days before D7. Proadrenomedulin (MR pro-ADM), Pro-ANP, renin, angiotensin II, aldosterone, cortisol, epinephrine, and epinephrine, CT-pro-arginine vasopressin (copeptin), and proendothelin were measured as biomarkers that could potentially be associated with extracellular or plasma volume and decrease (MR pro-ADM and Pro-ANP) or increase in blood pressure. Erythropoietin (EPO) was measured for its ability to change with RBCV. The standards and methods used are presented in Table 3.

Table 3 Methods, units, and lower and upper normal values for all plasma biomarkers studied.

options ways units sensitivity Lower level Upper level Pro-ADM Crypto+ nmol/l 0.25 0.39 Pro-ANP Crypto+ pmol/l 4.5 85.2 renin immunoluminescence pg/ml one 3 16 angiotensin II Chromatography + radioimmunology pmol/l 2 19 38 Aldosterone radioimmunology pg/ml ten 42 201 cortisol immunoluminescence ng/dl one 9 22 epinephrine HPLC pg/ml twenty 80 norepinephrine HPLC pg/ml twenty 450 Copeptin Crypto+ pmol/l 0.5 1.1 16.4 Proendothelin Crypto+ pmol/l one 44.3±10.6 EPO ELISA MUI/ml 1.2 6.4 63.8

Statistical analysis

Power analysis. The required number of patients was calculated using the published range of brain natriuretic peptide (BNP) and erythropoietin (EPO) concentrations in similar patients (Dorhout Mees et al., 2011). An 80% power was considered with an expected difference of 50% between groups.

The normality of data distributions was tested using quantile-quantile (qq) plots and the Shapiro test. When the data were normally distributed, statistical comparisons were made on the transformed data. Data are presented as mean ± standard deviation (SD) or median (25th to 75th percentile) or number of samples and frequency (in percent).

Correlations were studied using linear regression or Spearman regression depending on the normality of the data. In addition, since most of the data were measured with error, Deming regression with equal variances was used to calculate the slope of the regression curve. For within- and between-group comparisons, ANOVA (factorial for repeated measures) or non-parametric (Kruskal-Wallis or Friedman) tests were used, followed by Tukey or Mann-Whitney/Wilcoxon tests, the latter corrected for multiple comparisons using the Bonferroni correction.

True/false detection ratio (ROC) curves were constructed to calculate the performance of biomarkers for predicting fluid and sodium balance. The best sensitivity/specificity cutoffs were calculated using the unweighted Youden index.

Finally, concordance analysis was used using a four-quadrant plot with and without gray zone exclusion of 15% to compare marker variation and fluid and sodium balance (Perrino et al., 1998)

Analysis was 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.

An independent relationship was found between fluid balance and blood volumes and biomarkers. A mathematical predictive tool was constructed using biomarkers and other relevant parameters to determine clinically useful predictors of fluid status in critically ill patients using a selection of “reasonable” independent variables. The SOFA score, a measure of the severity of the patient's condition, which is a multilevel ordered variable, is considered as an additional dependent variable.

For fluid balance (ΔNa⁺ and ΔH₂O) a pooled predictor was constructed to identify critically ill patients with ΔNa⁺>36 g and ΔH₂O>4 l. To do this, we first jointly considered the residuals resulting from elimination one at a time for (ΔNa⁺ and ΔH₂O), whose distribution turned out to be Gaussian (centered) with a covariance matrix. Therefore, for each pair of predicted values (ΔNa⁺ and ΔH₂O) you can get the critical probability (Pcritical), which is easily calculated using the mvtnorm package (Genz et al., 2009; Genz et al., 2014),

results

During the first 7 days after admission to the intensive care unit, 67 patients were studied, divided between SBT (n=21), HSA (n=20), PT (n=20) and P (n=6). The overall demographics and number of patients studied in each clinical setting are shown in Table 4. The SOFA scores at D0, D2, D5, and D7 are presented in Table 5.

Table 4. General and demographic data. Data are presented as mean ± standard deviation (M ± SD), except for the Glasgow Outcome Scale (GOS), which is presented as median

Total (n=67) SBT (n=21) SAH (n=20) PT (n=20) P (n=6) Age (years) 46±19 38±16 53±14 39±18 69±16 Weight, kg) 75±18 73±14 53±14 84±22 71±9 Height (cm) 172±10 178±9 169±9 173±11 161±8 IGS II 43±13 49±9 42±12 37±11 54±10 Gender F/M 24/43 18/3 11/9 6/14 4/2 Length of stay (days) 27±22 34±3 27±16 22±13 21±6 GOS 4(2) 4(1) 4(1) 5(1) NP

Table 5. SOFA score in D0, D2, D5 and D7 for each clinical group. SBT: severe brain injury, SAH: subarachnoid bleeding, PT: polytrauma, P: peritonitis

SBT (n=21) SAH (n=20) PT (n=20) P (n=6) MED ET MED ET MED ET MED ET SOFA D0 12 2 eight 2 12 3 fifteen one SOFA D2 eight 3 7 3 ten 3 fourteen 2 SOFA D5 6 four eight 3 6 four 13 5 SOFA D7 5 four 6 2 four four 9 3

The study was carried out for all patients. The set of extracellular volume was recorded as changes in salt (ΔNa ⁺ ) and changes in water (ΔH ₂ O) at D2 and D7 for each clinical situation. Almost all patients showed a positive fluid balance, that is, an increase in extracellular volume in D2 (64/67 for ΔNa ⁺ or 63/67 for ΔH ₂ O), and most in D7 (42/67 for ΔNa ⁺ or 41/ 67 for ΔH ₂ O). A positive balance of hydrous soda in D2 and D7 was observed for all pathologies with higher salt increases for PT and P than for SHT and SAH. For example, in D2, PT and P show a total increase in salt of 70±32 g and 77±28 g as STB, while SAH shows 43±24 g and 28±24 g. ΔNa ⁺ is associated with ΔH ₂ O, and this shows that retained water is closely related to retained salt (r ² =0.67; p<0.0001). As well-known indicators of extracellular space, plasma protein concentration and weight change are associated with ΔNa ⁺ and ΔH ₂ 0, but these relationships are weak (r ² =0.44 for plasma proteins and ΔNa ⁺ , r ² =0.35 for plasma proteins and ΔH ₂ O, and r ² =0.33 for weight and ΔH ₂ O). Like plasma proteins, [Hb] has a weak relationship with ΔNa ⁺ (r ² =0.15) and ΔH20 (r ² =0.24).

Measured volumes with Cr51 (total blood volume (TBV), erythrocyte volume (RBCV) and plasma volume (PV)) were studied in 62 patients in D2 and 63 patients in D7. Most patients experienced a decrease in TBV. Only 21 patients in D2 and 25 in D7 were in the normal 20% range. Hypovolaemia with TBV less than 20% of normal was observed in 46 patients (74%) in D2 and 42 (66%) in D7. All patients were found to have low RBCV except 1 patient in D2 and 2 patients in D7. These patients with normal RBCV were also transfused. Even in non-hemorrhagic conditions (SBT or SAH), the reduction in RBCV could be significant with a deficit of 50% or less of normal RBCV in 25 patients (40%) in D2 and 21 (33%) in D7. There is no statistical relationship between TBV or RBCV and fluid balance, ie, ΔNa or ΔH ₂ O.

The PV distribution is in the normal range. There is no relationship between PV and fluid balance, ie, ΔNa or ΔH ₂ O, and we also found no relationship between changes in PV and changes in ΔH ₂ O between D2 or D7. The change in PV is not associated with changes in the plasma concentration of proteins. Hemoglobin concentration is weakly related to RBCV (r ² =0.33) and not related to VP (r ² =0.026).

In D7 VP 58 patients were measured by I ₁₂₅ (PVI ₁₂₅ ). There is a statistically significant relationship between both PVs (PV Cr51 and PVI ₁₂₅ ). Deming's regression found a slope of 0.852 (CI 95% 0.610-1.08) and an intersection at 780 ml (CI 95% 103-1485 ml) (confidence interval denoted herein as CI) towards PVI ₁₂₅ with r ² =0.752.

No relationship was found between blood volumes and variations in the inferior vena cava (ΔIVC) or E/E'.

Biomarkers

Detailed kinetics of all biomarkers are given in Appendix I. Most of them increase in D2 and then decrease statistically in D5 and D7 (copeptin, angio II, renin); some statistically decreased only in D7 (MR pro-ADM, EPO). Other biomarkers did not change during the three days examined (cortisol, aldosterone, Pro-ANP, proendothelin). Plasma norepinephrine was excluded from the study due to a diversion due to external infusion used as treatment in patients.

Among all tested biomarkers, we found only two biomarkers, renin and MR pro-ADM, having a statistical relationship with ΔNa ⁺ and/or ΔH ₂ O (table 6). The relationship between MR pro-ADM and ΔNa ⁺ and/or ΔH ₂ O was found to be very strong and completely independent of the type of clinical situation.

Table 6 Probability of association between plasma concentrations of biomarkers and measure of fluid balance (p) derived from mixed-purpose modeling.

biomarkers ΔH2O _{_} ∆ Na ⁺ renin 0.048 0.0001 MP-proADM <0.0001 <0.0001

Then, 4 L for ΔH₂O and/or 36 g for ΔNa⁺ (4 liters at 9‰ yields 36 g of salt) were considered thresholds for positive fluid balance (Bjerregaard et al., 2005). The ROC curve allowed us to find the best threshold for MR-proADM suggesting the best sensitivity/specificity trade-off with an unbalanced Youden index (Figure 4).

Since a reasonable probability has been found that MR-proADM can predict salt balance and fluid balance (ΔNa⁺and ΔH₂O), then the predictive estimate ΔNa⁺and/or ΔH₂O was built with MR-proADM and other simple independent variables. For ΔNa⁺ and/or ΔH₂O The best predictive model requires, from strongest to weakest, MR-proADM, BMI at D0, weight at D0, age, sex, [Hb], IGS, and fluid intake at D0. Removing MR-proADM from the model reduces its predictive value. The two models can independently account for about 70% of the variance and have good discrimination power with an AUC of 88% for ΔNa⁺ and 92% for ΔH₂A. In addition, the lack of IGS and fluid intake in D0 have very limited consequences in these two models with only a 2-3% loss of r², and do not affect AUC.

If the joint predictor for defining critically ill patients who have both Na ⁺ >36 g and ΔH ₂ O >4 L was built as described earlier in the statistical paragraph, then the performance improved significantly. The discrimination power of this joint predictor was high with AUC=0.9987 (95% CI [0.9964-1]). For example, with a threshold of 0.4 for critical P, a sensitivity of 0.988 and a specificity of 0.949 were obtained. Moreover, this predictor of fluid balance is significantly associated with SOFA scores for patients.

In addition, biomarkers were tested and blood volumes were measured. Interestingly, among all biomarkers, with respect to fluid balance, the same two biomarkers, renin and MR pro-ADM, had a statistical relationship with blood volumes. However, if blood volumes were again analyzed by regression for mixed targets on the logarithm of biomarker values, then the predictive value of the markers was poor with low AUC under the ROC curve (for renin: AUC TBV=0.5798 and AUC PV=0.6159, and for MR pro-ADM: AUC TBV=0.5967 and AUC PV=0.6277). Moreover, despite the high probability (p=0.0002), it was not possible to plot an ROC curve and measure the AUC for RBCV because the values were too uncharacteristic and low. In these situations, neither thresholds nor predictive models were tested.

Discussion

This study showed that a simple biomarker, MR-proADM, known as an indicator of endothelial permeability (Christ-Crain et al., 2005 and Koyama et al., 2013), is a good indicator of the increase in salt and water in the extracellular volume during the first week after admission. critically ill patients. In addition, no relationship was found between an increase in salt balance or fluid balance and direct measurement of blood volumes in D2 and D7.

It is now clear that excessive salt or fluid balance can be considered as a risk factor for morbidity and mortality in critically ill patients (Boyd et al., 2011; Kelm et al., 2015; Acheampong et al., 2015; Malarian et al., 2015 al., 2014). This situation often occurs because the goal of cardiac output efficiency involves increasing fluid volume to achieve efficiency (Cecconi et al., 2011 and Vincent et al., 2011). Often in conditions of severe inflammation in the first days after the intervention (with sepsis, trauma, or cerebral aneurysm), many patients are at high risk of accumulation of salt with water in the extracellular volume due to excessive capillary permeability, increased fluid, decreased diuresis, and difficulty maintaining salt in the plasma volume. Patients respond differently to fluid resuscitation. Accordingly, a clear and rapid indicator marker is needed to improve patient stratification and identification of patients with a positive interstitial salt balance in order to personalize treatment: choose more fluid for perfusion or adjust with catecholamine or diuretics. This marker should also be independent of the type of reason for admission to the intensive care unit.

While MR-proADM appears to be a particularly good indicator of salt balance and fluid, it cannot be an indicator of capillary permeability alone. Na ⁺ balance is controlled not only by the kidneys (Titze et al., 2014). Interstitial Na ⁺ excretion appears to be mediated by immune cells, especially macrophages, which control Na ⁺ release through the interstitial lymphatic capillary system (Titze et al., 2014). In inflammatory states, the loss of homeostatic control of immune cells by macrophages, including their inability to excrete Na ⁺ , may be an explanation for the accumulation of Na ⁺ in the interstitium (Jantsch et al., 2014). Interestingly, the adrenomedullin peptide is known to play a role in the organization of lymphatic channels, especially in organogenesis (Kahn et al., 2008).

The total amount of excess in the interstitium is associated not only with inflammation and decreased excretion of Na ⁺ . Deterioration may be due to physiological causes such as increased hydrostatic pressure caused by high mean arterial pressure (MAP) or high infusion rate (Bark et al., 2013) or increased blood distension caused by excessive cardiac output. A high MR-proADM (greater than 1 nmol/L) indicates sodium overload, and a set of, for example, more than 36 g of Na ⁺ and 4 L of water may be a warning to the clinician to take appropriate action immediately.

In many intensive care units, nurses systematically measure fluid balance by daily weight or daily calculation of fluid input and output. These methods are inaccurate. For example, a weak relationship was found between weight and fluid balance in our observational study (r ² = 0.33). Common markers of extracellular volume such as plasma proteins or [Hb] have little association with salt and fluid balances (r ² =0.35 and r ² =0.24). In this study, salt and fluid balance measurements required multiple biological samples, and measurements were always performed and monitored by two physicians. In daily practice, nurses cannot allocate the time required to collect the necessary information. MR-proADM appears to provide a more accurate measure of salt balance and extracellular status, and can also be used as an emergency indicator after an emergency. In some acute situations, in the first days after a shock, time can be of the essence. In real life, the presence of overload is detected too late, often after organ damage (acute lung injury, abdominal compression syndrome, or kidney dysfunction). The relationship between the SOFA score and the salt and fluid balance prediction model supported the notion that MR-proADM could be an interesting bedside tool.

No markers were found to measure total blood volume (TBV), plasma volume (PV), or red blood cell volume (RBCV). Renin and MR-proADM were found to be significant but not sufficient to construct a useful predictive model. Even ΔNa ⁺ or ΔH ₂ O are not predictors of blood volumes.

Another interesting result of this study is the demonstration that there is no relationship between PV and fluid balance. This was especially unexpected since volume replacement is the main reason for fluid perfusion. However, PV is the only volume for which the majority of patients were in the normal ranges at D2 and D7. The lack of correlation between fluid balance and PV supported the hypothesis of Na and water trapped in the interstitial volume. In daily practice it would be useful to have more control over this plasma volume, but we did not find any associations with biomarkers, proteins, [Hb], nor with signs associated with compressed volume measured by echography. To study this problem, it is necessary to conduct other studies with a combination of biomarkers or other elements.

The correlation found between the two PV measurement modes (Cr51 and I125) in D7 confirmed the result stated in the present description. Albumin may have a larger volume of distribution than erythrocytes, especially if capillary permeability is increased in pathological situations. At D7, the difference was quite large (780 ml), indicating that capillary permeability had not fully recovered. It may be interesting to discuss this comparison in D2 as well. Unfortunately, measurement using I125 in D2 could not be performed due to interactions with some biomarker measurement methods (Table 1).

A low RBCV level, anemia, is quite common and explains the low results for TBV. RBCV is difficult to assess and requires CR51 processing and radioactivity block management. It is evaluated only in rare cases, such as Vasquez disease. In daily practice, a [Hb] threshold test (<7-8 g/dl) is used to determine the need for a blood transfusion. Since [Hb] is the ratio between PV and RBCV, [Hb] is a poor indicator for RBCV (Takanishi et al., 2008). Patients may have an [Hb] of 11 g/dL and only 50% of a normal RBCV. The absence of 50% of normal red blood cells can have an important impact on the health of patients. Some studies have suggested that total RBC count may be a predictive marker of cognitive recuperation (Naidech et al., 2007). However, RBC transfusions are not only used to replace missing cells. In this study, EPO is independent of RBCV levels, indicating that EPO is not a good indicator for RBCV, and that EPO stimulation is due not only to RBC quantity, but also to RBC quality.

The clinical situations described in the study reflect the daily practice of our service. Severe trauma, traumatic brain injury, and neurological situations are common; septic shocks are less common. However, group as an independent variable does not interfere with the prediction of fluid balance or blood volume, suggesting that volume disturbances are rather independent of pathology. Other studies will be required to confirm the presented results.

Overload after shock is an important and underestimated survival factor. A positive salt balance is actually documented as an important prognostic marker. Blood volumes are not automatically associated with volume expansion. MR-proADM is an interesting indicator for assessing salt and fluid balance in the first week after an acute inflammatory condition in critically ill patients. A rupture is suspected with frequent excessive volume expansion.

Example 2 Predicting fluid balance and/or salt balance using MR-proADM

1. Introduction

The objectives of this study were to answer the following questions:

Can changes in Na and H ₂ O be predicted using biomarkers and/or other independent variables?

Can changes in volumes be predicted using biomarkers and/or other independent variables?

To this end, the predictive value of the presented biomarkers and independent variables was assessed using data collection and model selection techniques.

2 Material and methods

Note that the following Material and Methods section describes the material and methods used in Examples 2-4, which serve as exemplary embodiments only.

2 Material and methods

2.1 Data

Below is a description of the variables and datasets used in this report. Only 3.5% missing data was observed (54% for max.lactate) and this value is low. As a consequence, missing data was not really a major problem in this study. For this reason, we decided to conditionally set all missing values using the mean of the respective column.

Dependent variables

A total of 2 dependent variables were considered:

delta.H₂O (D2, D5, D7): water change (H₂O) expressed in liters (l). Change ≥4.0 l is considered critical.

delta.na (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 of ≤60 ml/kg is considered critical (the classic threshold of 72 has been lowered in order to increase the number of controls).

VP (D2, D7): plasma volume expressed in ml/kg. Plasma volume ≤40 ml/kg is considered critical.

VG (D2, D7): globular volume expressed in ml/kg. A globular volume 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): dynamic assessment of organ failure. Multilevel ordered dependent variable: the score increases with the severity of the patient's condition. Note that the 4 SOFA scores above 15 are set to 15 to avoid SOFA scores based on only one observation.

note: for D7, second volume measurements using iodine instead of CR51 were discarded.

Independent variables

• Patient independent variables (8): age, sex, weight.D0, BMI.D0 (body mass index), IGS.II (IGS II score), GOS (Glasgow Outcome Scale), fluid.D0 (fluid intake in D0) , Na.D0 (sodium consumption in D0)

• daily independent variables (5): max.temp (maximum temperature), max.lactate (lactate), min.PAM (minimum mean arterial pressure), FC (heart rate), max.cathe (catecholamine).

• biomarker independent variables (11): Hb, Prot.D0 (total serum protein on day 0), Prot (total serum protein), Angio (angiotensin II), Renin, Aldo (aldosterone), Pro.ANP, Adre (adrenaline) , Pro.Endo (proendothelin-1), CT.proAVP, MR.proADM, cortisol, Nor (norepinephrine), EPO (all on a logarithmic scale, log1p conversion).

Note: BMI was calculated from height and weight using the formula: BMI.J0=weight.J0/(height.J0) ² . The independent variable height.D0 was dropped to avoid redundancy with weight.D0 and imm.D0.

2.2. Statistical Methods

All statistical calculations were performed using R software (Rmanual) version 3.0.2.

random forests

Random forests (Breiman, 2001; Breiman, 2002) have been used to predict dependent variables from independent variables. This approach consists in the cyclic construction of decision trees based on bootstrapping data. Each time the algorithm was run, 5000 trees were built (500 for the "eliminate one by one" procedure). This algorithm is a powerful data collection technique known to be able to detect even non-linear effects. For a good introduction to the application of random forests in a biomedical context, see Boulesteix, 2012.

Importance

Sensitivity analysis was performed to rank the independent variables in order of decreasing importance. For each independent variable, random forest prediction was performed with and without the analyzed independent variable and studied the consequences of its absence in terms of the quality of the prediction.

Selection with inclusion and selection with exclusion

Due to the high correlation between the independent variables, choosing the best model by simply using the k most important variables will not necessarily lead to the most accurate prediction. To overcome this problem, the classical approach (Diaz 2006, Nguyen, 2013) is to perform an elimination selection procedure using a random forest.

In the case of inclusion selection, the idea is to start with an empty model, perform a sensitivity analysis (one RF per variable), add a variable providing the minimum improvement in the criterion of interest (R2 was used in the present description, see description below), and start again with extended model. Due to the high stochasticity of the random forest, the results of the inclusion selection procedure may vary in different iterations of the procedure. Therefore, a total of five repetitions with a large number of trees (tree=50,000) were systematically performed and a consensus model was selected that showed stability across all repetitions.

In the case of exclusion selection, the idea is to start with the full model, perform a sensitivity analysis (one RF per variable), remove the variable providing the minimum improvement in the criterion (in this description R2 (also denoted r ² ) was used, see description below ), and start over with a reduced model. Due to the high stochasticity of the random forest, the results of the elimination selection procedure may vary in different iterations of the procedure. Therefore, a total of five repetitions with a large number of trees (tree=50000) were systematically performed and a consensus model was selected that showed stability across all repetitions.

Linear regressions

Classical linear regression was also performed on the selected models. The study of linear coefficients also provides an easy way to understand the individual effects of independent variables on dependent variables.

Exception one by one

Since the data should not be separated into training data and test data, a classic cross-validation technique called “one-by-one elimination” was applied to avoid overfitting. With this approach, we looped one item out of the data, trained our model (linear regression or random forest) on the reduced dataset, and then used the resulting model to predict the value of the item that was dropped.

R2

The correlation between the dependent variable and the predicted response is measured in terms of the quadratic correlation R2 (or r ² ). In the context of a linear model, it exactly corresponds to the proportion of the explained variance. R2 is always between 0 and 100%, the higher the better.

AUC

Classification scores are measured in terms of the area under the ROC curve (AUC). This classic criterion is often preferred over power because it simultaneously considers all possible thresholds and does not even require control of the error rate H0. AUC is always between 0 and 100%. An AUC of about 50% is pure noise, an AUC below 70% is considered poor, an AUC between 70% and 80% is considered correct, between 80% and 90% is good, and more than 90% is excellent. AUC estimation is performed herein using the pROC R package (robin2011proc).

Survival Analysis

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

3 Results and discussion

3.1. Fluid balance

MR.proADM performs well for predicting delta.H ₂ O. MR.proADM alone achieves good classification power (AUC≃0.82) (Figure 2A) with the dependent variable explaining 35% of the variance.

A selection procedure for delta.H ₂ O was performed. Importance analysis showed the importance of the following independent variables for patients and daily independent variables: BMI.D0, weight.D0, age, sex, BMI, total protein, Hb, fluid intake.D0 (fluid .D0), group of patients (group). The biomarker MR.proADM received the highest importance. Other biomarkers played a role: Pro.Endo, CT.proAVP, EPO, total serum protein and Hb.

3.2 Salt balance

As noted for delta.H ₂ O, MR.proADM has good classification power (AUC≃0.79) with an r ² of 0.42 (FIG. 2B).

The importance analysis performed indicated the same markers/parameters as observed for delta.H ₂ O, with the difference that sodium intake at D0 (Na.D0) replaced fluid intake at D0 (liquid.D0).

four. conclusions

The key role of MR-proADM for the prediction of delta.H ₂ O and delta.Na has obviously been confirmed. However, the combination of MR-proADM with other markers and/or parameters may improve the prediction of fluid and/or salt balance.

Example 3: Improving Prediction by Including Additional Parameters

The aim of this study is to construct predictors suitable for clinical use using a choice of independent variables (additional markers or parameters) (see Table 7).

Table 7. Used markers and parameters.

primary MR.proADM; BMI.J0 (BMI on day 0); weight.J0 (weight in D0); Liquid.J0 (fluid consumption in D0); age; floor secondary Pro.Endo (pro-endothelin-1); CT.proAVP; Na.J0 (sodium intake on day 0); Adre (adrenaline); IGS.II Pro.ANP FC (heart rate); max.temp (maximum temperature); min.PAM (minimum mean arterial pressure); max.lactate (lactate) max.cath (catecholamine); Prot.J0 (total whey protein on day 0); Prot (total whey protein); Hb; the weight

1.1 Fluid balance and sodium balance

Reference model for delta.H ₂ O prediction:

delta.H ₂ O ~ MR.proADM+BMI.D0+Wt.D0+Age+Gender+Hb+Prot+IGS.II+Liquid.D0

where IGS.II and Fluid.D0 are optional due to the practical difficulty of obtaining them in a clinical context.

Table 8: Brief description of the delta.H ₂ O and delta.Na models.

Change Model R2 (lm) R2 (rf) SD CV AUC [95% CI] _H2O Reference 0.501 0.703 2.718 0.512 0.920 [0.884-0.956] no Liquid.D0 0.497 0.699 2.753 0.518 0.924 [0.890-0.958] no IGS.II 0.504 0.699 2.742 0.516 0.921 [0.886-0.957] no Liquid.D0/IGS.II 0.501 0.680 2.815 0.530 0.924 [0.890-0.958] H ₂ O(-D5) Reference 0.433 0.509 3.451 0.674 0.891 [0.835-0.946] Prot weight 0.298 0.430 3.710 0.725 0.793 [0.715-0.871] the weight 0.251 0.194 4.575 0.894 0.712 [0.624-0.801] Prot 0.076 0.178 4,710 0.920 0.629 [0.534-0.724] Na Reference 0.566 0.713 23.738 0.691 0.886 [0.841-0.931] no Liquid.D0 0.557 0.708 23.864 0.695 0.887 [0.882-0.931] no IGS.II 0.569 0.704 24.001 0.699 0.880 [0.834-0.927] no Liquid.D0/IGS.II 0.561 0.704 24.141 0.703 0.881 [0.835-0.927] Na(-D5) Reference 0.553 0.541 29.571 0.876 0.851 [0.786-0.917] Prot weight 0.238 0.316 35.983 1.066 0.749 [0.665-0.834] the weight 0.206 0.171 40.894 1.212 0.661 [0.565-0.756] Prot 0.053 0.194 40.979 1.214 0.634 [0.537-0.731]

As shown in Table 8 (change in "H ₂ O"), this model explained about 70% of the variance and had very good discrimination power with an AUC of 92% (95% CI lower bound is about 89%). The absence of IGS.II and/or Liquid.D0 has very limited consequences with a loss of only 2-3% of r ² and does not affect AUC at all.

This model was also compared with a predictor built from patient weight and Prot. Since there was no data available in D5 for weight, this point in time was completely removed, resulting in a smaller data set. The results were presented in table 8 ("H ₂ O(-D5)" response). The predictor built from weight and Prot achieved an AUC of only 80%. Note that the reduction was significant relative to our reference model (90% AUC). Moreover, the loss was even more pronounced when considering only weight (AUC=71%) or Prot (AUC=63%), further confirming the limited benefit of these two independent variables. It should also be noted that r ² was very limited in these models, especially when using only linear regressions.

sodium balance

Reference model for delta.Na prediction:

delta.Na ~ MR.proADM+BMI.D0+Wt.D0+Age+Sex+Hb+Prot+Na.D0+IGS.II+Fluid.D0

As noted for delta.H ₂ O, IGS.II and Liquid.D0 are optional.

Table 8 (change in "Na") shows that this model explained approximately 70% of the variance and had good discrimination power with an AUC of 88% (the lower bound of the 95% CI was approximately 84%). As noted for delta.H ₂ O, the absence of IGS.II and/or Liquid.D0 had very limited consequences. Again, as observed for delta.H ₂ O, weight and Prot appear to be of limited interest in predicting dependent variables as they have both low R ² (~20%) and low AUC (~63-75%).

Building a pooled predictor

In this section, a predictor is developed for "critical" patients, that is, patients with delta.H₂O>4 l and delta.Na>36 g. For this purpose, the residuals are eliminated one by one for (delta.H₂O, delta.Na) we consider jointly, and it was found that the distribution is Gaussian (centered) with a covariance matrix:

Therefore, for each pair (x, y) of predicted values (delta.H ₂ O, delta.Na) one can calculate the probability that they are critical, as:

this probability can be easily calculated using the mvtnorm R package (Genz and Bretz, 2009; Genz et al., 2014).

A graphical representation of the two-dimensional (p-critical) function would allow distinguishing between non-critical and critical patients using only the predicted values of delta.H ₂ O and delta.Na. Note that the discriminatory power of this pooled predictor is high with AUC=0.92 (see also Example 4).

2.2 Volume changes

Total volume and plasma volume

delta.H ₂ O and delta.Na were unexpectedly found to be suboptimal predictors for VT and VP (Table 9). In fact, they explained at best 7% of the variance and barely outperformed the random classifier in terms of discrimination power (best AUC=64%). This clearly demonstrated that the clinical idea of "filling patients" with salt water did not produce the expected plasma response.

Table 9: Brief description of volume change models

change Model lmR2 R2 SD CV AUC [95% CI] VT H2O _Na 0.009 0.040 12.370 0.215 0.582 [0.481-0.682] _H2O 0.007 0.037 12.896 0.224 0.579 [0.475-0.684] NA 0.017 0.069 12.533 0.218 0.636 [0.540-0.733] VP H2O _Na 0.031 0.029 9.101 0.224 0.589 [0.492-0.686] _H2O 0.095 0.021 9.674 0.239 0.571 [0.474-0.669] Na 0.017 0.037 9.458 0.233 0.573 [0.475-0.671] VG reference 0.408 0.455 3.263 0.192 0.824 [0.747-0.900] no MR.proADM 0.407 0.484 3,300 0.194 0.839 [0.764-0.914] Hb only 0.304 0.230 4.052 0.239 0.698 [0.593-0.802]

Globular volume

For globular volume (VG), a correlation was observed with delta.H ₂ O and delta.Na. It was possible to build a predictive (reference) model:

VG ~ MR.proADM+Hb+bm.D0+gender+age+Prot

As can be seen from Table 9, this model explains 45% of the variance (which is a low value) but still provides an AUC of 82% (the lower bound of the 95% CI is 75%). This obviously represented an improvement over the simple Hb-only model, which gave an r ² of only 30% (with a linear model, not from random forests) and an AUC of 70% (the lower bound of 95% was 60 %). This result was interesting as it marked the limits of Hb as the only biomarker for change in globular volume, which is the current clinical standard.

Table 9 shows that the "reference" model without MR-proADM achieved similar performance with r², constituting 48%, and an AUC of 84%. A reference model including MR-proADM identifies patients with low (<20 or 15) VG or RBCV.

When looking at the one-by-one elimination residuals in the best VG models, we clearly observed that these residuals are distributed according to a centered Gaussian distribution with a standard deviation of 3.2-3.3. Although much of the observed variance was not explained by this model, it can already be applied to identify critical patients: patients with VG<20 ml/kg. Indeed, if x is the predicted VG, we have:

Figure 7 shows the Pcritical function. This decision function can serve as a valid alternative to Hb for reliable detection of critical patients.

2.3 SOFA

Figure 5 illustrates the predicted SOFA values using the model chosen by the experts and the elimination procedure one at a time. The correlation was very high, even if the prediction accuracy was not great. Indeed, in Table 10, the exact predictions (difference=0) only had 19% accuracy, but this number increased dramatically when a large difference between the predicted and the exact SOFA was allowed. For a difference of 4, 82% SOFA was obtained.

SOFA ~ delta.H ₂ O+delta.Na+age+bm.D0+gender

Table 10: SOFA Prediction Accuracy

difference 0 one 2 3 four accuracy 0.19 0.47 0.69 0.77 0.82

Note that we also tried to regroup SOFA into three classes, but this did not provide a significant improvement (33% errors).

2.4 Duration of edema

Table 11 shows the available data.

Table 11: Edema duration data. A total of 21 observed endpoints (no edema) and 31 censored durations are shown.

time eight 5 7 7 four 7 7 7 eight 6 eight eight four eight 5 ten eight 9 delta 0 one one 0 one one 0 one 0 0 0 0 one 0 one 0 0 0 time 3 6 eight 6 6 2 6 eight 7 6 6 9 four 7 four 3 7 eight delta one one 0 one 0 one 0 0 0 one one 0 one 0 one one 0 0 time 7 7 6 6 7 6 6 6 eight eight 9 5 ten 9 7 7 delta 0 0 one 0 0 one one 0 0 0 0 one one 0 0 0

Table 12: Cox model for edema duration. Significant independent variables are marked with an asterisk.

Coef. Average (coefficient) Std off (coefficient) Z p age -0.05 0.95 0.02 -2.54 0.01* floor M 0.94 2.55 0.93 1.01 0.31 wt.D0 -0.07 0.93 0.05 -1.62 0.11 bmt.D0 0.14 1.14 0.13 1.03 0.30 IGS.II 0.00 1.00 0.02 0.13 0.90 SOFA -0.21 0.81 0.10 -2.13 0.03* MR.proADM 0.64 1.90 0.88 0.73 0.46

Cox model

The analysis was initiated by fitting a proportional hazard model to these data (Table 12). Among the independent variables tested, only age and SOFA were significant. In particular, MR.proADM does not appear to play a key role. We constructed the corrected edema duration by fitting the Cox model for age and SOFA only, with a reference of 41.5 for age (median) and 9 for SOFA (median).

Kaplan-Meier estimates

Survival curve estimates were analyzed, stratified by low (MR.proADM<1.5) and high (MR.proADM>1.5) values in D2. The difference between the two curves was apparently not significant, confirming the results of Table 12. Note that when using the uncorrected edema duration, there was a significant difference (p=0.01) between the two curves (data not shown), but this result disappeared when adjusted. by age and SOFA.

random forests

Random forest analyzes were performed on the same (unadjusted) data. The procedure achieved an overall error rate of 41%, which is high (the exact nature of this error rate was unclear). From the point of view of the importance of variables, the key role of age was confirmed, but SOFA and MR.proADM were found to have little effect on the result of this non-linear structure. Random forests were used to predict survival curves for each patient based on independent variables (age, SOFA and MR.proADM) using a one-by-one elimination technique. The difference between the two datasets is mainly due to the fact that patients with high MR-proADM in D2 were mostly not cured by the end of the study. However, patients with lower MR-proADM at D2 did not show a clear trend towards cure when the effects of age and SOFA were included.

3. conclusions

For globular volume, MR.proADM is of moderate significance. In addition, our study shows the limits of the current biomarker (Hb) and proposes a new model that may be useful for clinicians in the future to monitor patients with critical VG.

A more interesting achievement of this study is the edema predictor, which combines delta.H ₂ O and delta.Na predictions to very effectively detect (AUC>0.99) patients at critical risk for edema using only the lung in obtaining clinically independent variables (BMI .D0, weight.D0, age, sex and possibly IGS.II and Fluid.D0) with three biomarkers: MR.proADM, Hb and Prot.

Example 4. Further improvement of the prediction

Models

In order to simplify the models presented herein, IGS.II, Fluid.J0, and Na.J0 were removed from the original models to provide one simple model to predict both delta.H ₂ O and delta.Na ( hereinafter referred to as "model 2"). Including age2= ^age2 and age3= ^age3 in the model further increased the predictive value. This was the only addition of the transformed independent variable that had a significant effect. The models presented in Example 3 for predicting fluid balance and salt balance, ie, including parameters IGS.II, fluid.J0 and Na.J0, are referred to as "model 1".

• “Model 2”: ~ bm.J0+weight.J0+age+age2+age3+gender+MR.proADM+Hb+Prot

• “Without biomarkers”: ~ BMI.J0+weight.J0+age+age2+age3+sex

• Biomarkers only: ~ MR.proADM+Hb+Prot

Note. Note that MR.proADM, Hb and Prot refer to the log1p transformation of the original measurements.

For each of these models, we can either make a prediction using a one-by-one elimination approach or use the full dataset. Not surprisingly, in the latter case, the performance will always be higher than in the former. For reliable and replicable results, single exclusion scores should be preferred, for a more optimistic point of view, and for comparison with very rough methods (e.g. using MR.proADM directly to distinguish between regular and critical patients), scores should be used. for the full set of data.

AUC results

Table 13: AUC in the one-by-one elimination approach.

AUC [95% CI] _delta.H2O delta.na Critical model 2 0.923[0.887-0.959] 0.917[0.881-0.953] 0.926[0.882-0.960] model 1 0.922[0.886-0.959] 0.919[0.883-0.955] 0.922[0.888-0.957] no biomarkers 0.823[0.763-0.882] 0.815[0.751-0.878] 0.825[0.764-0.886] only biomarkers 0.884[0.840-0.929] 0.881[0.835-0.927] 0.886[0.841-0.930]

Table 14: AUC using all available data

_delta.H2O delta.na Critical model 2 0.948[0.922-0.974] 0.981[0.967-0.996] 0.990[0.981-0.999] model 1 0.947[0.921-0.974] 0.983[0.968-0.997] 0.990[0.981-0.999] no biomarkers 0.879[0.832-0.926] 0.902[0.857-0.946] 0.911[0.870-0.952] only biomarkers 0.942[0.913-0.970] 0.976[0.959-0.993] 0.977[0.961-0.993]

Model 2 herein, using only simple explanatory variables and selected biomarkers, achieved similar or even better results than Model 1 presented in Example 3. In all situations, Pcritical was found to effectively combine delta.H ₂ O and delta prediction. Na with little improvement over the best of the two. When considering a model without biomarkers, there was a significant decrease in performance. However, it should be noted that this model still shows patients with high levels of edema. When considering only biomarkers, performance was similar compared to the best model, but it was slightly worse.

In comparison, the performance of MR.proADM alone to distinguish between regular and critical patients reached an AUC of 0.845 [0.791-0.898], which should be compared with the AUC of Table 14 (0.990 for the best model) to obtain consistent data. Therefore, the combination of additional markers and/or parameters provided an even greater improvement in predictive value.

Details of the best model

Compared ROC obtained in three different ways, that is, 1) using only the predicted delta.H ₂ O; 2) using only the predicted delta.Na; 3) with the union of both predictions in Pcritical. If delta.H ₂ O is much less efficient than the other two (which is consistent with Table 13 and Table 14), then the ROCs for delta.Na and Pcritical are very similar. However, Pcritical outperforms delta.Na due to its high specificity (eg: Spe>0.95). This can be emphasized by considering the (adjusted) partial AUC for Spe∈[1.00;0.95]. A value of 0.781 was obtained for delta.H ₂ O, a value of 0.882 for delta.Na, and a value of 0.948 for Pcritical. These results suggest that Pcritical was even more reliable than delta.Na (and delta.H ₂ O) when high specificity was required.

Table 15: False positives (FP), false negatives (FN), sensitivity (Sen) and specificity (Spe) for the best model (all data) for different Pcritical cutoffs.

threshold FP FN Sen Spe 0.10 40 0 1.00 0.68 0.20 28 0 1.00 0.78 0.30 23 one 0.99 0.82 0.40 fifteen 3 0.96 0.88 0.50 ten four 0.95 0.92 0.60 four 6 0.92 0.97 0.70 one 9 0.88 0.99 0.80 one ten 0.87 0.99 0.90 0 twenty 0.73 1.00

Table 15 summarizes how prediction is performed using various thresholds. Depending on the cost of false positives and false negatives, this should allow a threshold to be selected that provides the best compromise between two simultaneous risks.

The relationship between predicted delta.H ₂ O and predicted delta.Na was investigated for 201 patients included in the study (figure 6). Both predictions had a high correlation (cor≃0.9), which is consistent with the observed delta.H ₂ O and delta.Na (cor≃0.8). The Pcritical presentation demonstrated that high-risk areas were represented almost exclusively by "critical" patients, and low-risk areas almost exclusively by "normal" patients.

Correlation between Pcritical, SOFA and VG

In this section, we compare the new Pcritical estimate with SOFA and VG (globular volume).

In Table 16 and Table 17 we see that there is a good correlation between SOFA and Pcritical.

From now on, we focus on two specific groups of interest: the "low risk" group, which included a total of 86 patients (out of 201) with Pcritical<0:1, and the "high risk" group, which collected 55 patients with Critical>0:9.

Table 16: Breakdown of SOFA into Pcritical groups.

SOFA 0 one 2 3 four 5 6 7 eight 9 ten eleven 12 13 fourteen fifteen 17 twenty 21 0.00<Pcritical<1.0 eight 13 12 25 21 16 17 13 twenty fourteen 12 eight ten 3 5 one one one one 0.00<Pcritical<0.25 6 eleven eleven fifteen fourteen ten 7 2 6 eight 6 3 2 one 0 0 0 0 0 0.25<Pcritical<0.50 one 0 0 3 one one four 3 one 0 one one one 0 0 one 0 0 0 0.50<Pcritical<0.75 one 0 0 3 2 2 0 one 0 one 2 one one 0 0 0 0 0 0 0.75<Pcritical<1.0 0 2 one 3 four 2 6 7 13 four 3 3 6 2 5 0 one one one

Table 17: Distribution of SOFA by Pcritical groups

choice Min. 1 quantile Median Average 3 quantile Max. 0.00<Pcritical<1.0 0.00 3.00 6.00 6.29 9.00 21.00 0.00<Pcritical<0.25 0.00 2.00 4.00 4.80 7.75 13.00 0.25<Pcritical<0.50 0.00 4.25 6.00 6.61 7.75 15.00 0.50<Pcritical<0.75 0.00 3.25 5.00 6.14 9.75 12.00 0.75<Pcritical<1.0 1.00 6.00 8.00 8.64 11.25 21.00

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.

Finally, Pcritical was used to distinguish between patients with VG<15 (91 cases) and 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 due to the fact that we only have 134 VG measurements in the data set.

conclusions

The model presented here using only biomarkers (MR.proADM, Hb, Prot) and simple explanatory variables (BMI, weight, age, sex) achieved a maximum AUC of 0.926 under a one-by-one exclusion approach, and an AUC equal to 0.990 when using all data. This performance is excellent. From a clinical perspective, charts such as those shown in Figure 6 can provide useful information to the clinician.

All references herein are incorporated herein by reference in their entirety. Upon full examination of the complete description of the invention, one skilled in the art will appreciate that the invention may be practiced under a wide and equivalent range of conditions, parameters, and the like, which do not affect the form or scope of the invention or any embodiment thereof.

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Claims (48)

1. A method for determining the status of an extracellular fluid volume in a subject, the method comprising determining, in a sample obtained from said subject, a level of proadrenomedullin (proADM) or a fragment thereof, the method comprising: (a1) comparing said proADM level value or fragment thereof with values of a reference proADM level or fragment thereof from at least one reference subject or population of reference subjects; or (a2) comparing said proADM level value or fragment thereof with a proADM level value or fragment thereof with a value obtained in a previous analysis of the same subject; and (b) identifying the extracellular fluid volume status of said subject based on the comparison in step (a1) or (a2), respectively, where an elevated level of proADM or a fragment thereof in a subject compared to the indicated reference level values of at least 0.5 nmol/l or more in the sample indicates that the subject has a critical extracellular fluid volume status, which is an increase of at least per 4 liters of fluid volume during hospitalization, where reference subjects are healthy subjects. 2. A method for determining fluid balance in a subject, wherein the method includes determining, in a sample obtained from said subject, a level value of proadrenomedullin (proADM) or a fragment thereof, the method comprising: (a1) comparing said proADM level value or fragment thereof with values of a reference proADM level or fragment thereof from at least one reference subject or population of reference subjects; or (a2) comparing said proADM level value or fragment thereof with a proADM level value or fragment thereof obtained in a previous analysis of the same subject; and (b) identifying the fluid balance of said subject based on the comparison in step (a1) or (a2), respectively, where (i) an elevated level of proADM or a fragment thereof in the subject compared to the indicated reference level values of at least 0.5 nmol/L or more in the sample indicates that the subject has a positive fluid balance of an increase of at least per 4 liters of fluid volume during hospitalization; (ii) an identical or similar level of proADM or a fragment thereof in a subject compared to said reference level indicates that said subject has identical or similar fluid balance; and/or (iii) a decreased level of proADM or a fragment thereof in the subject compared to the specified reference level indicates that the specified subject has a negative fluid balance, where reference subjects are healthy subjects. 3. A method for determining the salt balance status of a subject, the method comprising determining in a sample obtained from said subject a level of proadrenomedullin (proADM) or a fragment thereof, the method comprising: (a1) comparing said proADM level value, or fragment thereof, with values of a reference proADM level, or fragment thereof, of at least one reference subject or population of reference subjects; or (a2) comparing said proADM level value or fragment thereof with a proADM level value or fragment thereof obtained in a previous analysis of the same subject; and (b) identifying the salt balance in said subject based on the comparison in step (a1) or (a2), respectively, where (i) an elevated level of proADM or a fragment thereof in the subject compared to the indicated reference level values of at least 0.5 nmol/l or more in the sample indicates that the subject has a positive salt balance, which is an increase in salt by at least 36 g per day; (ii) an identical or similar level of proADM or a fragment thereof of the subject compared to the specified reference level value indicates that the specified subject has an identical or similar salt balance; and/or (iii) a reduced level of proADM or a fragment thereof in the subject compared to the specified reference level value indicates that the specified subject has a negative salt balance, where reference subjects are healthy subjects. 4. A method for determining the status of a globular volume in a subject, the method comprising determining, in a sample obtained from said subject, a level of proadrenomedullin (proADM) or a fragment thereof, the method comprising: (a1) comparing said proADM level value, or fragment thereof, with values of a reference proADM level, or fragment thereof, of at least one reference subject or population of reference subjects; or (a2) comparing said proADM level, or said fragment thereof, with a proADM level, or fragment thereof, obtained in a previous assay of the same subject; and (b) identifying the globular volume status of said subject based on the comparison in step (a1) or (a2), respectively, where (i) an elevated level of proADM or a fragment thereof in the subject compared to the specified reference level value of at least 0.5 nmol/L or more in the sample indicates that the specified subject has a critical globular volume status that is at least least below 20 mg/kg; (ii) an identical or similar level of proADM or a fragment thereof in the subject compared to the specified reference level indicates that the specified subject has a normal globular volume status, where reference subjects are healthy subjects. 5. The method according to any one of the preceding claims, wherein the level of proADM or said fragment thereof, which is 1 nmol/l or more in the sample, indicates that the subject has a critical extracellular fluid volume status, a critical globular volume status, a positive fluid balance and/or positive salt balance. 6. The method according to any one of the preceding claims, wherein said subject suffers from traumatic brain injury, aneurysm, head trauma, multiple traumatic injuries, and/or wherein said subject is a subject after surgery. 7. The method according to any one of the preceding claims, wherein said sample is blood, blood plasma, blood serum or urine. 8. A method according to any one of the preceding claims, wherein said level of proADM or said fragment thereof is determined by immunoassay, said assay being carried out in a homogeneous phase or in a heterogeneous phase. 9. An in vitro method for predicting deterioration in a subject's condition, wherein the extracellular fluid volume status of said subject is determined by the method of claim 1, wherein the disorder or medical condition is selected from the group consisting of severe brain injury (SBT), aneurysmal subarachnoid hemorrhage (SAH), severe trauma without head injury (PT), and post-surgical peritonitis with shock (P). 10. The method of any one of the preceding claims, wherein the proADM fragment is selected from the group consisting of MR-proADM, PAMP, adrenotensin, and mature adrenomedullin. 11. The method of claim 10 wherein the fragment is MR-proADM. 12. The use of a kit to determine the status of a subject's extracellular fluid volume, wherein the use of the kit includes determining the level of proADM or a specified fragment using one or more detection reagents in a sample of the specified subject, wherein one of the antibodies is labeled and the other antibody is bound to a solid phase or may selectively bind to the solid phase. 13. The use of a kit to determine the fluid balance of a subject, while the use of the kit includes determining the level of proADM or a specified fragment using one or more detecting reagents in a sample of the specified subject, while one of the antibodies is labeled and the other antibody is associated with a solid phase or can selectively bind to the solid phase. 14. The use of a kit to determine the salt balance of a subject, while the use of the kit includes determining the level of proADM or a specified fragment using one or more detecting reagents in a sample of the specified subject, while one of the antibodies is labeled and the other antibody is associated with a solid phase or can selectively bind to the solid phase. 15. The use of a kit to determine the globular volume status of a subject, wherein the use of the kit includes determining the level of proADM or a specified fragment using one or more detection reagents in a sample of the specified subject, while one of the antibodies is labeled and the other antibody is associated with a solid phase or may bind selectively to the solid phase. 16. Application according to any one of paragraphs. 12-15, where said fragment is MR-proADM. 17. Application according to any one of paragraphs. 12-15, wherein said detection reagents contain antibodies. 18. Application according to any one of paragraphs. 12-15, in which the first and second antibodies are present in dispersed form in a liquid reaction mixture and in which the first labeling component, which is part of a labeling system based on quenching or enhancing fluorescence or chemiluminescence, binds to the first antibody, and the second labeling component of this the labeling system binds to the second antibody so that after binding of both antibodies to proADM or the specified fragment, a detectable signal is generated that allows the detection of sandwich complexes formed in the measuring solution, optionally, while the specified labeling system contains rare earth cryptates or chelates in combination with a fluorescent or chemiluminescent dye, in particular of the cyanine type.

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