WO2009153706A1

WO2009153706A1 - Biomarkers for sepsis

Info

Publication number: WO2009153706A1
Application number: PCT/IB2009/052461
Authority: WO
Inventors: Chamindie Punyadeera; Wilhelmus F. J. Verhaegh; James D. Schaffer
Original assignee: Koninklijke Philips Electronics N. V.
Priority date: 2008-06-18
Filing date: 2009-06-10
Publication date: 2009-12-23

Abstract

The present invention relates to the identification and use of biomolecules (analytes) or biomarkers related to sepsis. More specifically, the invention relates to the distinction of SIRS and sepsis based on a change in analyte or biomolecule concentration in a test sample of a subject diagnosed with or suspected of suffering from SIRS or sepsis. Moreover, the invention relates to, based a change in analyte or biomolecule concentration is a test sample, assigning a specific sepsis state or condition in terms of sepsis, severe sepsis and sepsis shock; as well as to assigning the expected outcome of the sepsis condition.

Description

BIOMARKERS FOR SEPSIS

FIELD OF THE INVENTION

The present invention relates to the identification and use of biomolecules (analytes) or biomarkers related to sepsis. In various aspects the invention relates to a method, a detection system, a kit for performing an assay and computer readable code for assigning the condition of a subject based on an analysis of at least one biomolecule (analyte).

BACKGROUND OF THE INVENTION

In medicine, systemic inflammatory response syndrome (SIRS) is an inflammatory state of the whole body without a proven source of infection. Sepsis is SIRS with a known or suspected infection. Sepsis can be further divided into sepsis, severe sepsis and septic shock in accordance with the consensus definitions, as defined by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit Care Med. 20:864-874, 1992.

Sepsis is generally diagnosed either by clinical criteria or by culture of microorganisms from the blood of patients. The culturing of micro-organisms is time- and labor consuming.

Statistics on incidents have been collected and have established that sepsis is the 10^th leading cause of death in the USA with over 700,000 cases a year and with mortality rates above 30%, cf. the Society of Critical Care Medicine Consensus Conference reference, as cited above. The incidents continue to increase, with unacceptably high mortality rates, despite the use of a wide variety of therapies and continuing research. During the onset of sepsis, a massive inflammatory reaction takes place involving chemical mediators, such as cytokines and chemokines, and inflammatory cells, such as the polymorphonuclear neutrophils and macrophages. The reasons the body is unable to regulate the inflammatory response are still unknown.

In the published US patent application 2005/0196817 Al the use of biomarkers to determine sepsis in patients is disclosed. Furthermore, it is described how changes in the concentration of biomarkers in the blood can be used to indicate sepsis, risk of sepsis, progression of sepsis, remission of sepsis and risk of mortality.

However, there is still a need for identifying new biomarkers that will improve the ability to discriminate sepsis from SIRS as well as the different conditions in sepsis in terms of the biology rather than the externally observable physiology.

SUMMARY OF THE INVENTION

The present invention relates to the identification and use of biomarkers for the discrimination of sepsis from other causes of SIRS as well as to the identification and use of biomarkers for the assessment of a specific state of sepsis of a human subject. In particular it is an object of the present invention to identify specific biomarkers that have both high sensitivity and specificity in order to identify biomarkers with improved predictability or improved correlation to a specific state of a subject's condition.

According to a first aspect of the present invention there is provided, a method for indicating the condition of a subject to be SIRS or sepsis in a subject diagnosed with or suspected of suffering from SIRS or sepsis, the method comprising: determining a concentration of at least one analyte in a test sample, the at least one analyte being selected from a first subsample being selected from the group consisting of: MMP-I, MMP-2, MMP-7, MMP- 13 and E-selectin; assigning the condition of the subject as being septic if the concentration of at least one of the analytes of the test sample is lower than predefined reference concentrations.

The group of MMP-I, MMP-2, MMP-7, MMP- 13 and E-selectin may be referred to as a marker panel. In a given marker panel the test sample may be analysed with respect to determining the concentration of at least one of the analytes in the marker panel. The test sample may be analysed with respect to any number of the analytes in the marker panel, in any combination, such as at least two, at least three, at least four or even all five analytes may be analysed. Even though the condition of a subject may be determined based on a single analyte, it may, nevertheless, be advantageous to use two, three or even more analytes to assign the condition of the subject, since a high degree of certainty may be achieved when using more markers in the panel. In the context of the present invention, analytes are in the form of one or more biomolecules where a change in concentration level as compared to a reference level indicates a particular disease state. The analytes are thus biomarkers indicating or correlated to the subject's condition in relation to SIRS and sepsis. Reference is made to biomolecules and analytes. In general, the biomolecules can be derived from any available patient material as long as the desired biomolecules are detectable in the test sample. Typically the patient material is in the form of whole blood, serum or as in the present study plasma, however other types of body samples, such as fractioned test samples can be used as well. Any suitable detection method can be used, relevant methods are known to the skilled person.

In embodiments, the concentration of a given analyte is compared to the concentration of a predefined reference concentration. This predefined reference concentration may be derived from healthy individuals or individuals who are ill, but do not suffer from SIRS or sepsis. Reference concentrations may be in the form of ranges or values, so that the comparison is based on whether or not the concentration of an analyte falls within a range, as well as whether or not the analyte concentration is elevated or declined with respect to the reference range or reference value. Thus for each analyte used in the marker panel, a corresponding reference concentration is known or has been determined. In the event the condition of the subject has been assigned as sepsis, the severity of sepsis may further be assigned based on determining the concentration of at least one analyte being selected from a second subsample, the second subsample comprises the analytes: IL-Ia, IP-IO, Fas and TNF-R2. Once a subject develops sepsis as opposed to SIRS, it may be critical to determine the state of the sepsis in order to design the optimal treatment of the subject.

A subject developing a sepsis condition is in a high risk of death. In the event that the condition of the subject has been assigned as sepsis, the condition of the subject may further be assigned as fatal based on determining the concentration of at least one analyte being selected from a third subsample, the third subsample comprises the analytes: MMP-3, MMP-IO, IL-Ia, IP-IO, IL-2R, Fas, TNF-Rl, TNF-R2, RAGE, GM-CSF, IL-Ib and Eotaxin. In embodiments of the present invention, three subsamples of biomarkers have been identified that are relevant to the three clinical aspects, 1) differential diagnosis of SIRS vs. sepsis, 2) sepsis disease severity, and 3) the outcome in terms of survival or death of the subject. The biomarkers are proteins and/or soluable factors that are involved in regulating leukocyte apoptosis as well as proteins that are involved in extracellular matrix remodeling and chemokines.

In dependence upon the intended use of the marker panel, marker panels may be designed which comprise only analytes from a single subsample, from two subsamples or even from all three subsamples. Moreover, from each subsample one or more analytes may be selected in order to tailor the intended use of the marker panel. In accordance with a second aspect of the invention there is provided a detection system for detecting one or more analytes in a test sample and for indicating the condition of a subject to be SIRS or sepsis in a subject diagnosed with or suspected of suffering from SIRS or sepsis, the detection system comprising: a receptor unit for receiving a probe unit having immobilized thereon probe molecules that specifically bind to an analyte; a detector for detecting the presence of resultant binding complexes on the probe unit to determine whether the analyte is present in the test sample; a determination unit for determining a concentration of at least one analyte in the test sample, the at least one analyte being selected from a first subsample being selected from the group consisting of: MMP-I, MMP-2, MMP-7, MMP- 13 and E- selectin; and an assigning unit for assigning the condition of the subject as being septic if the concentration of at least one of the analytes of the test sample is lower than predefined reference concentrations. The second aspect of the present invention may provide a detection system for detecting the presence, and quantity in the form of a concentration, of one or more analytes. In a third aspect, the invention relates to a kit for performing an assay. The kit comprising: a probe unit having immobilized thereon probe molecules that specifically bind to at least one analyte being selected from a first subsample being selected from the group consisting of: MMP-I, MMP-2, MMP-7, MMP- 13 and E-selectin; and instructions for using the probe unit in a method according to claim 1.

In further embodiments of the second and third aspects, the probe unit may be adapted to comprise any of the analytes of the second and third subsamples and likewise the determination unit, the assigning unit and the instructions may be adapted to deal with these analytes as well. A detection system and kit may thereby be provided which can deal with differentiation of SIRS vs. sepsis, sepsis disease severity, and the outcome in terms of survival or death of the subject.

In a fourth aspect, the invention relates to a computer program product, when in use on a computer, to cause a system to perform the method of the first aspect.

The fourth aspect may be implemented into a detection system of the second aspect to operate one or more parts of the system.

In general the various aspects of the invention may be combined and coupled in any way possible within the scope of the invention. These and other aspects, features and/or advantages of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will be described, by way of example only, with reference to the drawings, in which

FIGS. IA-E show graphs of the ranges of the log measurements for analytes of the first subsample;

FIGS. 2A-D show graphs of the ranges of the log measurements for analytes of the second subsample;

FIGS. 3A-C show bi-plots between selected analytes;

FIG. 4 shows computed Pearson correlations for pairs of analytes; FIG. 5 shows Receiver Operating Characteristic (ROC) curves for predicting mortality;

FIG. 6 shows ROC curves for the distinction of SIRS and sepsis;

FIG. 7 schematically illustrates a detection system in accordance with embodiments of the present invention; and

FIG. 8 schematically illustrates steps involved in a method for indicating and differentiating the sepsis conditions.

DETAILED DESCRIPTION OF EMBODIMENTS The present invention is based on statistical analysis of biomarker molecules present in test samples of human subjects.

The invention is based on a study involving 16 patients diagnosed with or suspected of suffering from SIRS or sepsis, of the patients 6 died and 10 survived. In addition, four normal healthy control subjects (no SIRS symptoms) were also included in the study. During the study, each day, each patient's condition was classified as one of the states given in TABLE 1 , using the consensus definitions, as defined by the Society of Critical Care Medicine Consensus Conference, which is cited in the section "Background of the invention".

TABLE 1. The coded disease states. code disease state

0 -

1 bacterimia

2 infection

3 SIRS

4 sepsis

5 severe sepsis

6 septic shock

In the study, plasma samples were collected on a daily basis for 4 - 14 days, yielding a total of 118 samples. The plasma samples of 28 biomolecules were analyzed using the Luminex system obtained from the Luminex Corporation (www. luminexcorp . com) . The list of biomolecules used in the study comprises: 10 cytokines (TNFa, IL-Ib, Eotaxin, IL-13, MIP-Ia, IL-IO, IL-Ia, IP-IO, GM-CSF, and IFNg), 8 matrix metalloproteinases (MMPs -1, -2, -3, -7, -8, -9, -10, and -13), and 10 soluble factors (gpl30, IL-2R, ICAM, E-selectin, Fas, TNF-Rl, TNF-R2, RAGE, VCAM, and MIF). The inter- and intra-assay variation was <10%. Furthermore, the

Sequential Organ Failure Assessment (SOFA) score and the New Simplified Acute Physiology Score (SAPSII) were calculated to evaluate the severity of sepsis.

In an embodiment the invention is related to differentiating between the condition of a subject as being SIRS (code 3) or being sepsis (code 4-6). In further embodiments, the invention is directed to the differentiation of the severity of the sepsis, in that a differentiation between the conditions sepsis, severe sepsis and septic shock is made, thereby assigning a specific state of sepsis to the subject. In an even further embodiment, the invention is directed to the risk of mortality of the subject, that is a prognosis of an outcome of the subject condition is determined, in that the expected outcome is assigned as fatal or non- fatal.

In embodiments, the conditions of the subjects are assigned in accordance with the consensus definitions as defined in TABLE 1. The condition of the subject may be assigned in accordance with any available definition. It is within the skilled person to transform the disclosure of the present invention to any available sepsis definition.

Variance analysis

In a first aspect of the study an analysis of the variance on each biomolecule is made in order to discover differences among the four disease codes (3- SIRS, 4-sepsis, 5-severe sepsis, 6-septic shock). The distributions of measured values were not close to a normal distribution (assumed by ANOVA), so the data were log transformed before the analysis as is normal in the art. The numbers of observations available in each condition varied so much among the patients, that no design was found that permitted the rigorous separation of the patient and day effects. For instance, a patient contributed observations only to the SIRS condition, while another patient contributed only to septic shock. As a consequence each observation was treated as independent, but adjusted to the degrees of freedom of the F test to correct for these effects. Bonferroni correction was used to correct for the multiple tests as 28 biomolecules were tested simultaneously. The F test and the Bonferroni correction are known to the skilled person.

Corresponding p-values after Bonferroni correction are below 0.01. It is noted that the p-values may be biased due to the complications caused by the unbalanced observations from different patients in different conditions. The use of p-values is known to the skilled person.

FIGS. IA-E show graphs of the ranges of the log measurements for the biomolecules MMP-I (FIG. IA), -2 (FIG. IB), -7 (FIG. 1C), -13 (FIG. ID), and E- selectin (FIG. IE) for the coded disease states (0 - control, 3 - SIRS, 4 - sepsis, 5 - severe sepsis, 6 - septic shock), except for FIG. IE that do not show the measurements for code 0 - control. The vertical axes show the log 10 values of the measured concentrations in nanogram per millilitre (ng/ml), except for FIG. IE where the vertical axis refer to measured concentrations in picogram per millilitre (pg/ml).

FIGS. 2A-D show graphs of the ranges of the log measurements for the biomolecules IL-Ia (FIG. 2A), IP-10 (FIG. 2B), TNF-R2 (FIG. 2C) and Fas (FIG. 2D) for the coded disease states. The vertical axes show the log 10 values of the measured concentrations in picogram per millilitre (pg/ml).

The results of the variance analysis showed that:

• the concentration of MMP-I, -2, -7, -13, and E-selectin are significantly lower in septic cases than in SIRS cases.

• the concentration of IP-10, Fas, and TNF-R2 were elevated most in the plasma of patients in septic shock.

In accordance with these findings it is possible to differentiate between the SIRS condition and one of the sepsis conditions of a subject diagnosed with or suspected of suffering from SIRS or sepsis by determining the concentration of at least one of the analytes MMP-I, MMP-2, MMP-7, MMP- 13 and E-selectin in a test sample, and assigning the condition of the subject as being septic if the concentration of at least one of the analytes is lower than predefined reference concentrations.

Moreover, the severity of sepsis may be assigned as septic shock, if the concentration of at least one of the analytes IP-10, Fas or TNF-R2 is higher than a predefined reference concentration. Correlation analysis

In a second aspect of the study a correlation analysis was performed. In the analysis the Pearson correlation was computed, the computation of the Pearson correlation is known to the skilled person. The Pearson correlation was computed between the log-transformed measurements of each of the biomolecules on the one hand, and each of the following eight labels on the other hand:

• SIRS (code 3) vs. sepsis (code 4);

• SIRS (code 3) vs. sepsis, severe sepsis and septic shock (code 4,5,6);

• sepsis (code 4) vs. severe sepsis and septic shock (code 5,6); • sepsis (code 4) vs. septic shock (code 6);

• sepsis (code 4) vs. severe sepsis (code 5);

• the SOFA score;

• the SAPSII score; and

• the outcome (survived/died). As a reference significance level for each of the computed correlations, it was determined what correlations that can be obtained by chance, by randomly permuting each label 10,000 times and computing the correlation to each of the biomolecules. In this way, expected correlations and variances that were used to compute p-values were obtained. In addition, the Bonferroni-correction for multiple testing was again applied. Note again that the obtained p-values are biased, as each sample is treated as independent, whereas they come from a limited number of patients. The correlation analysis resulted in the following findings:

• MMP-I, -2, -7, and -13 plasma concentrations showed to be significantly lower in sepsis (4) as compared to SIRS (3), with absolute correlation values around 0.8.

• In addition to these four, IL-Ia, IP-10, and TNF-R2 were higher in sepsis, severe sepsis, and septic shock (4,5,6) as compared to SIRS (3).

• Fas and TNF-R2 were higher in severe sepsis and septic shock (5,6) as compared to sepsis (4), and also in septic shock (6) as compared to sepsis (4). • MMP-2, MMP-7 and IL-Ib were negatively correlated to the SAPSII score, and MMP-3, IL-Ia, IP-10, Fas, TNF-Rl and TNF-R2 were positively correlated to the SAPS2 score. The correlation of TNF-Rl was quite high (0.78). • Many biomolecules were correlated with the SOFA score, with however slightly lower absolute correlations (up to 0.50). MMP-2, -7, IL-Ib, Eotaxin, GM-CSF and IFNg were negatively correlated, and MMP-3, -8, -10, IL-10, IP- 10, Fas, TNF-Rl and TNF-R2 were positively correlated. • The patients who died from septic complications had elevated levels of

MMP-3, MMP-10, IL-Ia, IP-10, IL-2R, Fas, TNF-Rl, TNF-R2, and RAGE as compared to the surviving patients. In contrast, the former patients showed low levels of GM-CSF, IL-Ib, and Eotaxin.

In accordance with these findings it is possible to further differentiate between the sepsis conditions of a subject diagnosed with sepsis. The sepsis condition may be differentiated by an assignment of a severity of the sepsis. The assignment may be made in accordance with the consensus sepsis conditions: sepsis, severe sepsis and septic shock. The findings show that the severity of sepsis may be assigned as severe sepsis or septic shock, if the concentration of at least one of the analytes Fas or TNF-R2 is higher than predefined reference concentrations.

Even further, it is possible to determine a prognosis of the outcome of the sepsis condition, i.e. to determine or assign a risk of mortality for the subject. The condition of the subject may be assigned as being fatal if the concentration of at least one of the analytes MMP-3, MMP-10, IL-Ia, IP-10, IL-2R, Fas, TNF-Rl, TNF-R2 and RAGE is higher than a predefined reference concentration, or if the concentration of at least one of the analytes GM-CSF, IL-Ib and Eotaxin is lower than a predefined reference concentration.

To get more insight into biomarkers that may serve as a classifier for sepsis vs. SIRS, biplots of MMP- 13 with MIP-Ia, MMP-8 with MMP- 13 and MMP-2 with MMP-8 were made. These bi-plots are shown in FIG. 3A-C. As can be seen in the Figures, the main separators are MMP- 13 in the FIG. A and B, and MMP-2 in FIG. C. This corresponds to the fact that they correlate strongly to the label. MIP-Ia and MMP- 8 seem to only make the separation slightly better.

Correlations between biomolecules

In a third aspect of the study, Pearson correlations were computed between the log measurements for each pair of biomolecules. The results are shown in FIG. 4, where dark colours indicate a correlation of +1 (see e.g. the diagonal), or a correlation of-1. Furthermore, black dots indicate correlations with an absolute value greater than 0.8.

Three groups of strongly correlated compounds appear in the figure: • MMP-I, MMP-2, MMP-7, and MMP- 13;

• TNFa, IL-Ib, Eotaxin, IL-13, and GM-CSF; and

• IL-Ia and IP-IO.

The correlation of these groups support the findings of the variance analysis and the correlation analysis.

ROC curves

In a fourth aspect of the study, Receiver Operating Characteristic (ROC) curves for predicting mortality is illustrated. In FIG. 5, the panel of the biomolecules identified as correlating to survival (MMP-3, MMP-IO, IL-Ia, IP-IO, IL-2R, Fas, TNF- Rl , TNF-R2, RAGE, GM-CSF, IL-Ib and Eotaxin) are used in a simple naive Bayesian classification (i.e. assuming independent features and Gaussian distributions), and doing a train-on-all, test-on-all. A high degree of true positives and a high probability that the used molecules will rank a randomly chosen positive instance higher than a randomly chosen negative one are found. FIG. 5 lists sensitivity vs. specificity for the above- mentioned panel (ref. numeral 50) as well as for the SAPSII 51 and SOFA 52 scores. In FIG. 6 ROC curve for the distinction of SIRS vs. sepsis, and for the four correlated MMPs (-1, -2, -7 and -13) (ref. numerals 60-63) are shown.

It is seen that each of the individual biomolecules exhibits a very high degree of true positives, and that MMP- 13 alone already reaches an AUC of 0.99. In embodiments of the present invention, one or more biomolecules are correlated to the presence of SIRS as well as to the differentiation of sepsis states. In embodiments, thresholds in terms of ranges or values are established in order to compare measured concentrations to reference concentrations. The sensitivity and specificity of a diagnostic and/or prognostic test depends on the definition of what constitutes an abnormal result. That is an abnormal result is based on a proper determinations of reference values as well as the specific biomolecules selected for a given marker panel. ROC curves may be used for selecting proper marker panels and reference values. The biomolecules or analytes identified from the statistical analysis of the present invention may be used for developing improved medical equipment for use in the intensive care unit, or other places, for assisting the clinicians in performing improved diagnosis of SIRS and sepsis. The identified analytes may be used in a probe unit with the ability to specifically bind the analytes in a way so that the analyte species may be identified and so that a concentration of the analyte may be derived.

Such probe unit may contain a biomarker panel comprising probe spots or receptor spots for one or more of the analytes of a first subsample: MMP-I, MMP-2, MMP-7, MMP- 13 and E-selectin; and optionally one or more analytes of a second subsample: IL-Ia, IP-IO, Fas and TNF-R2; and optionally one or more analytes of a third subsample: MMP-3, MMP-IO, IL-Ia, IP-IO, IL-2R, Fas, TNF-Rl, TNF-R2, RAGE, GM-CSF, IL-Ib and Eotaxin. Some of the analytes of the first, second and third subsamples may overlap. In such cases typically only one receptor spot for the analyte in question is provided.

The probe unit may be based on a microarray type probe, e.g. as used in the Luminex system or as used in a chip-based array. It is within the ability of the skilled person to design appropriate probe units.

In a further aspect, the invention relates to a detection system for detecting one or more analytes in a test sample and for indicating the condition of a subject to be SIRS or sepsis in a subject diagnosed with or suspected of suffering from SIRS or sepsis.

FIG. 7 schematically illustrates a detection system 70 comprising a receptor unit 71 for receiving a probe unit 72 having immobilized thereon probe molecules that specifically binds to an analyte; a detector 73 for detecting the presence of resultant binding complexes on the probe unit to determine whether the analyte is present in the sample; a determination unit 74 for determining a concentration of at least one analyte in the test sample; and an assigning unit 75 for assigning the condition of the subject as being septic if the concentration of at least one of the analytes of the test sample is lower than predefined reference concentrations. The assigning unit may be implemented as a, or as part of a, decision support system.

The detection system may e.g. be implemented as a single unit, e.g. as hand held device or as a device which is distributed over a number of devices or device parts. For example the analysis and determination part may be performed at dedicated equipment, whereas the assignment may be performed in connection with a computer, either stand-alone equipment or the resulting concentrations may be inputted into a general purpose computer, such as a laptop.

FIG. 8 schematically illustrates steps involved in a method for indicating the condition of a subject to be SIRS or sepsis. In a first step, a test sample is obtained 80. The test sample may be a plasma sample extracted from a blood sample of a human subject in intensive care diagnosed with or suspected of suffering from SIRS or sepsis. In a further step, a concentration of at least one analyte of the test sample is determined 81. The at least one analyte is selected from a first subsample. The concentration of the at least one analyte is compared to at least one predefined reference concentration, and if the concentration of at least one of the analytes of the test sample is lower than the predefined reference concentration. The condition of the subject is assigned 82 as being septic. Otherwise, new tests 83 may be made, e.g. by repeating the same test at a later stage.

Having determined the condition as septic, the severity of septic may be assigned.

To determine the severity of sepsis, the concentration of the at least one analyte of the test sample is determined 84, the at least one analyte is selected from a second subsample. The at least one analyte is compared to at least one predefined reference concentration. If the concentration of the at least one analytes of the second subsample is higher than the predefined reference concentration. The severity of sepsis is assigned 85 in accordance with the consensus definitions. Having assigned the condition of the subject to be septic 82, it may be of extreme importance to determine the risk of mortality of the subject.

To determine the risk of mortality, the concentration of at least one analyte from the test sample is determined 86, the at least one analyte being selected from a third subsample. The at least one analyte is compared to at least one predefined reference concentration. If the concentration of the at least one of the analytes of a first group of the third subsample is higher than a specific predefined reference concentration or if the at least one of the analytes of a second group of the third subsample is lower than a specific predefined reference concentration. The condition of the subject is assigned 87 as fatal.

Kits for use in detection assays are also provided. The kits at least include a probe unit having immobilized thereon probe molecules that specifically binds to at least one analyte being selected from a first subsample being selected from the group consisting of: MMP-I, MMP-2, MMP-7, MMP- 13 and E-selectin. The kits may further include one or more additional components to be used when carrying out a detection assay, such as one or more substrates, sample preparation reagents, buffers, labels, etc. The instructions for use may be provided in paper format, be recorded on a suitable recording medium, be provided in the form of directions as how to access the instructions via a remote source, e.g. the Internet, etc.

The invention can be implemented in any suitable form including hardware, software, firmware or any combination of these. The invention or some features of the invention can be implemented as computer software running on one or more data processors and/or digital signal processors. The elements and components of an embodiment of the invention may be physically, functionally and logically implemented in any suitable way. Indeed, the functionality may be implemented in a single unit, in a plurality of units or as part of other functional units. As such, the invention may be implemented in a single unit, or may be physically and functionally distributed between different units and processors.

Although the present invention has been described in connection with the specified embodiments, it is not intended to be limited to the specific form set forth herein. Rather, the scope of the present invention is limited only by the accompanying claims. In the claims, the term "comprising" does not exclude the presence of other elements or steps. Additionally, although individual features may be included in different claims, these may possibly be advantageously combined, and the inclusion in different claims does not imply that a combination of features is not feasible and/or advantageous. In addition, singular references do not exclude a plurality. Thus, references to "a", "an", "first", "second" etc. do not preclude a plurality. Furthermore, reference signs in the claims shall not be construed as limiting the scope.

Claims

CLAIMS:

1. A method for indicating the condition of a subject to be SIRS or sepsis in a subject diagnosed with or suspected of suffering from SIRS or sepsis, the method comprising: determining (81) a concentration of at least one analyte in a test sample, the at least one analyte being selected from a first subsample being selected from the group consisting of: MMP-I, MMP-2, MMP-7, MMP- 13 and E-selectin; assigning (82) the condition of the subject as being septic if the concentration of at least one of the analytes of the test sample is lower than predefined reference concentrations.

2. The method according to claim 1, wherein the severity of sepsis is further assigned, and wherein the test sample further comprising at least one analyte being selected from a second subsample being selected from the group: IL-Ia, IP-IO, Fas and TNF-R2; in the event the condition of the subject has been assigned as sepsis, assigning (85) a severity of sepsis in accordance with sepsis, severe sepsis or septic shock, if the concentration of at least one of the analytes of the second subsample is higher than a predefined reference concentration.

3. The method according to claim 2, wherein the severity of sepsis is assigned as septic shock, if the concentration of at least one of the analytes IP-IO, Fas or TNF-R2 is higher than a predefined reference concentration.

4. The method according to claim 2, wherein the severity of sepsis is assigned as severe sepsis or septic shock, if the concentration of at least one of the analytes Fas or TNF-R2 is higher than a predefined reference concentration.

5. The method according to claim 1, wherein the risk of mortality is further assigned, and wherein the test sample further comprising at least one analyte being selected from a third subsample being selected from the group: MMP-3, MMP-IO, IL- Ia, IP-IO, IL-2R, Fas, TNF-Rl , TNF-R2, RAGE, GM-CSF, IL-Ib, and Eotaxin; in the event the condition of the subject has been assigned as sepsis, assigning (87) the condition of the subject as being fatal if the concentration of at least one of the analytes: MMP-3, MMP-IO, IL-Ia, IP-IO, IL-2R, Fas, TNF-Rl, TNF-R2 of the test sample is determined 81 or RAGE, is higher than a predefined reference concentration or if the concentration of at least one of the analytes: GM-CSF, IL-Ib or Eotaxin, is lower than a predefined reference concentration.

6. An detection system (70) for detecting one or more analytes in a test sample and for indicating the condition of a subject to be SIRS or sepsis in a subject diagnosed with or suspected of suffering from SIRS or sepsis, the detection system comprising: a receptor unit (71) for receiving a probe unit (72) having immobilized thereon probe molecules that specifically bind to an analyte; a detector (73) for detecting the presence of resultant binding complexes on the probe unit to determine whether the analyte is present in the test sample; a determination unit (74) for determining a concentration of at least one analyte in a test sample, the at least one analyte being selected from a first subsample being selected from the group consisting of: MMP-I, MMP-2, MMP-7, MMP- 13 and E- selectin; and an assigning unit (75) for assigning the condition of the subject as being septic if the concentration of at least one of the analytes of the test sample is lower than predefined reference concentrations.

7. A kit for performing an assay, the kit comprising: a probe unit having immobilized thereon probe molecules that specifically binds to at least one analyte being selected from a first subsample being selected from the group consisting of: MMP-I, MMP-2, MMP-7, MMP- 13 and E-selectin; and instructions for using the probe unit in a method according to claim 1.

8. A computer program product, when in use on a computer, to cause a system to perform method of claim 1.