Abstract:
A method of estimating sepsis risk in an individual with infection comprises a step of assaying a biological sample from the individual for an IL-2 or IL-7 mRNA value, and correlating the mRNA value with sepsis risk. The IL-2 and IL-7 mRNA values are quantified by absolute quantification of mRNA copy number, wherein the copy numbers are normalised to a house keeping gene and corrected against a calibration curve for serial dilutions of the IL-2 and IL-7 cDNA. The method generally involves a step of assaying a biological sample from the individual for IL-2 and/or IL-7 mRNA values, optionally in combination with mRNA values for other cytokines, and correlating a sum or difference of the values with sepsis risk using a regression analysis curve against outcome.

Description:
INTRODUCTION 
       [0001]    The invention relates to a method of estimating the risk of an individual with infection developing severe sepsis. The invention also relates to a method of discriminating between a patient with infection who is unlikely to develop severe sepsis, and a patient with infection who is likely to develop severe sepsis. 
       STATEMENT OF INVENTION 
       [0002]    According to the invention, there is provided a method of estimating sepsis risk in an individual comprising a step of assaying a biological sample from the individual for an IL-2 or IL-7 mRNA value, and correlating the mRNA value with sepsis risk. 
         [0003]    As used herein, the term “sepsis risk” should be understood to mean the risk that an individual will develop sepsis. Generally, the individual will have an infection, in which case the term should be taken to mean the risk that the individual will develop sepsis in response to infection. In other cases, the method may be employed to monitor an at-risk patient to determine whether their risk of developing sepsis is changing. This may be carried out, for example, in the situation where an individual has received an at-risk prognosis, and is being treated to avoid development of sepsis, wherein the method of the invention is employed to monitor sepsis risk. 
         [0004]    In this specification, the term “infection” should be taken to include any disease or illness which is induced or caused by the presence of organisms in tissue, bodily fluid or cavity. In this specification, the term “sepsis” or “severe sepsis” are used interchangeably, and should be taken to mean the occurrence of an overwhelming illness with failure of bodily organ systems, which may be remote from the site of infection. These failing organ systems include but are not limited to the respiratory system, the cardiovascular system, the renal system, the hepatic, coagulation systems and the central nervous system. This definition is in accordance with consensus definition of severe sepsis and sepsis (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,  1992. 20(6): p. 864-74.) 
         [0005]    The term “biological sample” may be any sample obtained from an individual such as, for example, blood, serum, saliva, urine, cerebrospinal fluid, tissue, cells, etc. In a preferred embodiment of the invention, the sample will be a lymphocyte preparation such as lymphocytes from a peripheral blood sample, especially lymphocytes derived from the buffy coat layer of a peripheral blood sample (which is rich in T-cells and monocytes). In many cases, the individual will be a person with an established infection, or a person at risk of developing an infection, such as a patient who is immunocompromised due to disease, surgery or other factors. In other cases, the individual may be a person known to have an infection, or severe sepsis, and who is under going a therapeutic treatment regime, in which case the method of the invention may be employed to monitor the effectiveness of the treatment. In most cases, the individual will be human, however the use of the invention with higher mammals is not excluded. 
         [0006]    Suitably, the IL-2 and IL-7 mRNA value is quantified by absolute quantification of mRNA copy number (or a function of copy number), wherein the copy numbers are normalised to a house keeping gene. PCR is generally employed, especially quantitative PCR (i.e. real time PCR), in which the PCR process is typically calibrated against serial dilutions of a known quantity of the cDNA of the respective cytokine. The mRNA value may be a normalised mRNA copy number, or a function of a normalised mRNA copy number, for example the Log 10 of the normalised mRNA copy number. The housekeeper gene employed for normalisation is selected generally selected from β-actin and GAPDH, although other housekeeping genes will be known to the person skilled in the art. Generally, the values for mRNA are normalised against a housekeeping gene and corrected against a calibration curve for serial dilutions of the respective cytokine cDNA. 
         [0007]    The present application is based on the surprising finding that IL-2 and IL-7 mRNA levels vary between individuals with infection, individuals with sepsis, and healthy individuals and, as such, may be used as prognostic biomarkers to predict the risk of development of sepsis. Thus, IL-2 and/or IL-7 levels may be used as prognostic variables of sepsis, optionally in combination with other cytokine mRNA levels. Cytokine levels may be represented in a number of different ways, for example mRNA copy number, or a function of the mRNA copy number, for example, Log 10 of the mRNA copy number, and the mRNA copy number for a given cytokine will vary depending on the housekeeping gene employed to normalise the copy number. Moreover, the present invention encompasses a number of different ways of correlating the mRNA values with sepsis risk, including: correlating IL-2 or IL-7 absolute mRNA copy number with risk of sepsis; correlating the sum of IL-2 and IL-7 absolute mRNA copy number (or a function of copy number) with risk of sepsis; correlating a sum of at least two pro-inflammatory cytokine mRNA values (including at least one of IL-2 and IL-7, and preferably at least one of IL-23 and Interferon-γ) with risk of sepsis; and correlating the difference between (a) a mRNA value of at least one pro-inflammatory cytokine (including at least one of IL-2 and IL-7) and (b) a mRNA value of at least one anti-inflammatory cytokine, with risk of sepsis. A number of different algorithms are provided herein for correlating IL-2 and/or IL-7 mRNA values, optionally in combination with mRNA values for other cytokines, with risk of sepsis. 
         [0008]    In one embodiment, the method involves a step of assaying a biological sample from the individual for IL-2 and IL-7 mRNA values, and correlating a sum of the values with sepsis risk. Typically, the mRNA values are provided in the form of a function of the (normalised) mRNA copy number, for example the Log 10 of the normalised copy number. Thus, the IL-2 mRNA value is represented by the Log 10 of the IL-2 mRNA copy number, and the IL-7 mRNA value is represented by the Log 10 of the IL-7 mRNA copy number, wherein the sum of the mRNA values is correlated with a numerical scale, for example 3 to 8.5, to provide sepsis risk, wherein 8.5 typically represents low sepsis risk and 3.5 typically represents high sepsis risk. 
         [0009]    In another embodiment, the method involves a step of assaying a biological sample from the individual for a mRNA value of at least two pro-inflammatory cytokines including at least one of IL-2 and IL-7, and optionally at least one of IL-23 and Interferon-γ (INF), and correlating a sum of the mRNA values with sepsis risk. Typically, the step of correlating the sum of mRNA values with sepsis risk comprises the step of correlating the sum using a logistic regression analysis curve against outcome. Suitably, the mRNA value is a normalised mRNA copy number or a function of the normalised mRNA copy number, for example, the Log 10 of the mRNA copy number. 
         [0010]    Preferably, the at least two pro-inflammatory cytokines are selected from the group consisting of: IL-2 and IL-23; IL-2 and INF; IL-7 and IL-23; IL-7 and INF; IL-2, IL-7 and IL-23; IL-2, IL-7 and INF; IL-2, IL-23, and INF; and IL-7, IL-23, and INF. 
         [0011]    Thus, for example, the at least two pro-inflammatory cytokines may be IL-2 and IL-23, and wherein the sum of the Log 10 of the mRNA values is correlated with a numerical scale, suitably of 5 to 9 or 4 to 8, to provide sepsis risk, in which 9 typically represents low sepsis risk and 5 typically represents high sepsis risk. 
         [0012]    Alternatively, the at least two pro-inflammatory cytokines may be IL-2 and INF, and wherein the sum of the Log 10 of the mRNA values is correlated with a numerical scale, suitably of 2.5 to 8 to 3.5 to 7, to provide sepsis risk, in which 8 typically represents low sepsis risk and 2.5 typically represents high sepsis risk. 
         [0013]    Alternatively, the at least two pro-inflammatory cytokines may be IL-7 and IL-23, and wherein the sum of the Log 10 of the mRNA values is correlated with a numerical scale, suitably of 6 to 10.5 or 7 to 10, to provide sepsis risk, wherein 10.5 typically represents low sepsis risk and 6 typically represents high sepsis risk. 
         [0014]    Alternatively, the at least two pro-inflammatory cytokines may be IL-7 and INF, and wherein the sum of the Log 10 of the mRNA values is correlated with a numerical scale, typically of 3.5 to 8.5, to provide sepsis risk, wherein 8.5 typically represents low sepsis risk and 3.5 typically represents high sepsis risk. 
         [0015]    In a further embodiment, the method of the invention involves a step of assaying a biological sample from the individual for a mRNA value of at least one pro-inflammatory cytokine, including at least one of IL-2 and IL-7, and at least one anti-inflammatory cytokine, preferably (but not necessarily) selected from IL-10 and IL-27, calculating the difference between the pro-inflammatory mRNA value and the anti-inflammatory mRNA value, and correlating the difference with sepsis risk. The difference is the mRNA value of the pro-inflammatory cytokine minus the mRNA value of the ant-inflammatory cytokine. Where there is more than one pro-inflammatory cytokine, a composite mRNA value is provided (i.e. mRNA value of one cytokine plus the mRNA value of the other cytokine(s). Likewise, where there is more than one anti-inflammatory cytokine, a composite mRNA value is provided (i.e. mRNA value of one cytokine plus the mRNA value of the other cytokine(s). 
         [0016]    Thus, two or more pro-inflammatory cytokines are typically employed, wherein the mRNA value for each of the two or more pro-inflammatory cytokines are summated to provide a composite pro-inflammatory mRNA value. Likewise, where two or more anti-inflammatory cytokines are employed, the mRNA values for each of the two or more anti-inflammatory cytokines are summated to provide a composite anti-inflammatory mRNA value. 
         [0017]    Preferably, the two more pro-inflammatory cytokines includes at least one of IL-2 or IL-7, and one or more of IL-23 and INF. 
         [0018]    Suitably, the step of correlating the sum of mRNA values with sepsis risk comprises the step of correlating the sum using a logistic regression analysis curve against outcome. Typically, the mRNA value is a normalised mRNA copy number or a function of the normalised mRNA copy number, such as a Log 10 of the mRNA copy number. 
         [0019]    The pro-inflammatory and anti-inflammatory combination is suitably selected from the group consisting of: IL-2 and IL-10; IL-2 and IL-27; IL-2, IL-23, INF, and IL-10; IL-2, IL-23, INF, and IL-27; IL-7 and IL-10; IL-7 and IL-27; IL-7, IL-23, INF, and IL-10; IL-7, IL-23, INF, and IL-27. 
         [0020]    Thus, for example, the pro-inflammatory cytokine may comprises IL-2 and the anti-inflammatory cytokine may comprise IL-10, and wherein the difference of the Log 10 of the pro-inflammatory and anti-inflammatory mRNA values is correlated with a numerical numerical scale, typically of −3.5 to 1.5 or −2.5 to 1, to provide sepsis risk, in which 1.5 typically represents low sepsis risk and −3.5 typically represents high sepsis risk. Alternatively, the pro-inflammatory cytokine may comprise IL-7 and the anti-inflammatory cytokine may comprise IL-10, and wherein the difference of the Log 10 of the pro-inflammatory and anti-inflammatory mRNA values is correlated with a numerical scale, typically of −1 to 2.5 or 0 to 1.5, to provide sepsis risk, in which 2.5 typically represents low sepsis risk and −1 typically represents high sepsis risk. 
         [0021]    Alternatively, the pro-inflammatory cytokines may comprise IL-2, IL-23, and INF, and the anti-inflammatory cytokine may comprise IL-10, and wherein the difference of the Log 10 of the pro-inflammatory and anti-inflammatory mRNA values is correlated with a numerical numerical scale, typically of 3.5 to 9.5 or 4 to 8, to provide sepsis risk, in which 9.5 typically represents low sepsis risk and 3.5 typically represents high sepsis risk. For the avoidance of doubt, the pro-inflammatory mRNA value is the sum of the Log 10 of the IL-2, IL-23 and INF mRNA copy numbers, which copy numbers are typically normalised against β-actin. 
         [0022]    Alternatively, the pro-inflammatory cytokines may comprise IL-7, IL-23, and INF, and the anti-inflammatory cytokine may comprise IL-10, and wherein the difference of the Log 10 of the pro-inflammatory and anti-inflammatory mRNA values is correlated with a numerical scale, typically of 4.5 to 10.5, to provide sepsis risk, in which 10.5 typically represents low sepsis risk and 4.5 typically represents high sepsis risk. For the avoidance of doubt, the pro-inflammatory mRNA value is the sum of the Log 10 of the IL-2, IL-23 and INF mRNA copy numbers. 
         [0023]    Alternatively, the pro-inflammatory cytokines may comprise IL-7, IL-23, and INF, and the anti-inflammatory cytokines comprise IL-10 and Il-27, and wherein the difference of the Log 10 of the pro-inflammatory and anti-inflammatory mRNA values is correlated with a numerical scale, typically of 1.5 to 7, to provide sepsis risk, in which 7 typically represents low sepsis risk and 1.5 typically represents high sepsis risk. For the avoidance of doubt, the pro-inflammatory mRNA value is the sum of the Log 10 of the IL-2, IL-23 and INF mRNA copy numbers, and the anti-inflammatory mRNA value is the sum of the Log 10 of the IL-7 and IL-10 mRNA copy numbers. 
         [0024]    In a preferred embodiment, the invention relates to a method of discrimination between a patient with infection who is unlikely to develop severe sepsis and a patient with infection who is likely to develop severe sepsis, the method comprising a step of assaying a biological sample from the individual for an expression level of IL-2 mRNA and/or IL-7 mRNA, wherein the level of expression of IL-2 mRNA and/or IL-7 mRNA correlates with a likelihood of the patient developing severe sepsis. Thus, for example, when a high level of IL-2 or IL-7 mRNA expression is identified, this correlates with a likelihood of the patient not developing severe sepsis. Alternatively, where a low level of IL-2 or IL-7 mRNA expression is determined, this correlates with likelihood that the patient will develop severe sepsis. Assessing the risk that a patient may develop severe sepsis is important as it will help a clinician decide whether the patient needs to be treated in an ICU, and whether aggressive antibiotic or other therapies are required. 
         [0025]    In one embodiment of the invention, the method of the invention involves a step of assaying a biological sample from the individual for an expression level of IL-2 and IL-7 mRNA, and correlating the determined levels with a likelihood of the patient developing/not developing severe sepsis. 
         [0026]    The levels of IL-2 and IL-7 mRNA present in biological samples, especially in patients with infection and severe sepsis, tend to be very low. Accordingly, the method employed in assaying a biological sample for the levels of these cytokines is required to be extremely sensitive. Cytokine mRNA values are quantified by absolute quantification of mRNA copy number, wherein the copy numbers are ideally normalised to a house keeping gene such as, for example, GAPDH or b-Actin, and corrected against a calibration curve for serial dilutions of the respective cytokine cDNA. Other suitable housekeeper genes will be known to those skilled in the art. Using this method, and β-actin as the housekeeping gene, a high level of IL-2 mRNA expression correlates with IL-2 copy number of at least 570 copy numbers of mRNA, and a low level of IL-2 expression correlates with an IL-2 copy number of less than 172 copy numbers of mRNA. Likewise, a high level of IL-7 expression correlates with IL-7 copy number of at least 1675 copy numbers of mRNA, and a low level of IL-2 expression correlates with an IL-7 copy number of less than 283 copy numbers of mRNA. 
         [0027]    In a preferred embodiment of the invention, the methods of the invention comprise a scoring system in which a patient is assigned a score of 1, 2 or 3 depending on whether the level of expression of IL-2 or IL-7 is high, medium or low. In the case of IL-2, a score of 1 correlates with a mRNA copy number of at least 570, a score of 2 correlates with a mRNA copy number of from 570 and 172, and a score of 3 correlates with a mRNA copy number of less than 172. A score of 3 correlates with a likelihood of developing severe sepsis, and a score of 1 correlates with a likelihood of not developing severe sepsis. 
         [0028]    In an analysis of the relation between IL-2 derived risk category, patients in group 3 with low IL-2 mRNA copy numbers were 3.25 times more likely to have severe sepsis rather than infection when compared with patients in group 1 and 2. 
         [0029]    In the case of IL-7, a score of 1 correlates with a mRNA copy number of at least 1675, a score of 2 correlates with a mRNA copy number of from 1675 and 283, and a score of 3 correlates with a mRNA copy number of less than 283. A score of 3 correlates with a likelihood of developing severe sepsis, and a score of 1 correlates with a likelihood of not developing severe sepsis. 
         [0030]    In an analysis of the relation between IL-7 derived risk category, patients in groups 2 and 3 were 3.5 times more likely to have severe sepsis rather than infection when compared with patients in group 1. 
         [0031]    Preferably, the scoring system involves assaying a patient for IL-2 and IL-7 mRNA levels, assigning a score of 1, 2 or 3 to the patient in respect of each of IL-2 and IL-7, and summating the score to provide a composite score for the patient of between 2 and 6. Typically, a score of 4, 5 or 6 indicates a likelihood that the patient has, or will develop, severe sepsis. Suitably, a score of 5 or 6 indicates a strong likelihood that the patient has, or will develop, severe sepsis. Typically, a score of 2 or 3 indicates a likelihood that the patient will not develop severe sepsis. Suitably, a score of 2 indicates a strong likelihood that the patient will not develop severe sepsis. 
         [0032]    With respect to the probability of developing severe sepsis, these scoring systems can be used to determine relative risk, or an odds ratio. Thus when patients with the combined IL-2 and IL-7 scores of 2 and 3 are considered as a low risk group, with patients with a score of 4 as an intermediate risk group, and patients with a score of 5 or 6 as a high risk group, there is an obvious relation between risk group and response to infection. In this analysis of patients with infection and patients with severe sepsis, intermediate risk patients were 8 times more likely to have severe sepsis than low risk patients, high risk patients were 13.7 times more likely to develop severe sepsis than intermediate risk patients, and high risk patients were 110 times more likely to develop severe sepsis than low risk patients. 
         [0033]    It will be appreciated that the values assigned above are informed by the choice of housekeeping gene against which the copy numbers are corrected, and that therefore a different housekeeping gene would likely result in a different set of values for determining “high” and “low” expression levels. It will be appreciated therefore that alternative methods of determining absolute quantification of mRNA copy numbers that employ different housekeeping genes likewise forms part of the invention. 
         [0034]    As mentioned above, the methods of the invention may be employed to estimate risk of an individual with infection developing severe sepsis, and to stratify patients with infection into those that are unlikely to develop severe sepsis and those that are likely to develop severe sepsis. As such, the invention also relates to a method of treating or preventing severe sepsis in a patient comprising a step of determining whether the patient is likely to develop severe sepsis in response to infection according to the methods of the invention, and where it is determined that the patient is likely to develop severe sepsis, then treating the patient to treat or prevent severe sepsis. 
         [0035]    The invention also relates to a method of monitoring the efficacy of a therapeutic or prophylactic treatment of severe sepsis, the method comprising a step of assaying a biological sample from the individual for an expression level of IL-2 mRNA and/or IL-7 mRNA, and correlating the level of expression of IL-2 mRNA and/or IL-7 mRNA with sepsis risk. Thus, for example, when the levels of the mRNA for each cytokine increase (i.e. from low to high, or from low to medium), this would be indicative of the treatment having a positive effect. Likewise, if initially high levels of the mRNA for each cytokine decrease during a course of treatment, then the likelihood would be that the treatment is not working. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0036]      FIG. 1  shows a restriction map and multiple cloning sites for pDNR-LIB vector 
           [0037]      FIG. 2  is a map of the pDNR-LIB vector MCS, multiple cloning site. The IL2 cDNA insert replaces the stuffer fragment. Unique restriction sites are shown in bold or in colour. 
           [0038]      FIG. 3  shows a restriction map and multiple cloning sites for pCMV-SPORT6 vector 
           [0039]      FIG. 4  shows a DNA gel Single Digest gel with Ecor I and Xba I for IL2 and IL7 respectively—1% agarose DNA Gel—Lane 1 contains a 1 kb ladder. Lane 2 contains linear plasmid IL2 DNA following single restriction enzyme digestion with EcorI. Lane 3 contains linear plasmid IL7 DNA following single restriction enzyme with XbaI. 
           [0040]      FIG. 5  shows a DNA gel Double Digest gel with Ecor I and HINDIII for IL2 and EcorI and XbaI IL7 respectively—1% agarose DNA Gel—Lane 1 contains a 1 kb ladder. Lane 2 contains linear plasmid IL2 DNA following double restriction enzyme digestion with EcorI and HINDIII. Lane 3 contains linear plasmid IL7 DNA following double restriction enzyme digestion with XbaI and EcorI. 
           [0041]      FIG. 6  shows an absorbance Spectrum of plasmid DNA 
           [0042]      FIG. 7  shows a Standard Curve for IL2. 
           [0043]      FIG. 8  shows a Standard Curve for IL7. 
           [0044]      FIG. 9  shows a Standard Curve for the house-keeping gene β-Actin. 
           [0045]      FIG. 10 : logistic Fit of Response to infection By Combined IL-2 and IL-7 mRNA Log base 10 
           [0046]      FIG. 11A  analysis of Summated Score A By Groups (IL-7, IL-23, INF and IL-10) 
           [0047]      FIG. 11B : logistic Fit of Response to Infection By Summated Score A, with 0 representing sepsis and 1 representing infection. 
           [0048]      FIG. 12A  analysis of Summated Score B By Groups (IL-2, IL-23, INF and IL-10) 
           [0049]      FIG. 12B  logistic Fit of Response to Infection By Summated Score B, with 0 representing sepsis and 1 representing infection. 
           [0050]      FIG. 13A  analysis of Summated Score C By Groups (IL-7, IL-23, INF, IL-10 and IL-27) 
           [0051]      FIG. 13B  logistic Fit of Response to Infection By Summated Score C, with 0 representing sepsis and 1 representing infection. 
           [0052]      FIG. 13C  ROC Curve for Patient response to infection by Summated Score C. ROC value=0.88. 
           [0053]      FIG. 14A  analysis of Summated Score E By Groups (IL-2 and IL-10) 
           [0054]      FIG. 14B  logistic Fit of Response to Infection By Summated Score E, with 0 representing sepsis and 1 representing infection. 
           [0055]      FIG. 15A  analysis of Summated Score F By Groups (IL-2 and INF) 
           [0056]      FIG. 15B  logistic Fit of Response to Infection By Summated Score F, with 0 representing sepsis and 1 representing infection 
           [0057]      FIG. 16A  analysis of Summated Score G By Groups (IL-2 and IL-23) 
           [0058]      FIG. 16B  logistic Fit of Response to Infection By Summated Score G, with 0 representing sepsis and 1 representing infection 
           [0059]      FIG. 17A  analysis of Summated Score H By Groups (IL-7 and IL-10) 
           [0060]      FIG. 17B  logistic Fit of Response to Infection By Summated Score H, with 0 representing sepsis and 1 representing infection 
           [0061]      FIG. 18A  analysis of Summated Score I By Groups (IL-7 and INF) 
           [0062]      FIG. 18B  logistic Fit of Response to Infection By Summated Score I, with 0 representing sepsis and 1 representing infection. 
           [0063]      FIG. 19A  analysis of Summated Score J By Groups (IL-7 and IL-23) 
           [0064]      FIG. 19B  logistic Fit of Response to Infection By Summated Score J, with 0 representing sepsis and 1 representing infection. 
           [0065]      FIG. 20  is a graph showing the IL-2 mRNA copy numbers at day 1 for the three patients groups. 
           [0066]      FIG. 21  is a graph showing the IL-7 mRNA copy numbers at day 1 for the three patients groups. 
           [0067]      FIG. 22  is a graph showing the IL-2 mRNA copy numbers at day 1 for the infection and severe sepsis patients groups. 
           [0068]      FIG. 23  is a graph showing the IL-7 mRNA copy numbers at day 1 for the infection and severe sepsis patients groups. 
           [0069]      FIG. 24  is a graph showing the IL-2 mRNA copy numbers at day 1 for the control and infection patient groups. 
           [0070]      FIG. 25  is a graph illustrating the scoring system of the invention in which patients are scored as 1, 2 or 3 according to their expression levels of IL-2, and the scores are correlated with the severe sepsis and infection patient groups. 
           [0071]      FIG. 26  is a graph illustrating the scoring system of the invention in which patients are scored as 1, 2 or 3 according to their expression levels of IL-2, and the scores are correlated with the control, severe sepsis and infection patient groups. 
           [0072]      FIG. 27  is a graph illustrating the scoring system of the invention in which patients are scored as 1, 2 or 3 according to their expression levels of IL-7, and the scores are correlated with the control, severe sepsis and infection patient groups. 
           [0073]      FIG. 28  is a graph illustrating the scoring system of the invention in which patients are scored as 1, 2 or 3 according to their expression levels of IL-7, and the scores are correlated with the severe sepsis and infection patient groups. 
           [0074]      FIG. 29  is a graph illustrating the scoring system of the invention in which the scores for IL-2 and IL-7 are summated to provide a composite score of from 2 to 6 for the patient, and the scores are correlated with the control, infection and severe sepsis groups. 
           [0075]      FIG. 30  is a graph illustrating the scoring system of the invention in which the scores for IL-2 and IL-7 are summated to provide a composite score of from 2 to 6 for the patient, and the scores are correlated with the infection and severe sepsis groups. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     1. Characterisation of Patients Groups 
       [0076]    The data contained herein is based on two distinct cohort of patients, each cohort comprising three patient groups, namely a control group of healthy volunteers, a group of patients with bacteraemia who did not develop severe sepsis and a group of patient with obvious infection who developed severe sepsis. The Bacteraemic patients that were recruited had been hospitalised with infection, and then grew gram negative organisms in blood cultures, but did not have any evidence of organ failure. The septic group of patients had obvious infection, such as pneumonia, peritonitis or cellulitis, and developed an overwhelming illness in response to infection, with the majority developing septic shock and multiple organ failure, and requiring admission to intensive care for support of failing organ systems. 
         [0077]    Patients with obvious cause of immune compromise, such as HIV/AIDS, neutropoenia, and or high steroid dosage, were excluded from this study. 
       2. Description of Methods of Determining Copy Number 
       [0078]    The method described below relate to determining mRNA copy number (or a function of copy number) for IL-2 and IL-7. The methods may likewise be applied for the determination of copy numbers for IL-10, IL-23, IL-27, Interferon-γ, and other cytokine (WO2007/060647) 
         [0079]    The DNA standards for quantitative real-time per may be prepared by either cloning a PCR product that encompasses the quantified amplicon. This can be prepared by PCR from a cDNA population containing the target mRNA. Alternatively, as described below, DNA standards may be prepared by culturing  E. Coli  with the enclosed vector containing the relevant gene sequence, in this case IL-2 and IL-7. The ensuing DNA harvested from the plasmid is purified, IL-2 and IL-7 sequence verification and quantified, and finally the volume of plasmid DNA corresponding to copy numbers of target nucleic acid sequences is determined. 
       2.1.1 Preparation of the IL2 Standard 
       [0080]    IL2 plasmid was purchased from Open Biosystems (MHS1011-98053730 Human MGC Verified FL cDNA IRAU). It consisted of an 894 bp cDNA clone inserted into a 4.161 kb pDNR-LIB vector. This is illustrated in  FIGS. 1 and 2 . 
       2.1.2 Preparation of the IL7 Standard 
       [0081]    IL7 plasmid was purchased from Open Biosystems (MHS1010-9205095 Human MGC Verified FL cDNA IRAT). It consisted of a 2125 bp cDNA clone inserted into a 4396 bpDNR-LIB vector. This is illustrated in  FIG. 3 . 
       2.1.3 Plasmid Culture Conditions 
       [0082]    An  E. coli  culture harbouring the pDNR-LIB vector containing the IL-2 gene was streaked onto a chloramphenicol (25 μg/ml) containing LB agar plate and incubated at 37° C. overnight. A similar  E. coli  culture harbouring the pCMV-SPORT6 vector containing the IL-7 gene was streaked onto an ampicillin (100 μg/ml). A single colony was isolated from each plate and streaked onto another plate. A well-isolated colony from this second plate was then used to inoculate a liquid L-broth culture grown overnight at 37° C. for each plasmid. These steps ensure the isolation of a clone of a single bacterium. 
       2.1.4 Purification of DNA 
       [0083]    The Fast Ion Plasmid Midi kit Fast Ion™ (Cat No YP125/YPM10), was used to purify plasmid DNA from 100 ml overnight cultures of  E. coli  according to the manufacturers&#39; instructions. Bacteria were lysed and the cleared lysate is passed through a cation-exchange column, which binds the re-natured plasmid DNA. The column with bound DNA was washed repeatedly and the DNA is eluted in a high-salt buffer. The DNA is then further purified and desalted by precipitation with isopropanol and resuspended in ddH 2 O. Purified plasmid DNA was visualized on a 1% agarose gel as shown in  FIG. 4 . 
       2.1.5 Agarose Gel Electrophoresis 
       [0084]    DNA samples were visualised following separation on a 1% agarose gel. Briefly, for a 1% gel, agarose (1 g) was added to 100 ml of 0.5×TBE buffer (44.5 mM tris borate, pH 8.3, 1 mM E DTA) and heated to 100° C. to dissolve the agarose. Ethidium bromide was added to a final concentration of 1 μg/ml and the molten gel was poured into a gel mould and allowed to set. DNA samples were prepared by adding an appropriate volume of 5× sample loading buffer (25 mM tris pH 7.6, 30% (v/v) glycerol, 0.125% (w/v) bromophenol blue) and these samples were electrophoresed through the gel at 135 V for 45 min in 0.5×TBE buffer. The separated DNA fragments were photographed while illuminated under UV light ( FIG. 5 ). 
       2.1.6 Restriction Endonuclease Digestion of DNA 
       [0085]    All restriction digests were carried out using enzymes supplied by New England Biolabs (NEB) according to the manufacturers&#39; instructions. Briefly, 0.1-2 μg of purified DNA was incubated with 10-20 U of restriction enzyme in the appropriate NEB buffer for 2 h at the appropriate temperature. Digests with double enzymes were carried out in the recommended double digest buffer, in which all enzymes had 100% activity ( FIG. 2.4 ). 
       2.1.7 Clone Verification 
       [0086]    The IL2 and IL7 clone was end sequenced by MWG-Biotech, Ebersberg, Germany. This was verified against the GeneBank sequence for IL2 and IL7 using BLAST. 
       2.1.8 Quantification of DNA 
       [0087]    DNA was quantified and qualified using the Nanodrop® ND 8000 (220-750 nm) full spectrum spectrophotometer. Briefly a 1 μl sample of DNA was placed on the measuring pedestal. The pedestal is actually the end of a fiber optic cable (receiving fibers). A second set of fiber optic cables (the source fibers) are then brought in contact with the liquid sample, causing the liquid to bridge the gaps between the fiber optic ends. A pulsed xenon flash lamp provides the light source and a spectrometer using a linear CCD array is used to analyse the light that passes through the samples. Absorbance measurements, measure any molecules absorbing at a specific wavelength. Nucleotides, RNA, ssDNA and dsDNA all absorb at 260 nm and contribute to the overall absorbance. The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA. A ratio of ˜1.8 is accepted as “pure” for DNA; a ratio ˜2.0 is accepted as “pure” for RNA. If the ratio is lower it indicates the presence of contaminants. An additional measure of nucleic acid incorporates absorbance at 230 nm, with the 260/230 ratio is used as a secondary measure of nucleic acid purity. The 260/230 values for “pure” nucleic acid are expected to be in the range of 2.0-2.2. The absorbance spectrum in  FIG. 2.5  indicates a DNA concentration of 3250.1 ng/μl, with a 260-280 ratio of 1.86 and a 260-230 ratio of 2.16. The absorbance spectrum for IL2 and IL7 are presented in table 1 and  FIG. 6 . 
         [0000]                                                                        TABLE 1                   Results from absorbance spectrum                A260   A280           Conc       Sample   10 mm path   10 mm path   260/280   260/230   ng/μl                    IL2   10.314   5.548   1.86   2.05   515.7       IL7   67.852   37.271   1.83   2.08   3365.6                    
2.1.9 Determining the Volume of Plasmid DNA Corresponding to Copy Numbers of Target Nucleic Acid Sequences, i.e. Creating a Standard Curve with a Plasmid DNA Template.
 
         [0088]    To prepare a standard curve from in which both, the cloned IL2 and IL7 sequence is present in 10*8 to 10*0 copies correspondingly. This standard curve is utilised to calculate absolute copy numbers of IL2 and IL7 in patient samples. Our quantitative real time per reactions are set up such that 1.5 μl of plasmid DNA is pipetted into each QRT-PCR reaction. 
       2.1.9.1 IL2 Standard Curve 
       [0089]    The stock of IL2 plasmid DNA was determined to be 515.7 ng/μl by spectrophotometric analysis. The vector size for pDNR-LIB is 4161 bp. The IL2 cloned insert is 814 bp. The size of the vector+insert=4975 bp. 
         [0090]    First we calculate the mass of a single plasmid molecule. The size of the entire plasmid (plasmid+insert) is used in this calculation using DNA Mass Formula. 
         [0000]    m=(n)(1.096×10 −21  g/bp);
 
m=mass
 
n=plasmid size (bp)
 
         [0091]    In the case of IL2: 
         [0000]    m=4975 bp (1.096×10 −21 ) g/bp
 
m=5.453×10 −18  g=mass of a single plasmid molecule.
 
         [0092]    We then calculate the mass of plasmid containing the copy numbers of interest, in this example 10 8 : 
       E.g. 
       [0093]      Copy number (CN) of interest×mass of single plasmid=mass of plasmid DNA needed for Copy Number of interest
 
         [0000]      (10*8 CN)×(5.453×10 −18  g)=5.453×10 −10  g
 
         [0094]    Therefore, the mass of plasmid DNA needed for 10*8 copy numbers=5.453×10 −10  g We then calculate the concentrations of plasmid DNA needed to achieve the copy numbers of interest (table 2). 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 TABLE 2 
               
               
                   
                   
               
               
                   
                 Copy Number of Interest 
                 Mass of Plasmid DNA (g) 
               
               
                   
                   
               
             
             
               
                   
                 10*8 
                 5.453 × 10 −10   
               
               
                   
                 10*7 
                 5.453 × 10 −11   
               
               
                   
                 10*6 
                 5.453 × 10 −12   
               
               
                   
                 10*5 
                 5.453 × 10 −13   
               
               
                   
                 10*4 
                 5.453 × 10 −14   
               
               
                   
                 10*3 
                 5.453 × 10 −15   
               
               
                   
                 10*2 
                 5.453 × 10 −16   
               
               
                   
                 10*1 
                 5.453 × 10 −17   
               
               
                   
                   
               
             
          
         
       
     
         [0095]    We next calculate the concentrations of plasmid DNA needed to achieve the copy numbers of interest, dividing the mass needed for respective copy number of interest by the volume pipetted into each reaction (1.5 μl) see table 3. 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                   
                   
                   
                 Final Conc 
               
               
                 Copy Number 
                 Mass of Plasmid 
                 Volume used in 
                 of plasmid 
               
               
                 of Interest 
                 DNA needed (g) 
                 each QRT-PCR μl 
                 DNA(g/μl) 
               
               
                   
               
             
             
               
                 10*9 
                 5.453 × 10 −9    
                 1.5 
                 3.635 × 10 −9    
               
               
                 10*8 
                 5.453 × 10 −10   
                 1.5 
                 3.635 × 10 −10   
               
               
                 10*7 
                 5.453 × 10 −11   
                 1.5 
                 3.635 × 10 −11   
               
               
                 10*6 
                 5.453 × 10 −12   
                 1.5 
                 3.635 × 10 −12   
               
               
                 10*5 
                 5.453 × 10 −13   
                 1.5 
                 3.635 × 10 −13   
               
               
                 10*4 
                 5.453 × 10 −14   
                 1.5 
                 3.635 × 10 −14   
               
               
                 10*3 
                 5.453 × 10 −15   
                 1.5 
                 3.635 × 10 −15   
               
               
                 10*2 
                 5.453 × 10 −16   
                 1.5 
                 3.635 × 10 −16   
               
               
                 10*1 
                 5.453 × 10 −17   
                 1.5 
                 3.635 × 10 −17   
               
               
                   
               
             
          
         
       
     
         [0096]    The final step is to prepare a serial dilution of the plasmid DNA. The cloned sequences are highly concentrated in purified plasmid DNA stocks. A series of serial dilutions are performed if necessary to achieve a working stock of plasmid DNA for quantitative RT PCR applications. 
         [0097]    Once the plasmid is at a workable concentration, the following formula is used to calculate the volume needed to prepare the 10*8 copy standard dilution. (in case of IL2 dilution III) 
         [0000]      C 1 V 1 =C 2 V 2    
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                   
                 Volume of 
                   
                   
                   
                   
               
               
                   
                 Source of 
                 Initial Conc 
                 plasmid DNA 
                 Vol of 
                 Final 
                   
                 Resulting 
               
               
                   
                 Plasmid DNA 
                 (g/μl) 
                 (μl) 
                 Diluent 
                 Volume 
                 Final Conc 
                 Copy 
               
               
                 Dilution 
                 for dilution 
                 (C 1 ) 
                 (V 1 ) 
                 (μl) 
                 (μl) (V 2 ) 
                 (g/μl) (C 2 ) 
                 Numbers 
               
               
                   
               
             
             
               
                 I 
                 Stock 
                 51.57 × 10 −9   
                 10    
                 990    
                 1000 
                 5.157 × 10 −9   
                 N/A 
               
               
                 II 
                 Dil I 
                 5.157 × 10 −9   
                 70.486 
                 29.514 
                  100 
                 3.635 × 10 −9   
                 10* 9   
               
               
                 III 
                 Dil II 
                 3.635 × 10 −9   
                 10    
                 90    
                  100 
                 3.635 × 10 −10   
                 10* 8   
               
               
                 IV 
                 Dil III 
                 3.635 × 10 −10   
                 10    
                 90    
                  100 
                 3.635 × 10 −11   
                 10* 7   
               
               
                 V 
                 Dil IV 
                 3.635 × 10 −11   
                 10    
                 90    
                  100 
                 3.635 × 10 −12   
                 10* 6   
               
               
                 VI 
                 Dil V 
                 3.635 × 10 −12   
                 10    
                 90    
                  100 
                 3.635 × 10 −13   
                 10* 5   
               
               
                 VII 
                 Dil VI 
                 3.635 × 10 −13   
                 10    
                 90    
                  100 
                 3.635 × 10 −14   
                 10* 4   
               
               
                 VIII 
                 Dil VII 
                 3.635 × 10 −14   
                 10    
                 90    
                  100 
                 3.635 × 10 −15   
                 10* 3   
               
               
                 IX 
                 Dil VIII 
                 3.635 × 10 −15   
                 10    
                 90    
                  100 
                 3.635 × 10 −16   
                 10* 2   
               
               
                 X 
                 Dil IX 
                 3.6315 × 10 −16   
                 10    
                 90    
                  100 
                 3.635 × 10 −17   
                 10* 1   
               
               
                   
               
             
          
         
       
     
         [0098]    The dilutent used in these dilutions was sterile TE buffer (10 mM Tris HCL, 1 mM EDTA pH 8.0 with 10 μg/ml double stranded herring DNA (sigma). 
         [0099]    Dilutions II to X were used for quantitative PCR application. 
       2.1.9.2 IL 7 Standard Curve 
       [0100]    The stock of IL7 plasmid DNA was determined to be 3365.6 ng/μl by spectrophotometric analysis. The vector size for pCMV-SPORT6 is 4396 bp. The IL2 cloned insert is 2125 bp. The size of the vector+insert=6521 bp. 
         [0101]    First we calculate the mass of a single plasmid molecule. The size of the entire plasmid (plasmid+insert) is used in this calculation using DNA Mass Formula. 
         [0000]    m=(n)(1.096×10 −21  g/bp);
 
m=mass
 
n=plasmid size (bp)
 
         [0102]    In the case of IL7: 
         [0000]    m=6521 bp (1.096×10 −21 ) g/bp
 
m=7.147×10 18  g=mass of a single plasmid molecule.
 
         [0103]    We then calculate the mass of plasmid containing the copy numbers of interest, in this example 10 8 : 
         [0000]      Copy number (CN) of interest×mass of single plasmid=mass of plasmid DNA needed for Copy Number of interest
 
         [0000]      (10*8 CN)×(7.147×10 18  g)=7.147×10 −10  g
 
         [0104]    Therefore, the mass of plasmid DNA needed for 10*8 copy numbers=7.147×10 −10  g 
         [0105]    We then calculate the concentrations of plasmid DNA needed to achieve the copy numbers of interest (table 5). 
         [0000]    
       
         
               
               
               
             
           
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 Copy Number of Interest 
                 Mass of Plasmid DNA (g) 
               
               
                   
                   
               
             
             
               
                   
                 10*8 
                 7.147 × 10 −10   
               
               
                   
                 10*7 
                 7.147 × 10 −11   
               
               
                   
                 10*6 
                 7.147 × 10 −12   
               
               
                   
                 10*5 
                 7.147 × 10 −13   
               
               
                   
                 10*4 
                 7.147 × 10 −14   
               
               
                   
                 10*3 
                 7.147 × 10 −15   
               
               
                   
                 10*2 
                 7.147 × 10 −16   
               
               
                   
                 10*1 
                 7.147 × 10 −17   
               
               
                   
                   
               
             
          
         
       
     
         [0106]    We next calculate the concentrations of plasmid DNA needed to achieve the copy numbers of interest, dividing the mass needed for respective copy number of interest by the volume pipetted into each reaction (1.5 μl) see table 6. 
         [0000]    
       
         
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                   
                   
                   
                 Final Conc 
               
               
                 Copy Number 
                 Mass of Plasmid 
                 Volume used in 
                 of plasmid 
               
               
                 of Interest 
                 DNA needed (g) 
                 each QRT-PCR μl 
                 DNA(g/μl) 
               
               
                   
               
             
             
               
                 10*9 
                 7.147 × 10 −9    
                 1.5 
                 4.765 × 10 −9    
               
               
                 10*8 
                 7.147 × 10 −10   
                 1.5 
                 4.765 × 10 −10   
               
               
                 10*7 
                 7.147 × 10 −11   
                 1.5 
                 4.765 × 10 −11   
               
               
                 10*6 
                 7.147 × 10 −12   
                 1.5 
                 4.765 × 10 −12   
               
               
                 10*5 
                 7.147 × 10 −13   
                 1.5 
                 4.765 × 10 −13   
               
               
                 10*4 
                 7.147 × 10 −14   
                 1.5 
                 4.765 × 10 −14   
               
               
                 10*3 
                 7.147 × 10 −15   
                 1.5 
                 4.765 × 10 −15   
               
               
                 10*2 
                 7.147 × 10 −16   
                 1.5 
                 4.765 × 10 −16   
               
               
                 10*1 
                 7.147 × 10 −17   
                 1.5 
                 4.765 × 10 −17   
               
               
                   
               
             
          
         
       
     
         [0107]    The final step is to prepare a serial dilution of the plasmid DNA. The cloned sequences are highly concentrated in purified plasmid DNA stocks. A series of serial dilutions are performed if necessary to achieve a working stock of plasmid DNA for quantitative RT PCR applications. 
         [0108]    Once the plasmid is at a workable concentration, the following formula is used to calculate the volume needed to prepare the 10*8 copy standard dilution. (table 7) 
         [0000]      C 1 V 1 =C 2 V 2    
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
               
                   
               
               
                   
                 Source of 
                   
                 Volume of 
                   
                   
                   
                   
               
               
                   
                 Plasmid 
                 Initial Conc 
                 plasmid 
                 Vol of 
                 Final 
                 Final Conc 
                 Resulting 
               
               
                   
                 DNA for 
                 (μg/l) 
                 DNA 
                 Diluent 
                 Volume 
                 (μg/l) 
                 Copy 
               
               
                 Dilution 
                 dilution 
                 (C 1 ) 
                 (μl) (V 1 ) 
                 (μl) 
                 (μl) (V 2 ) 
                 (C 2 ) 
                 Numbers 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 I 
                 Stock 
                 33.659 × 10 −7    
                 14.156 
                 85.844 
                 100 
                 4.765 × 10 −7   
                 10* 11   
               
               
                 II 
                 Dil I 
                 4.765 × 10 −7   
                 10    
                 90    
                 100 
                 4.765 × 10 −8   
                 10* 10   
               
               
                 III 
                 Dil II 
                 4.765 × 10 −8   
                 10    
                 90    
                 100 
                 4.765 × 10 −9   
                 10* 9   
               
               
                 IV 
                 Dil III 
                 4.765 × 10 −9   
                 10    
                 90    
                 100 
                 4.765 × 10 −10   
                 10* 8   
               
               
                 V 
                 Dil IV 
                 4.765 × 10 −10   
                 10    
                 90    
                 100 
                 4.765 × 10 −11   
                 10* 7   
               
               
                 VI 
                 Dil V 
                 4.765 × 10 −11   
                 10    
                 90    
                 100 
                 4.765 × 10 −12   
                 10* 6   
               
               
                 VII 
                 Dil VI 
                 4.765 × 10 −12   
                 10    
                 90    
                 100 
                 4.765 × 10 −13   
                 10* 5   
               
               
                 VIII 
                 Dil VII 
                 4.765 × 10 −13   
                 10    
                 90    
                 100 
                 4.765 × 10 −14   
                 10* 4   
               
               
                 IX 
                 Dil VIII 
                 4.765 × 10 −14   
                 10    
                 90    
                 100 
                 4.765 × 10 −15   
                 10* 3   
               
               
                 X 
                 Dil IX 
                 4.765 × 10 −15   
                 10    
                 90    
                 100 
                 4.765 × 10 −16   
                 10* 2   
               
               
                 XI 
                 Dil X 
                 4.765 × 10 −16   
                 10    
                 90    
                 100 
                 4.765 × 10 −17   
                 10* 1   
               
               
                   
               
             
          
         
       
     
         [0109]    The dilutent used in these dilutions was sterile TE buffer (10 mM Tris HCL, 1 mM EDTA pH 8.0 with 10 μg/ml double stranded herring DNA (sigma). 
         [0110]    Dilutions IV to XI were used for quantitative PCR application. 
       2.2.0 Primers and Probes for IL2 and IL7 
       [0111]    All primers and probes for IL2 and IL7 were synthesized at Applied Biosystems (Foster City, Calif.). Both IL2 and IL7 were obtained as a precustomised primer and probe mix. (Taqman®Gene Expression Assays ID Hs00174114_ml and for IL7 is Taqman® Gene Expression Assays ID Hs00174202_ml). 
         [0112]    Expression of IL2 and IL7 in patient samples were normalised to 10*7 copy numbers of the house-keeping gene β-Actin. A house-keeping gene is a reference gene that acts as an internal standard or loading control. The ideal house-keeping gene should have various features: (1) The standard gene should have the same copy numbers in all cells and (2) It should be expressed in all cells. Commonly used housekeeping standards Glyceraldehyde-3-phosphate dehydrogenase mRNA, β-Actin mRNA, MHC (major histocompatibility complex I) mRNA, Cyclophilin mRNA, mRNAs for certain ribosomal proteins e.g. RPLPO (ribosomal protein, large P0), This is also known as 36B4, P0, L10E, RPPO, PRLP0, 60S acidic ribosomal protein P0, ribosomal protein L10, Arbp or acidic ribosomal phosphoprotein P0. However, the perfect housekeeping gene does not exist, therefore we used β-Actin as it has been validated for the tissue (PBMCs)—it does not change significantly in expression when PBMCs are subjected to the experimental variables used in these experiments. The β-Actin primers and probe were designed and customised as per Stordeur at al. (Stordeur et al, 2002). 
         [0113]    The probe stock for β-Actin (40 pmol/L) was stored at −20° C. and a working dilution of 4 pmol/L, with 200 nM probe used per 20 μL QRT-PCR reaction. 300 nM of forward and reverse primers were used per 20 μL QRT-PCR reaction. 
         [0114]    Sequences for β-Actin (J. Immune. Methods. 2002 Apr. 1; 262 (1-2):299) 
         [0000]    
       
         
               
               
             
           
               
                 Forward Primer 
                 GGATGCAGAAGGAGATCACTG 
               
               
                   
               
               
                 Reverse Primer 
                 CGATCCACACGGAGTACTTG 
               
               
                   
               
               
                 Probe 
                 6Fam-CCCTGGCACCCAGCACAATG-Tamra-p 
               
             
          
         
       
     
       2.2.1 Standard Curve IL2 and IL7 and the House-Keeping Gene β-Actin. (FIGS. 7,  8  and  9 ) 
       [0115]    The slope of the standard curve can be used to determine the exponential amplification a efficiency of the QRT-PCR reaction. A slope between −3.2 and −3.6 is an acceptable efficiency. The ideal QRT-PCR reaction has an efficiency of 1.0092 and amplification of 2.0092, this corresponds to a slope of −3.3. 
       3. Brief Description of Results 
       [0116]    The results from a first cohort of patients are provided in Examples 1 to 12 below. 
       Example 1 (to 12) 
       [0117]    In an additional data set of patients with sepsis, patients with bacterial infection who did not develop sepsis and a control group, IL-2 and IL-7 mRNA levels in peripheral blood leukocytes were reduced in patients with sepsis compared to control patients and patients with bacterial infection. 
         [0118]    Thus IL-2 mRNA copy numbers are lesser in sepsis than with infection, and are lesser in infection than in control patients. 
         [0000]    IL-2 mRNA Copy Numbers 
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Minimum 
                 10% 
                 25% 
                 Median 
                 75% 
                 90% 
                 Maximum 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 122.1559 
                 211.7746 
                 306.6765 
                 656.4726 
                 1152.576 
                 1679.923 
                 1682.266 
               
               
                 Infection 
                 16.71462 
                 51.78462 
                 144.302 
                 199.7228 
                 379.9402 
                 768.6838 
                 2072.529 
               
               
                 Sepsis 
                 16.78441 
                 29.46672 
                 43.98022 
                 104.2151 
                 178.094 
                 581.2909 
                 1551.134 
               
               
                   
               
             
          
         
       
     
       Wilcoxon/Kruskal-Wallis Tests (Rank Sums) 
       [0119]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 (Mean − 
               
               
                 Level 
                 Count 
                 Score Sum 
                 Score Mean 
                 Mean0)/Std0 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 20 
                 1615.00 
                 80.7500 
                 4.525 
               
               
                 Infection 
                 50 
                 2685.00 
                 53.7000 
                 0.221 
               
               
                 Sepsis 
                 35 
                 1265.00 
                 36.1429 
                 −4.007 
               
               
                   
               
             
          
         
       
     
       1-Way Test, ChiSquare Approximation 
       [0120]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 ChiSquare 
                 DF 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                 27.3547 
                 2 
                 &lt;.0001 
               
               
                   
               
             
          
         
       
     
         [0121]    Similarly IL-7 Copy numbers are less in patients with sepsis than infection, and are less in patients with infection than a control group. 
       IL-7 Copy Numbers 
       [0122]      
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Minimum 
                 10% 
                 25% 
                 Median 
                 75% 
                 90% 
                 Maximum 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 2430.134 
                 3640.706 
                 5317.928 
                 6518.609 
                 11323.74 
                 13046.01 
                 20571.62 
               
               
                 Infection 
                 27.93651 
                 2364.416 
                 3029.921 
                 5447.709 
                 8947.922 
                 13438.1 
                 211279.9 
               
               
                 Sepsis 
                 377.714 
                 1087.902 
                 1621.671 
                 3035.628 
                 5689.765 
                 7978.581 
                 17673.52 
               
               
                   
               
             
          
         
       
     
       Wilcoxon/Kruskal-Wallis Tests (Rank Sums) 
       [0123]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 (Mean − 
               
               
                 Level 
                 Count 
                 Score Sum 
                 Score Mean 
                 Mean0)/Std0 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 19 
                 1462.00 
                 76.9474 
                 2.806 
               
               
                 Infection 
                 53 
                 3373.00 
                 63.6415 
                 1.846 
               
               
                 Sepsis 
                 42 
                 1720.00 
                 40.9524 
                 −4.080 
               
               
                   
               
             
          
         
       
     
       1-Way Test, ChiSquare Approximation 
       [0124]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 ChiSquare 
                 DF 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                 18.9341 
                 2 
                 &lt;.0001 
               
               
                   
               
             
          
         
       
     
       Example 2 
       [0125]    The mRNA levels of these two cytokines, IL-2 and IL-7, can be combined to produce a risk score for sepsis. This score can be derived from the sum of the log to base 10 of the mRNA copy numbers. 
         [0126]    Thus for the three groups this data shows that the combines cytokine copy numbers are reduced in patients with sepsis compared with infection, and in turn are reduced in patients with infection compared with sepsis. 
       Combined IL-2 and IL-7 (Log Base 10 Copy Numbers). 
       [0127]      
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Number 
                 Mean 
                 Std Error 
                 Lower 95% 
                 Upper 95% 
               
               
                   
               
             
             
               
                 Control 
                 19 
                 6.59995 
                 0.16547 
                 6.2711 
                 6.9288 
               
               
                 Infection 
                 49 
                 6.07458 
                 0.10304 
                 5.8698 
                 6.2794 
               
               
                 Sepsis 
                 22 
                 5.60276 
                 0.15378 
                 5.2971 
                 5.9084 
               
               
                   
               
             
          
         
       
     
       Analysis of Variance 
       [0128]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Sum of 
                 Mean 
                   
                   
               
               
                 Source 
                 DF 
                 Squares 
                 Square 
                 F Ratio 
                 Prob &gt; F 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Groups 
                 2 
                 10.140035 
                 5.07002 
                 9.7455 
                 0.0002 
               
               
                 Error 
                 87 
                 45.261060 
                 0.52024 
               
               
                 C. Total 
                 89 
                 55.401095 
               
               
                   
               
             
          
         
       
     
       Means Comparisons 
     Comparisons for all Pairs Using Tukey-Kramer HSD 
       [0129]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Abs(Dif) − LSD 
                 Control 
                 Infection 
                 Sepsis 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 −0.55800 
                 0.06055 
                 0.45855 
               
               
                   
                 Infection 
                 0.06055 
                 −0.34747 
                 0.03044 
               
               
                   
                 Sepsis 
                 0.45855 
                 0.03044 
                 −0.51856 
               
               
                   
                   
               
             
          
         
       
     
         [0130]    Positive values show pairs of means that are significantly different. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Level 
                   
                   
                 Mean 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 A 
                   
                   
                 6.5999473 
               
               
                   
                 Infection 
                   
                 B 
                   
                 6.0745817 
               
               
                   
                 Sepsis 
                   
                   
                 C 
                 5.6027562 
               
               
                   
                   
               
             
          
         
       
     
         [0131]    Levels not connected by same letter are significantly different. 
       Example 3 
       [0132]    Furthermore from this scoring system a risk of developing sepsis can be derived by comparing infection and sepsis groups in a logistic regression analysis ( FIG. 10 ). 
       Whole Model Test 
       [0133]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Model 
                 -LogLikelihood 
                 DF 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
             
          
           
               
                 Difference 
                 2.756830 
                 1 
                 5.513659 
                 0.0189 
               
               
                 Full 
                 41.191313 
               
               
                 Reduced 
                 43.948143 
               
               
                   
               
             
          
           
               
                   
                 RSquare (U) 
                 0.0627 
               
               
                   
                 Observations (or Sum Wgts) 
                 71 
               
               
                   
                   
               
             
          
         
       
     
       Parameter Estimates 
       [0134]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Prob &gt; 
                   
               
               
                 Term 
                 Estimate 
                 Std Error 
                 ChiSquare 
                 ChiSq 
                 Odds Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Intercept 
                 −3.9083567 
                 2.1455026 
                 3.32 
                 0.0685 
                 . 
               
               
                 Combined 
                 0.80533408 
                 0.367884 
                 4.79 
                 0.0286 
                 60.19321 
               
               
                 IL-2 and 
               
               
                 IL-7 
               
               
                   
               
             
          
         
       
     
         [0135]    Over the range of this scale the risk for sepsis changes by 60.2 fold. That is the risk for sepsis is 60.2 times greater in a patient with the least score compared with a patient with the greatest score. The risk for sepsis increases by 2.24 fold for each unit change in the score. 
       Example 4 
     Score A 
       [0136]    However the mRNA copy numbers of the cytokines IL-2 and IL-7 can be incorporated, individually into scoring systems based on mRNA copy numbers of the cytokines IL-23, IL-10 and Interferon Gamma. 
         [0137]    In these scoring systems the log base 10 of the cytokine mRNA copy numbers are calculated. These values are the actual corrected read out from the PCR runs. 
         [0138]    In this algorithm the values for IL-2 or IL-7 are added to the value for IL-23 and Interferon gamma. This result is then subtracted from the value for IL-10. 
         [0139]    In this manner the values for pro inflammatory cytokines, IL-2, IL-7, IL-23 and interferon gamma are normalised to the value for an anti inflammatory cytokine such as IL-10. 
       Thus Consider the Combination of IL-7, IL-23, Interferon Gamma, and IL-10. Referred to as Summated Score A. 
       [0140]    This score is greater in controls than in patients with infection, and greater in patients with infection than in patients with Sepsis ( FIG. 11A ). 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Number 
                 Mean 
                 Std Error 
                 Lower 95% 
                 Upper 95% 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 18 
                 9.65626 
                 0.22228 
                 9.2156 
                 10.097 
               
               
                 Infection 
                 50 
                 8.49983 
                 0.13337 
                 8.2354 
                 8.764 
               
               
                 Sepsis 
                 41 
                 6.90635 
                 0.14728 
                 6.6143 
                 7.198 
               
               
                   
               
             
          
         
       
     
       Analysis of Variance 
       [0141]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Sum of 
                 Mean 
                   
                   
               
               
                 Source 
                 DF 
                 Squares 
                 Square 
                 F Ratio 
                 Prob &gt; F 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Groups 
                 2 
                 109.99705 
                 54.9985 
                 61.8388 
                 &lt;.0001 
               
               
                 Error 
                 106 
                 94.27484 
                 0.8894 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Level 
                   
                   
                 Mean 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 A 
                   
                   
                 9.6562613 
               
               
                   
                 Infection 
                   
                 B 
                   
                 8.4998250 
               
               
                   
                 Sepsis 
                   
                   
                 C 
                 6.9063481 
               
               
                   
                   
               
             
          
         
       
     
         [0142]    Levels not connected by same letter are significantly different. 
         [0143]    The risk for developing sepsis in patients with infection can be estimated with this score by using logistic regression analysis. 
         [0144]    In this scoring system, as the rated score increases the risk for sepsis is reduced ( FIG. 11B ). 
       Whole Model Test 
       [0145]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Model 
                 -LogLikelihood 
                 DF 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
               
                 Difference 
                 22.683792 
                 1 
                 45.36758 
                 &lt;.0001 
               
               
                 Full 
                 39.946818 
               
               
                 Reduced 
                 62.630610 
               
               
                   
               
             
          
           
               
                   
                 RSquare (U) 
                 0.3622 
               
               
                   
                 Observations (or Sum Wgts) 
                 91 
               
               
                   
                 Converged by Gradient 
               
               
                   
                   
               
             
          
         
       
     
       Parameter Estimates 
       [0146]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Term 
                 Estimate 
                 Std Error 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Intercept 
                 12.240006 
                 2.5639555 
                 22.79 
                 &lt;.0001 
               
               
                 Summated 
                 −1.6049533 
                 0.3279525 
                 23.95 
                 &lt;.0001 
               
               
                 Score B 
               
               
                   
               
             
          
         
       
     
         [0147]    The Odds ratio per unit change is as follows. 
         [0148]    For unit increase in the score the risk for sepsis increases by 0.2 fold, that is it decreases 5 fold. 
         [0149]    Over the range of the scoring system the overall risk of sepsis increases 5000 fold form highest score with the least risk, to lowest score with the greatest risk. 
       Example 5 
     Score B 
       [0150]    Now consider summated scoring system which includes IL-2, IL-23, Interferon Gamma and IL-10. 
         [0151]    This summated score, score B is clearly different in the three groups ( FIG. 12A ). 
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Number 
                 Mean 
                 Std Error 
                 Lower 95% 
                 Upper 95% 
               
               
                   
               
             
             
               
                 Control 
                 19 
                 8.55940 
                 0.22431 
                 8.1141 
                 9.0047 
               
               
                 Infection 
                 49 
                 7.10814 
                 0.13968 
                 6.8309 
                 7.3854 
               
               
                 Sepsis 
                 31 
                 5.67185 
                 0.17561 
                 5.3233 
                 6.0204 
               
               
                   
               
             
          
         
       
     
       Analysis of Variance 
       [0152]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Sum of 
                 Mean 
                   
                   
               
               
                 Source 
                 DF 
                 Squares 
                 Square 
                 F Ratio 
                 Prob &gt; F 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Groups 
                 2 
                 101.06520 
                 50.5326 
                 52.8590 
                 &lt;.0001 
               
               
                 Error 
                 96 
                 91.77488 
                 0.9560 
               
               
                 C. Total 
                 98 
                 192.84007 
               
               
                   
               
             
          
         
       
     
         [0153]    The risk for developing sepsis in patients with infection can be estimated with this score by using logistic regression analysis. 
         [0154]    In this scoring system, as the rated score increases the risk for sepsis is reduced (Figure Example 12B). 
       Whole Model Test 
       [0155]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Model 
                 -LogLikelihood 
                 DF 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
               
                 Difference 
                 15.591304 
                 1 
                 31.18261 
                 &lt;.0001 
               
               
                 Full 
                 37.818028 
               
               
                 Reduced 
                 53.409333 
               
               
                   
               
             
          
           
               
                   
                 RSquare (U) 
                 0.2919 
               
               
                   
                 Observations (or Sum Wgts) 
                 80 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Term 
                 Estimate 
                 Std Error 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Intercept 
                 8.32746821 
                 2.0157838 
                 17.07 
                 &lt;.0001 
               
               
                 Summated 
                 −1.3685629 
                 0.3136444 
                 19.04 
                 &lt;.0001 
               
               
                 score B 
               
               
                   
               
             
          
         
       
     
         [0156]    In this system as the score decreased by each unit, the risk for sepsis increased 4 fold. 
         [0157]    Over the range of the score, as the score decreased, the risk of sepsis increased by 2500 fold, i.e. when the risk associated with the highest score was compared with the risk associated with the lowest score. 
       Example 6 
     Score C 
       [0158]    Additional cytokines can be added to this algorithm. The best discrimination between patients with sepsis and infection is with a combination of IL-7, IL-23, Interferon gamma on one hand and IL-10 and IL-27. 
         [0159]    This score, Summated Score C is different in control, Infection and Sepsis groups (Figure Example 13A). 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Rsquare 
                 0.575831 
               
               
                   
                 Adj Rsquare 
                 0.567752 
               
               
                   
                 Root Mean Square Error 
                 1.023602 
               
               
                   
                 Mean of Response 
                 5.078865 
               
               
                   
                 Observations (or Sum Wgts) 
                 108 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Sum of 
                 Mean 
                   
                   
               
               
                 Source 
                 DF 
                 Squares 
                 Square 
                 F Ratio 
                 Prob &gt; F 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Groups 
                 2 
                 149.35089 
                 74.6754 
                 71.2714 
                 &lt;.0001 
               
               
                 Error 
                 105 
                 110.01495 
                 1.0478 
               
               
                 C. Total 
                 107 
                 259.36584 
               
               
                   
               
             
          
         
       
     
       Means for Oneway Anova 
       [0160]      
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Number 
                 Mean 
                 Std Error 
                 Lower 95% 
                 Upper 95% 
               
               
                   
               
             
             
               
                 Control 
                 17 
                 7.11602 
                 0.24826 
                 6.6238 
                 7.6083 
               
               
                 Infection 
                 50 
                 5.46727 
                 0.14476 
                 5.1802 
                 5.7543 
               
               
                 Sepsis 
                 41 
                 3.76053 
                 0.15986 
                 3.4436 
                 4.0775 
               
               
                   
               
             
          
         
       
     
         [0161]    The risk for developing sepsis in patients with infection can be estimated with this score by using logistic regression analysis. 
         [0162]    In this scoring system, as the rated score increases the risk for sepsis is reduced ( FIG. 13B ). 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Model 
                 -LogLikelihood 
                 DF 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
               
                 Difference 
                 23.020230 
                 1 
                 46.04046 
                 &lt;.0001 
               
               
                 Full 
                 39.610381 
               
               
                 Reduced 
                 62.630610 
               
               
                   
               
             
          
           
               
                   
                 RSquare (U) 
                 0.3676 
               
               
                   
                 Observations (or Sum Wgts) 
                 91 
               
               
                   
                   
               
             
          
         
       
     
       Parameter Estimates 
       [0163]      
         [0000]    
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                   
                 Unit Odds 
                 Odds 
               
               
                 Term 
                 Estimate 
                 Std Error 
                 ChiSquare 
                 Prob &gt; ChisSq 
                 Ratio 
                 Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Intercept 
                 7.07811881 
                 1.5455212 
                 20.97 
                 &lt;.0001 
                 — 
                 — 
               
               
                 Summated score 
                 −1.5482397 
                 0.3197876 
                 23.44 
                 &lt;.0001 
                 0.21262192 
                 0.00031881 
               
               
                 C 
                   
                   
                   
                   
                   
                   
               
               
                   
               
             
          
         
       
     
         [0164]    In this system as the score decreased by each unit, the risk for sepsis increased approximately fold. 
         [0165]    Over the range of the score, as the score decreased, the risk of sepsis increased by 3300 fold, i.e. when the risk associated with the highest score was compared with the risk associated with the lowest score. 
         [0166]    This scoring system had an ROC curve as follows ( FIG. 13C ). Area Under Curve=0.88927 
       Example 7 
     Score E 
       [0167]    IL-2 copy numbers can be combined with each of IL-10 and Interferon gamma and IL-23 mRNA copy numbers to produce a summated scoring system. In this method the Log to base 10 of copy numbers are added in the case of IL-2 and IL-23 and interferon gamma, or subtracted as in the case of IL-2 and IL-10. 
         [0168]    The difference between IL-2 and Il-10 mRNA copy numbers to base 10. 
         [0169]    This scoring system differentiates between controls, patients with Infection and patients with Sepsis: with a lower score in patients with infection compared to controls, and a lower score in patients with sepsis compared to patients with infection ( FIG. 14A ). 
       Analysis of Variance 
       [0170]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Sum of 
                 Mean 
                   
                   
               
               
                 Source 
                 DF 
                 Squares 
                 Square 
                 F Ratio 
                 Prob &gt; F 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Groups 
                 2 
                 28.445782 
                 14.2229 
                 31.2802 
                 &lt;.0001 
               
               
                 Error 
                 97 
                 44.105204 
                 0.4547 
               
               
                 C. Total 
                 99 
                 72.550986 
               
               
                   
               
             
          
         
       
     
       Means for Oneway Anova 
       [0171]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Number 
                 Mean 
                 Std Error 
                 Lower 95% 
                 Upper 95% 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 19 
                 0.2838 
                 0.15470 
                 −0.023 
                 0.591 
               
               
                 Infection 
                 50 
                 −0.7887 
                 0.09536 
                 −0.978 
                 −0.599 
               
               
                 Sepsis 
                 31 
                 −1.2612 
                 0.12111 
                 −1.502 
                 −1.021 
               
               
                   
               
             
          
         
       
     
       Means Comparisons 
     Comparisons for all Pairs Using Tukey-Kramer HSD 
       [0172]      
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 q* 
                 Alpha 
               
               
                   
                   
               
             
             
               
                   
                 2.38024 
                 0.05 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Abs(Dif) − LSD 
                 Control 
                 Infection 
                 Sepsis 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 −0.5207 
                 0.6399 
                 1.0773 
               
               
                   
                 Infection 
                 0.6399 
                 −0.3210 
                 0.1056 
               
               
                   
                 Sepsis 
                 1.0773 
                 0.1056 
                 −0.4077 
               
               
                   
                   
               
             
          
         
       
     
         [0173]    Positive values show pairs of means that are significantly different. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Level 
                   
                   
                 Mean 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 A 
                   
                   
                 0.283774 
               
               
                   
                 Infection 
                   
                 B 
                   
                 −0.788677 
               
               
                   
                 Sepsis 
                   
                   
                 C 
                 −1.261182 
               
               
                   
                   
               
             
          
         
       
     
         [0174]    Levels not connected by same letter are significantly different. 
         [0175]    This score, SCORE E, can be used in a logistic regression analysis to determine the risk for sepsis in patients with infection ( FIG. 14B ). 
       Whole Model Test 
       [0176]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Model 
                 -LogLikelihood 
                 DF 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
             
          
           
               
                 Difference 
                 4.500243 
                 1 
                 9.000487 
                 0.0027 
               
               
                 Full 
                 49.395384 
               
               
                 Reduced 
                 53.895628 
               
               
                   
               
             
          
           
               
                   
                 RSquare (U) 
                 0.0835 
               
               
                   
                 Observations (or Sum Wgts) 
                 81 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Term 
                 Estimate 
                 Std Error 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Intercept 
                 −1.5240247 
                 0.4509522 
                 11.42 
                 0.0007 
               
               
                 Score E 
                 −1.0416064 
                 0.3744524 
                 7.74 
                 0.0054 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Unit Odds Ratio 
                 Odds Ratio 
               
               
                   
                   
               
             
             
               
                   
                 0.35288733 
                 0.01038771 
               
               
                   
                   
               
             
          
         
       
     
         [0177]    Thus as the score increases the risk for sepsis decreases. Patients with the greatest score have 0.01 times the risk of sepsis as patients with the least score. For each unit increase in SCORE E the risk for sepsis decreased by 0.35, or approximately by a third. Conversely a unit decrease in SCORE E was associated with an approximate 2.8 fold increase in risk for sepsis in patients with infection, while the risk for sepsis in patients with infection was approximately 96 times greater in patients with the least score compared with patients with the greatest score. 
       Example 8 
     Score F 
       [0178]    This score system is based on the sum of IL-2 and Interferon mRNA copy numbers to the base 10. 
         [0179]    This scoring system differentiates between controls, patients with Infection and patients with Sepsis: with a lower score in patients with infection compared to controls, and a lower score in patients with sepsis compared to patients with infection ( FIG. 15A ). 
       Analysis of Variance 
       [0180]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Sum of 
                 Mean 
                   
                   
               
               
                 Source 
                 DF 
                 Squares 
                 Square 
                 F Ratio 
                 Prob &gt; F 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Groups 
                 2 
                 33.341650 
                 16.6708 
                 31.4574 
                 &lt;.0001 
               
               
                 Error 
                 98 
                 51.934979 
                 0.5299 
               
               
                 C. Total 
                 100 
                 85.276629 
               
               
                   
               
             
          
         
       
     
       Means for Oneway Anova 
       [0181]      
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Number 
                 Mean 
                 Std Error 
                 Lower 95% 
                 Upper 95% 
               
               
                   
               
             
             
               
                 Control 
                 20 
                 5.84048 
                 0.16278 
                 5.5175 
                 6.1635 
               
               
                 Infection 
                 49 
                 5.18889 
                 0.10400 
                 4.9825 
                 5.3953 
               
               
                 Sepsis 
                 32 
                 4.26004 
                 0.12869 
                 4.0047 
                 4.5154 
               
               
                   
               
             
          
         
       
     
       Means Comparisons 
     Comparisons for all Pairs Using Tukey-Kramer HSD 
       [0182]      
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 q* 
                 Alpha 
               
               
                   
                   
               
             
             
               
                   
                 2.37986 
                 0.05 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Abs(Dif) − LSD 
                 Control 
                 Infection 
                 Sepsis 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 −0.5479 
                 0.1919 
                 1.0866 
               
               
                   
                 Infection 
                 0.1919 
                 −0.3500 
                 0.5351 
               
               
                   
                 Sepsis 
                 1.0866 
                 0.5351 
                 −0.4331 
               
               
                   
                   
               
             
          
         
       
     
         [0183]    Positive values show pairs of means that are significantly different. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Level 
                   
                   
                 Mean 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 A 
                   
                   
                 5.8404839 
               
               
                   
                 Infection 
                   
                 B 
                   
                 5.1888890 
               
               
                   
                 Sepsis 
                   
                   
                 C 
                 4.2600360 
               
               
                   
                   
               
             
          
         
       
     
         [0184]    Levels not connected by same letter are significantly different. 
         [0185]    This score, SCORE F, can be used in a logistic regression analysis to determine the risk for sepsis in patients with infection ( FIG. 15B ). 
       Whole Model Test 
       [0186]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Model 
                 -LogLikelihood 
                 DF 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
               
                 Difference 
                 12.095017 
                 1 
                 24.19003 
                 &lt;.0001 
               
               
                 Full 
                 42.252621 
               
               
                 Reduced 
                 54.347638 
               
               
                   
               
             
          
           
               
                   
                 RSquare (U) 
                 0.2225 
               
               
                   
                 Observations (or Sum Wgts) 
                 81 
               
               
                   
                   
               
             
          
         
       
     
       Parameter Estimates 
       [0187]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Term 
                 Estimate 
                 Std Error 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Intercept 
                 6.90303226 
                 1.8259727 
                 14.29 
                 0.0002 
               
               
                 Score F 
                 −1.5419465 
                 0.3814791 
                 16.34 
                 &lt;.0001 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Unit Odds Ratio 
                 Odds Ratio 
               
               
                   
                   
               
             
             
               
                   
                 0.21396421 
                 0.00067978 
               
               
                   
                   
               
             
          
         
       
     
         [0188]    Thus as the score increases, the risk for sepsis decreases. Patients with the greatest score have 0.0006 times (6 ten thousandths, or approximately 1472 fold times less) the risk of sepsis as patients with the least score. For each unit increase in SCORE F the risk for sepsis decreased by 0.21, or to approximately one fifth of the prior risk. Conversely a unit decrease in SCORE F was associated with approximately a five-fold increase in risk for sepsis in patients with infection, while the risk for sepsis in patients with infection was approximately 1472 times greater in patients with the least score compared with patients with the greatest score. 
       Example 9 
     Score G 
       [0189]    This score system is based on the sum of IL-2 and IL-23 mRNA copy numbers to the base 10. 
         [0190]    This scoring system differentiates between controls, patients with Infection and patients with Sepsis: with a lower score in patients with infection compared to controls, and a lower score in patients with sepsis compared to patients with infection ( FIG. 16A ). 
       Analysis of Variance 
       [0191]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Sum of 
                 Mean 
                   
                   
               
               
                 Source 
                 DF 
                 Squares 
                 Square 
                 F Ratio 
                 Prob &gt; F 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Groups 
                 2 
                 24.016167 
                 12.0081 
                 27.9328 
                 &lt;.0001 
               
               
                 Error 
                 99 
                 42.559324 
                 0.4299 
               
               
                 C. Total 
                 101 
                 66.575491 
               
               
                   
               
             
          
         
       
     
       Means for Oneway Anova 
       [0192]      
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Number 
                 Mean 
                 Std Error 
                 Lower 95% 
                 Upper 95% 
               
               
                   
               
             
             
               
                 Control 
                 20 
                 7.96964 
                 0.14661 
                 7.6787 
                 8.2605 
               
               
                 Infection 
                 50 
                 7.17054 
                 0.09272 
                 6.9866 
                 7.3545 
               
               
                 Sepsis 
                 32 
                 6.57532 
                 0.11591 
                 6.3453 
                 6.8053 
               
               
                   
               
             
          
         
       
     
       Means Comparisons 
     Comparisons for all Pairs Using Tukey-Kramer HSD 
       [0193]      
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 q* 
                 Alpha 
               
               
                   
                   
               
             
             
               
                   
                 2.37950 
                 0.05 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Abs(Dif) − LSD 
                 Control 
                 Infection 
                 Sepsis 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 −0.49336 
                 0.38632 
                 0.94961 
               
               
                   
                 Infection 
                 0.38632 
                 −0.31203 
                 0.24202 
               
               
                   
                 Sepsis 
                 0.94961 
                 0.24202 
                 −0.39004 
               
               
                   
                   
               
             
          
         
       
     
         [0194]    Positive values show pairs of means that are significantly different. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Level 
                   
                   
                 Mean 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 A 
                   
                   
                 7.9696387 
               
               
                   
                 Infection 
                   
                 B 
                   
                 7.1705385 
               
               
                   
                 Sepsis 
                   
                   
                 C 
                 6.5753222 
               
               
                   
                   
               
             
          
         
       
     
         [0195]    Levels not connected by same letter are significantly different. 
         [0196]    This score, SCORE G, can be used in a logistic regression analysis to determine the risk for sepsis in patients with infection ( FIG. 16B ). 
       Whole Model Test 
       [0197]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Model 
                 -LogLikelihood 
                 DF 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
             
          
           
               
                 Difference 
                 7.203572 
                 1 
                 14.40714 
                 0.0001 
               
               
                 Full 
                 47.642707 
               
               
                 Reduced 
                 54.846279 
               
               
                   
               
             
          
           
               
                   
                 RSquare (U) 
                 0.1313 
               
               
                   
                 Observations (or Sum Wgts) 
                 82 
               
               
                   
                   
               
             
          
         
       
     
       Parameter Estimates 
       [0198]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Term 
                 Estimate 
                 Std Error 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Intercept 
                 8.79047124 
                 2.7130131 
                 10.50 
                 0.0012 
               
               
                 Score G 
                 −1.3463521 
                 0.3980357 
                 11.44 
                 0.0007 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Unit Odds Ratio 
                 Odds Ratio 
               
               
                   
                   
               
             
             
               
                   
                 0.26018768 
                 0.01036251 
               
               
                   
                   
               
             
          
         
       
     
         [0199]    Thus as the score increases, the risk for sepsis decreases. Patients with the greatest score have 0.01 times (one hundredth) the risk of sepsis as patients with the least score. For each unit increase in SCORE G the risk for sepsis decreased by 0.26, or to approximately one quarter of the prior risk. Conversely a unit decrease in SCORE G was associated with an approximate four-fold increase in risk for sepsis in patients with infection, while the risk for sepsis in patients with infection was approximately 100 times greater in patients with the least score compared with patients with the greatest score. 
       Example 10 
     Score H 
       [0200]    This score system is based on the difference between IL-7 and IL-10 mRNA copy numbers to the base 10. 
         [0201]    This scoring system differentiates between controls, patients with Infection and patients with Sepsis: with a lower score in patients with infection compared to controls, and a lower score in patients with sepsis compared to patients with infection ( FIG. 17A ). 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Rsquare 
                 0.41947 
               
               
                   
                 Adj Rsquare 
                 0.408619 
               
               
                   
                 Root Mean Square Error 
                 0.526087 
               
               
                   
                 Mean of Response 
                 0.637382 
               
               
                   
                 Observations (or Sum Wgts) 
                 110 
               
               
                   
                   
               
             
          
         
       
     
       Analysis of Variance 
       [0202]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Sum of 
                 Mean 
                   
                   
               
               
                 Source 
                 DF 
                 Squares 
                 Square 
                 F Ratio 
                 Prob &gt; F 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Groups 
                 2 
                 21.398124 
                 10.6991 
                 38.6572 
                 &lt;.0001 
               
               
                 Error 
                 107 
                 29.614169 
                 0.2768 
               
               
                 C. Total 
                 109 
                 51.012293 
               
               
                   
               
             
          
         
       
     
       Means for Oneway Anova 
       [0203]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Number 
                 Mean 
                 Std Error 
                 Lower 95% 
                 Upper 95% 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 18 
                 1.34295 
                 0.12400 
                 1.097 
                 1.5888 
               
               
                 Infection 
                 51 
                 0.80489 
                 0.07367 
                 0.659 
                 0.9509 
               
               
                 Sepsis 
                 41 
                 0.11926 
                 0.08216 
                 −0.044 
                 0.2821 
               
               
                   
               
             
          
         
       
     
       Means Comparisons 
     Comparisons for all Pairs Using Tukey-Kramer HSD 
       [0204]      
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 q* 
                 Alpha 
               
               
                   
                   
               
             
             
               
                   
                 2.37679 
                 0.05 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Abs(Dif) − LSD 
                 Control 
                 Infection 
                 Sepsis 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 −0.41680 
                 0.19525 
                 0.87014 
               
               
                   
                 Infection 
                 0.19525 
                 −0.24762 
                 0.42334 
               
               
                   
                 Sepsis 
                 0.87014 
                 0.42334 
                 −0.27617 
               
               
                   
                   
               
             
          
         
       
     
         [0205]    Positive values show pairs of means that are significantly different. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Level 
                   
                   
                 Mean 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 A 
                   
                   
                 1.3429480 
               
               
                   
                 Infection 
                   
                 B 
                   
                 0.8048855 
               
               
                   
                 Sepsis 
                   
                   
                 C 
                 0.1192622 
               
               
                   
                   
               
             
          
         
       
     
         [0206]    This score, SCORE H, can be used in a logistic regression analysis to determine the risk for sepsis in patients with infection ( FIG. 17B ). 
       Whole Model Test 
       [0207]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Model 
                 -LogLikelihood 
                 DF 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
               
                 Difference 
                 17.686604 
                 1 
                 35.37321 
                 &lt;.0001 
               
               
                 Full 
                 45.538383 
               
               
                 Reduced 
                 63.224987 
               
               
                   
               
             
          
           
               
                   
                 RSquare (U) 
                 0.2797 
               
               
                   
                 Observations (or Sum Wgts) 
                 92 
               
               
                   
                   
               
             
          
         
       
     
       Parameter Estimates 
       [0208]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Term 
                 Estimate 
                 Std Error 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Intercept 
                 1.06669221 
                 0.37366 
                 8.15 
                 0.0043 
               
               
                 Score H 
                 −2.7845425 
                 0.602742 
                 21.34 
                 &lt;.0001 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Unit Odds Ratio 
                 Odds Ratio 
               
               
                   
                   
               
             
             
               
                   
                 0.06175734 
                 0.00024141 
               
               
                   
                   
               
             
          
         
       
     
         [0209]    Thus as the score increases the risk for sepsis decreases. Patients with the greatest score have 0.0002 times (one five thousandth) the risk of sepsis as patients with the least score. For each unit increase in SCORE H the risk for sepsis decreased by 0.06, or to one sixteenth of the prior risk. Conversely a unit decrease in SCORE H was associated with a sixteen-fold increase in risk for sepsis in patients with infection, while the risk for sepsis in patients with infection was approximately 4000 times greater in patients with the least score compared with patients with the greatest score. 
       Example 11 
     Score I 
       [0210]    This score system is based on the sum of IL-7 and Interferon mRNA copy numbers to the base 10. 
         [0211]    This scoring system differentiates between controls, patients with Infection and patients with Sepsis: with a lower score in patients with infection compared to controls, and a lower score in patients with sepsis compared to patients with infection ( FIG. 18A ). 
       Analysis of Variance 
       [0212]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Sum of 
                 Mean 
                   
                   
               
               
                 Source 
                 DF 
                 Squares 
                 Square 
                 F Ratio 
                 Prob &gt; F 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Groups 
                 2 
                 24.715614 
                 12.3578 
                 21.9466 
                 &lt;.0001 
               
               
                 Error 
                 107 
                 60.250212 
                 0.5631 
               
               
                 C. Total 
                 109 
                 84.965826 
               
               
                   
               
             
          
         
       
     
       Means for Oneway Anova 
       [0213]      
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Number 
                 Mean 
                 Std Error 
                 Lower 95% 
                 Upper 95% 
               
               
                   
               
             
             
               
                 Control 
                 19 
                 6.94545 
                 0.17215 
                 6.6042 
                 7.2867 
               
               
                 Infection 
                 50 
                 6.59176 
                 0.10612 
                 6.3814 
                 6.8021 
               
               
                 Sepsis 
                 41 
                 5.74361 
                 0.11719 
                 5.5113 
                 5.9759 
               
               
                   
               
             
          
         
       
     
       Means Comparisons 
     Comparisons for all Pairs Using Tukey-Kramer HSD 
       [0214]      
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 q* 
                 Alpha 
               
               
                   
                   
               
             
             
               
                   
                 2.37679 
                 0.05 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Abs(Dif) − LSD 
                 Control 
                 Infection 
                 Sepsis 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 −0.57865 
                 −0.12698 
                 0.70686 
               
               
                   
                 Infection 
                 −0.12698 
                 −0.35670 
                 0.47238 
               
               
                   
                 Sepsis 
                 0.70686 
                 0.47238 
                 −0.39391 
               
               
                   
                   
               
             
          
         
       
     
         [0215]    Positive values show pairs of means that are significantly different. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Level 
                   
                 Mean 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 A 
                   
                 6.9454503 
               
               
                   
                 Infection 
                 A 
                   
                 6.5917621 
               
               
                   
                 Sepsis 
                   
                 B 
                 5.7436135 
               
               
                   
                   
               
             
          
         
       
     
         [0216]    Levels not connected by same letter are significantly different. 
         [0217]    This score, SCORE I, can be used in a logistic regression analysis to determine the risk for sepsis in patients with infection ( FIG. 18B ). 
       Whole Model Test 
       [0218]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Model 
                 -LogLikelihood 
                 DF 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
               
                 Difference 
                 11.557743 
                 1 
                 23.11549 
                 &lt;.0001 
               
               
                 Full 
                 51.072867 
               
               
                 Reduced 
                 62.630610 
               
               
                   
               
             
          
           
               
                   
                 RSquare (U) 
                 0.1845 
               
               
                   
                 Observations (or Sum Wgts) 
                 91 
               
               
                   
                   
               
             
          
         
       
     
       Parameter Estimates 
       [0219]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Prob &gt; 
                   
               
               
                 Term 
                 Estimate 
                 Std Error 
                 ChiSquare 
                 ChiSq 
                 Odds Ratio 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Intercept 
                 8.28323342 
                 2.1110414 
                 15.40 
                 &lt;.0001 
                 . 
               
               
                 Score I 
                 −1.3688679 
                 0.3377283 
                 16.43 
                 &lt;.0001 
                 0.00235533 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Unit Odds Ratio 
                 Odds Ratio 
               
               
                   
                   
               
             
             
               
                   
                 0.25439481 
                 0.00235533 
               
               
                   
                   
               
             
          
         
       
     
         [0220]    Thus as the score increases the risk for sepsis decreases. Patients with the greatest score have 0.002 times (one five hundredth) the risk of sepsis as patients with the least score. For each unit increase in SCORE I the risk for sepsis decreased by 0.25, or to one approximately a quarter of the prior risk. Conversely a unit decrease in SCORE I was associated with an approximate four-fold increase in risk for sepsis in patients with infection, while the risk for sepsis in patients with infection was approximately 424 times greater in patients with the least score compared with patients with the greatest score. 
       Example 12 
     Score J 
       [0221]    This score system is based on the sum of IL-2 and IL-23 mRNA copy numbers to the base 10. 
         [0222]    This scoring system differentiates between controls, patients with Infection and patients with Sepsis: with a lower score in patients with infection compared to controls, and a lower score in patients with sepsis compared to patients with infection ( FIG. 19A ). 
       Analysis of Variance 
       [0223]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Sum of 
                 Mean 
                   
                   
               
               
                 Source 
                 DF 
                 Squares 
                 Square 
                 F Ratio 
                 Prob &gt; F 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Groups 
                 2 
                 16.559547 
                 8.27977 
                 22.3690 
                 &lt;.0001 
               
               
                 Error 
                 109 
                 40.345769 
                 0.37014 
               
               
                 C. Total 
                 111 
                 56.905316 
               
               
                   
               
             
          
         
       
     
       Means for Oneway Anova 
       [0224]      
         [0000]    
       
         
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Number 
                 Mean 
                 Std Error 
                 Lower 95% 
                 Upper 95% 
               
               
                   
               
             
             
               
                 Control 
                 19 
                 9.06886 
                 0.13958 
                 8.7922 
                 9.3455 
               
               
                 Infection 
                 52 
                 8.56451 
                 0.08437 
                 8.3973 
                 8.7317 
               
               
                 Sepsis 
                 41 
                 7.99171 
                 0.09502 
                 7.8034 
                 8.1800 
               
               
                   
               
             
          
         
       
     
       Means Comparisons 
     Comparisons for all Pairs Using Tukey-Kramer HSD 
       [0225]      
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 q* 
                 Alpha 
               
               
                   
                   
               
             
             
               
                   
                 2.37618 
                 0.05 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Abs(Dif) − LSD 
                 Control 
                 Infection 
                 Sepsis 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 −0.46903 
                 0.11681 
                 0.67594 
               
               
                   
                 Infection 
                 0.11681 
                 −0.28352 
                 0.27087 
               
               
                   
                 Sepsis 
                 0.67594 
                 0.27087 
                 −0.31929 
               
               
                   
                   
               
             
          
         
       
     
         [0226]    Positive values show pairs of means that are significantly different. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Level 
                   
                   
                 Mean 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Control 
                 A 
                   
                   
                 9.0688604 
               
               
                   
                 Infection 
                   
                 B 
                   
                 8.5645099 
               
               
                   
                 Sepsis 
                   
                   
                 C 
                 7.9917084 
               
               
                   
                   
               
             
          
         
       
     
         [0227]    Levels not connected by same letter are significantly different. 
         [0228]    This score, SCORE J, can be used in a logistic regression analysis to determine the risk for sepsis in patients with infection ( FIG. 19B ). 
       Whole Model Test 
       [0229]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Model 
                 -LogLikelihood 
                 DF 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
             
          
           
               
                 Difference 
                 8.743534 
                 1 
                 17.48707 
                 &lt;.0001 
               
               
                 Full 
                 54.481453 
               
               
                 Reduced 
                 63.224987 
               
               
                   
               
             
          
           
               
                   
                 RSquare (U) 
                 0.1383 
               
               
                   
                 Observations (or Sum Wgts) 
                 92 
               
               
                   
                   
               
             
          
         
       
     
       Parameter Estimates 
       [0230]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Term 
                 Estimate 
                 Std Error 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Intercept 
                 12.5106108 
                 3.5899849 
                 12.14 
                 0.0005 
               
               
                 Score J 
                 −1.5373514 
                 0.433562 
                 12.57 
                 0.0004 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Unit Odds Ratio 
                 Odds Ratio 
               
               
                   
                   
               
             
             
               
                   
                 0.21494967 
                 0.00147077 
               
               
                   
                   
               
             
          
         
       
     
         [0231]    Thus as the score increases the risk for sepsis decreases. Patients with the greatest score have 0.0014 times (approximately one seven hundredth) the risk of sepsis as patients with the least score. For each unit increase in SCORE J the risk for sepsis decreased by 0.21, or to approximately one fifth of the prior risk. Conversely a unit decrease in SCORE J was associated with an approximate five-fold increase in risk for sepsis in patients with infection, while the risk for sepsis in patients with infection was at an approximately 680 times greater in patients with the least score compared with patients with the greatest score. 
         [0232]    Examples 13 to 24 below are carried out using a cohort of patients different to those for which Examples 1 to 12 are based. Like the first cohort of patients, the second cohort of patients comprises three distinct groups; Healthy Controls, Patients with Infection, and Patients with Severe sepsis. 
       Example 13 and 14 
       [0233]    Examples 13 and 14 give the distribution of IL-2 and IL-7 mRNA levels in three groups of patients; Healthy Controls, Patients with Infection, and Patients with Severe sepsis. These two examples contain a statistical analysis of the comparison between the three groups and relate to  FIGS. 20 and 21 . 
       Example 13 
     IL-2 mRNA Levels in Three Patient Groups 
     Quantiles 
       [0234]      
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Minimum 
                 10% 
                 25% 
                 Median 
                 75% 
                 90% 
                 Maximum 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 222.7601 
                 227.2838 
                 270.8055 
                 514.5454 
                 722.5639 
                 1006.729 
                 1029.959 
               
               
                 Infection 
                 29.92869 
                 30.5752 
                 97.44937 
                 264.6081 
                 342.9062 
                 596.98 
                 653.0492 
               
               
                 Severe 
                 4.513578 
                 16.07357 
                 23.10027 
                 55.99495 
                 120.2476 
                 281.0151 
                 535.4691 
               
               
                 sepsis 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
             
          
         
       
     
       Wilcoxon/Kruskal-Wallis Tests (Rank Sums) 
       [0235]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 (Mean − 
               
               
                 Level 
                 Count 
                 Score Sum 
                 Score Mean 
                 Mean0)/Std0 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 10 
                 796.000 
                 79.6000 
                 4.372 
               
               
                 Infection 
                 16 
                 974.000 
                 60.8750 
                 2.591 
               
               
                 Severe sepsis 
                 64 
                 2325.00 
                 36.3281 
                 −5.221 
               
               
                   
               
             
          
         
       
     
       1-Way Test, ChiSquare Approximation 
       [0236]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 ChiSquare 
                 DF 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                 30.4678 
                 2 
                 &lt;.0001 
               
               
                   
               
             
          
         
       
     
       Example 14 
     IL-7 mRNA Levels in Three Patient Groups 
     Quantiles 
       [0237]      
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Minimum 
                 10% 
                 25% 
                 Median 
                 75% 
                 90% 
                 Maximum 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 283.0553 
                 300.9931 
                 1372.122 
                 2658.712 
                 3819.715 
                 14730.06 
                 15641.59 
               
               
                 Infection 
                 487.0186 
                 753.2133 
                 1791.384 
                 3008.941 
                 5924.231 
                 7239.475 
                 8124.894 
               
               
                 Severe 
                 38.02993 
                 172.5917 
                 407.9582 
                 737.0873 
                 1587.198 
                 3218.928 
                 211242 
               
               
                 sepsis 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
             
          
         
       
     
       Wilcoxon/Kruskal-Wallis Tests (Rank Sums) 
       [0238]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 (Mean − 
               
               
                 Level 
                 Count 
                 Score Sum 
                 Score Mean 
                 Mean0)/Std0 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 10 
                 617.000 
                 61.7000 
                 1.986 
               
               
                 Infection 
                 16 
                 1090.00 
                 68.1250 
                 3.685 
               
               
                 Severe sepsis 
                 65 
                 2479.00 
                 38.1385 
                 −4.485 
               
               
                   
               
             
          
         
       
     
       1-Way Test, ChiSquare Approximation 
       [0239]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 ChiSquare 
                 DF 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                 20.5175 
                 2 
                 &lt;.0001 
               
               
                   
               
             
          
         
       
     
       Examples 15 and 16 
       [0240]    Examples 15 and 16 give the distribution of IL-2 and IL-7 mRNA levels in two groups of patients; Patients with Infection, and Patients with Severe sepsis. These two examples contain statistical analyses of the comparison between the three groups and relate to  FIGS. 22 and 23 . 
       Example 15 
     IL-2 mRNA Levels in Two Patient Groups; Infection and Severe Sepsis 
     Quantiles 
       [0241]      
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Minimum 
                 10% 
                 25% 
                 Median 
                 75% 
                 90% 
                 Maximum 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Infection 
                 29.92869 
                 30.5752 
                 97.44937 
                 264.6081 
                 342.9062 
                 596.98 
                 653.0492 
               
               
                 Severe 
                 4.513578 
                 16.07357 
                 23.10027 
                 55.99495 
                 120.2476 
                 281.0151 
                 535.4691 
               
               
                 sepsis 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
             
          
         
       
     
       Wilcoxon/Kruskal-Wallis Tests (Rank Sums) 
       [0242]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 (Mean − 
               
               
                 Level 
                 Count 
                 Score Sum 
                 Score Mean 
                 Mean0)/Std0 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Infection 
                 16 
                 941.000 
                 58.8125 
                 3.518 
               
               
                 Severe sepsis 
                 64 
                 2299.00 
                 35.9219 
                 −3.518 
               
               
                   
               
             
          
         
       
     
       2-Sample Test, Normal Approximation 
       [0243]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 S 
                 Z 
                 Prob &gt; |Z| 
               
               
                   
               
             
             
               
                 941 
                 3.51823 
                 0.0004 
               
               
                   
               
             
          
         
       
     
       1-Way Test, ChiSquare Approximation 
       [0244]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 ChiSquare 
                 DF 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                 12.4203 
                 1 
                 0.0004 
               
               
                   
               
             
          
         
       
     
       Example 16 
     IL-7 mRNA Levels in Two Patient Groups; Infection and Severe Sepsis 
     Quantiles 
       [0245]      
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Minimum 
                 10% 
                 25% 
                 Median 
                 75% 
                 90% 
                 Maximum 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Infection 
                 487.0186 
                 753.2133 
                 1791.384 
                 3008.941 
                 5924.231 
                 7239.475 
                 8124.894 
               
               
                 Severe 
                 38.02993 
                 168.3913 
                 400.7392 
                 727.2953 
                 1531.579 
                 2875.682 
                 9531.648 
               
               
                 sepsis 
                   
                   
                   
                   
                   
                   
                   
               
               
                   
               
             
          
         
       
     
       Wilcoxon/Kruskal-Wallis Tests (Rank Sums) 
       [0246]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 (Mean − 
               
               
                 Level 
                 Count 
                 Score Sum 
                 Score Mean 
                 Mean0)/Std0 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Infection 
                 16 
                 998.000 
                 62.3750 
                 4.204 
               
               
                 Severe sepsis 
                 64 
                 2242.00 
                 35.0313 
                 −4.204 
               
               
                   
               
             
          
         
       
     
       2-Sample Test, Normal Approximation 
       [0247]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 S 
                 Z 
                 Prob &gt; |Z| 
               
               
                   
               
             
             
               
                 998 
                 4.20383 
                 &lt;.0001 
               
               
                   
               
             
          
         
       
     
       1-Way Test, ChiSquare Approximation 
       [0248]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 ChiSquare 
                 DF 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                 17.7228 
                 1 
                 &lt;.0001 
               
               
                   
               
             
          
         
       
     
       Examples 17 and 18 
       [0249]    Examples 17 and 18 give the distribution of IL-2 and IL-7 mRNA levels in two groups of patients; Healthy Controls and Patients with Infection. These two examples contain a statistical analysis of the comparison between the three groups. 
       Example 17 
     IL-2 mRNA Levels in Two Patient Groups; Infection and Control (FIG.  24 ) 
     Quantiles 
       [0250]      
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Minimum 
                 10% 
                 25% 
                 Median 
                 75% 
                 90% 
                 Maximum 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Con- 
                 222.7601 
                 227.2838 
                 270.8055 
                 514.5454 
                 722.5639 
                 1006.729 
                 1029.959 
               
               
                 trol 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Infec- 
                 29.92869 
                 30.5752 
                 97.44937 
                 264.6081 
                 342.9062 
                 596.98 
                 653.0492 
               
               
                 tion 
               
               
                   
               
             
          
         
       
     
       Wilcoxon/Kruskal-Wallis Tests (Rank Sums) 
       [0251]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 (Mean − 
               
               
                 Level 
                 Count 
                 Score Sum 
                 Score Mean 
                 Mean0)/Std0 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 10 
                 182.000 
                 18.2000 
                 2.451 
               
               
                 Infection 
                 16 
                 169.000 
                 10.5625 
                 −2.451 
               
               
                   
               
             
          
         
       
     
       2-Sample Test, Normal Approximation 
       [0252]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 S 
                 Z 
                 Prob &gt; |Z| 
               
               
                   
               
             
             
               
                 182 
                 2.45077 
                 0.0143 
               
               
                   
               
             
          
         
       
     
       1-Way Test, ChiSquare Approximation 
       [0253]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 ChiSquare 
                 DF 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                 6.1361 
                 1 
                 0.0132 
               
               
                   
               
             
          
         
       
     
       Example 18 
     IL-7 mRNA Levels in Two Patient Groups; Infection and Severe Sepsis 
     Quantiles 
       [0254]      
         [0000]    
       
         
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 Level 
                 Minimum 
                 10% 
                 25% 
                 Median 
                 75% 
                 90% 
                 Maximum 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 283.0553 
                 300.9931 
                 1372.122 
                 2658.712 
                 3819.715 
                 14730.06 
                 15641.59 
               
               
                 Infection 
                 487.0186 
                 753.2133 
                 1791.384 
                 3008.941 
                 5924.231 
                 7239.475 
                 8124.894 
               
               
                   
               
             
          
         
       
     
       Wilcoxon/Kruskal-Wallis Tests (Rank Sums) 
       [0255]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 (Mean − 
               
               
                 Level 
                 Count 
                 Score Sum 
                 Score Mean 
                 Mean0)/Std0 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Control 
                 10 
                 123.000 
                 12.3000 
                 −0.606 
               
               
                 Infection 
                 16 
                 228.000 
                 14.2500 
                 0.606 
               
               
                   
               
             
          
         
       
     
       2-Sample Test, Normal Approximation 
       [0256]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 S 
                 Z 
                 Prob &gt; |Z| 
               
               
                   
               
             
             
               
                 123 
                 −0.60610 
                 0.5444 
               
               
                   
               
             
          
         
       
     
       1-Way Test, ChiSquare Approximation 
       [0257]      
         [0000]    
       
         
               
               
               
             
           
               
                   
               
               
                 ChiSquare 
                 DF 
                 Prob &gt; ChiSq 
               
               
                   
               
             
             
               
                 0.4000 
                 1 
                 0.5271 
               
               
                   
               
             
          
         
       
     
       Example 19 
       [0258]    Example 19 gives the distribution of the IL-2 categories, 1 or 2 or 3, in two groups of patients; Patients with Infection, and Patients with Severe sepsis. This table contains a statistical analysis of the distribution of IL-2 categories between the two groups and relates to  FIG. 25 . 
         [0259]    IL-2 levels have been divided into three levels, indicated by the numerals 1, 2 and 3. 
         [0260]    The distribution of these IL-2 levels in patients with Infection and Severe sepsis is listed in the table. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Contingency Table 
               
             
          
           
               
                   
                 Count 
                   
                   
                   
               
               
                   
                 Total % 
               
               
                   
                 Col % 
               
               
                   
                 Row % 
                 Infection 
                 Severe sepsis 
               
               
                   
                   
               
             
          
           
               
                   
                 1 
                 2 
                 0 
                 2 
               
               
                   
                   
                 2.50 
                 0.00 
                 2.50 
               
               
                   
                   
                 12.50 
                 0.00 
               
               
                   
                   
                 100.00 
                 0.00 
               
               
                   
                 2 
                 10 
                 13 
                 23 
               
               
                   
                   
                 12.50 
                 16.25 
                 28.75 
               
               
                   
                   
                 62.50 
                 20.31 
               
               
                   
                   
                 43.48 
                 56.52 
               
               
                   
                 3 
                 4 
                 51 
                 55 
               
               
                   
                   
                 5.00 
                 63.75 
                 68.75 
               
               
                   
                   
                 25.00 
                 79.69 
               
               
                   
                   
                 7.27 
                 92.73 
               
               
                   
                   
                 16 
                 64 
                 80 
               
               
                   
                   
                 20.00 
                 80.00 
               
               
                   
                   
               
             
          
         
       
     
       Tests 
       [0261]      
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 N 
                 DF 
                 -LogLike 
                 RSquare (U) 
               
               
                   
                   
               
             
             
               
                   
                 80 
                 2 
                 9.9509790 
                 0.2486 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Test 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
                   
               
             
             
               
                   
                 Likelihood 
                 19.902 
                 &lt;.0001 
               
               
                   
                 Ratio 
                 21.492 
                 &lt;.0001 
               
               
                   
                 Pearson 
               
               
                   
                   
               
             
          
         
       
     
       Example 20 
       [0262]    Example 20 gives the distribution of the IL-2 categories, 1 or 2 or 3, in three groups of patients; Healthy Controls, Patients with Infection, and Patients with Severe sepsis. This table contains a statistical analysis of the distribution of IL-2 categories between the three groups and relates to  FIG. 26 . 
         [0263]    IL-2 levels have been divided into three levels, indicated by the numerals 1, 2 and 3. 
         [0264]    The distribution of these IL-2 levels in a control Group and in patients with Infection and Severe sepsis is listed in the table. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Count 
                   
                   
                   
                   
               
               
                 Total % 
               
               
                 Col % 
               
               
                 Row % 
                 Control 
                 Infection 
                 Severe sepsis 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 5 
                 2 
                 0 
                 7 
               
               
                   
                 5.56 
                 2.22 
                 0.00 
                 7.78 
               
               
                   
                 50.00 
                 12.50 
                 0.00 
               
               
                   
                 71.43 
                 28.57 
                 0.00 
               
               
                 2 
                 5 
                 10 
                 13 
                 28 
               
               
                   
                 5.56 
                 11.11 
                 14.44 
                 31.11 
               
               
                   
                 50.00 
                 62.50 
                 20.31 
               
               
                   
                 17.86 
                 35.71 
                 46.43 
               
               
                 3 
                 0 
                 4 
                 51 
                 55 
               
               
                   
                 0.00 
                 4.44 
                 56.67 
                 61.11 
               
               
                   
                 0.00 
                 25.00 
                 79.69 
               
               
                   
                 0.00 
                 7.27 
                 92.73 
               
               
                   
                 10 
                 16 
                 64 
                 90 
               
               
                   
                 11.11 
                 17.78 
                 71.11 
               
               
                   
               
             
          
         
       
     
       Tests 
       [0265]      
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 N 
                 DF 
                 -LogLike 
                 RSquare (U) 
               
               
                   
                   
               
             
             
               
                   
                 90 
                 4 
                 24.019811 
                 0.3363 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Test 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
                   
               
             
             
               
                   
                 Likelihood 
                 48.040 
                 &lt;.0001 
               
               
                   
                 Ratio 
               
               
                   
                 Pearson 
                 50.109 
                 &lt;.0001 
               
               
                   
                   
               
             
          
         
       
     
       Example 21 
       [0266]    Example 21 gives the distribution of the IL-7 categories, 1 or 2 or 3, in three groups of patients; Healthy Controls, Patients with Infection, and Patients with Severe sepsis. This example contains a statistical analysis of the distribution of IL-7 categories between the three groups and relates to  FIG. 27 . 
         [0267]    IL-7 levels have been divided into three levels, indicated by the numerals 1, 2 and 3. 
         [0268]    The distribution of these IL-2 levels in a control Group and in patients with Infection and Severe sepsis is listed in the table. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Count 
                   
                   
                   
                   
               
               
                 Total % 
               
               
                 Col % 
               
               
                 Row % 
                 Control 
                 Infection 
                 Severe sepsis 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 8 
                 13 
                 15 
                 36 
               
               
                   
                 8.89 
                 14.44 
                 16.67 
                 40.00 
               
               
                   
                 80.00 
                 81.25 
                 23.44 
               
               
                   
                 22.22 
                 36.11 
                 41.67 
               
               
                 2 
                 2 
                 3 
                 36 
                 41 
               
               
                   
                 2.22 
                 3.33 
                 40.00 
                 45.56 
               
               
                   
                 20.00 
                 18.75 
                 56.25 
               
               
                   
                 4.88 
                 7.32 
                 87.80 
               
               
                 3 
                 0 
                 0 
                 13 
                 13 
               
               
                   
                 0.00 
                 0.00 
                 14.44 
                 14.44 
               
               
                   
                 0.00 
                 0.00 
                 20.31 
               
               
                   
                 0.00 
                 0.00 
                 100.00 
               
               
                   
                 10 
                 16 
                 64 
                 90 
               
               
                   
                 11.11 
                 17.78 
                 71.11 
               
               
                   
               
             
          
         
       
     
       Tests 
       [0269]      
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 N 
                 DF 
                 -LogLike 
                 RSquare (U) 
               
               
                   
                   
               
             
             
               
                   
                 90 
                 4 
                 14.453386 
                 0.2024 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Test 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
                   
               
             
             
               
                   
                 Likelihood 
                 28.907 
                 &lt;.0001 
               
               
                   
                 Ratio 
               
               
                   
                 Pearson 
                 26.041 
                 &lt;.0001 
               
               
                   
                   
               
             
          
         
       
     
       Example 22 
       [0270]    Example 22 gives the distribution of the IL-7 categories, 1 or 2 or 3, in two groups of patients; Patients with Infection, and Patients with Severe sepsis. This example contains a statistical analysis of the distribution of IL-7 categories between the two groups and relates to  FIG. 28 . 
         [0271]    IL-7 levels have been divided into three levels, indicated by the numerals 1, 2 and 3. 
         [0272]    The distribution of these IL-2 levels in patients with Infection and Severe sepsis is listed in the table. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Count 
                   
                   
                   
               
               
                   
                 Total % 
               
               
                   
                 Col % 
               
               
                   
                 Row % 
                 Infection 
                 Severe sepsis 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 1 
                 13 
                 15 
                 28 
               
               
                   
                   
                 16.25 
                 18.75 
                 35.00 
               
               
                   
                   
                 81.25 
                 23.44 
               
               
                   
                   
                 46.43 
                 53.57 
               
               
                   
                 2 
                 3 
                 36 
                 39 
               
               
                   
                   
                 3.75 
                 45.00 
                 48.75 
               
               
                   
                   
                 18.75 
                 56.25 
               
               
                   
                   
                 7.69 
                 92.31 
               
               
                   
                 3 
                 0 
                 13 
                 13 
               
               
                   
                   
                 0.00 
                 16.25 
                 16.25 
               
               
                   
                   
                 0.00 
                 20.31 
               
               
                   
                   
                 0.00 
                 100.00 
               
               
                   
                   
                 16 
                 64 
                 80 
               
               
                   
                   
                 20.00 
                 80.00 
               
               
                   
                   
               
             
          
         
       
     
       Tests 
       [0273]      
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 N 
                 DF 
                 -LogLike 
                 RSquare (U) 
               
               
                   
                   
               
             
             
               
                   
                 80 
                 2 
                 10.119177 
                 0.2528 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Test 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
                   
               
             
             
               
                   
                 Likelihood 
                 20.238 
                 &lt;.0001 
               
               
                   
                 Ratio 
               
               
                   
                 Pearson 
                 19.166 
                 &lt;.0001 
               
               
                   
                   
               
             
          
         
       
     
       Example 23 
       [0274]    In example 23 the scores for both IL-2 and IL-7 categories have been summated, giving a scoring system which ranges from 2 to 6. The distribution of these scores in the three patient groups, Control, Infection, and Severe sepsis, is statistically analysed and relates to  FIG. 29 . 
         [0275]    In this table the summated score for IL-2 and IL-7 levels is presented as these scores are distributed within 3 groups of patients, Controls, Infection and Severe sepsis 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Count 
                   
                   
                   
                   
               
               
                 Total % 
               
               
                 Col % 
               
               
                 Row % 
                 Control 
                 Infection 
                 Severe sepsis 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 2 
                 5 
                 2 
                 0 
                 7 
               
               
                   
                 5.56 
                 2.22 
                 0.00 
                 7.78 
               
               
                   
                 50.00 
                 12.50 
                 0.00 
               
               
                   
                 71.43 
                 28.57 
                 0.00 
               
               
                 3 
                 3 
                 8 
                 4 
                 15 
               
               
                   
                 3.33 
                 8.89 
                 4.44 
                 16.67 
               
               
                   
                 30.00 
                 50.00 
                 6.25 
               
               
                   
                 20.00 
                 53.33 
                 26.67 
               
               
                 4 
                 2 
                 5 
                 16 
                 23 
               
               
                   
                 2.22 
                 5.56 
                 17.78 
                 25.56 
               
               
                   
                 20.00 
                 31.25 
                 25.00 
               
               
                   
                 8.70 
                 21.74 
                 69.57 
               
               
                 5 
                 0 
                 1 
                 35 
                 36 
               
               
                   
                 0.00 
                 1.11 
                 38.89 
                 40.00 
               
               
                   
                 0.00 
                 6.25 
                 54.69 
               
               
                   
                 0.00 
                 2.78 
                 97.22 
               
               
                 6 
                 0 
                 0 
                 9 
                 9 
               
               
                   
                 0.00 
                 0.00 
                 10.00 
                 10.00 
               
               
                   
                 0.00 
                 0.00 
                 14.06 
               
               
                   
                 0.00 
                 0.00 
                 100.00 
               
               
                   
                 10 
                 16 
                 64 
                 90 
               
               
                   
                 11.11 
                 17.78 
                 71.11 
               
               
                   
               
             
          
         
       
     
       Tests 
       [0276]      
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 N 
                 DF 
                 -LogLike 
                 RSquare (U) 
               
               
                   
                   
               
             
             
               
                   
                 90 
                 8 
                 29.204026 
                 0.4089 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Test 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
                   
               
             
             
               
                   
                 Likelihood 
                 58.408 
                 &lt;.0001 
               
               
                   
                 Ratio 
               
               
                   
                 Pearson 
                 60.253 
                 &lt;.0001 
               
               
                   
                   
               
             
          
         
       
     
       Example 24 
       [0277]    In example 24 the scores for both IL-2 and IL-7 categories have been summated, giving a scoring system which ranges from 2 to 6. The distribution of these scores in the two patient groups, Infection, and Severe sepsis, is statistically analysed. Example 24 relates to  FIG. 30 . 
         [0278]    In this table the summated score for IL-2 and IL-7 levels is presented as these scores are distributed within 2 groups of patients, with Infection and Severe sepsis 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                 Contingency Table 
               
               
                 Column 30 By Sepsiis v Infection 
               
             
          
           
               
                   
                 Count 
                   
                   
                   
               
               
                   
                 Total % 
               
               
                   
                 Col % 
               
               
                   
                 Row % 
                 Infection 
                 Severe sepsis 
               
               
                   
                   
               
             
          
           
               
                   
                 2 
                 2 
                 0 
                 2 
               
               
                   
                   
                 2.50 
                 0.00 
                 2.50 
               
               
                   
                   
                 12.50 
                 0.00 
               
               
                   
                   
                 100.00 
                 0.00 
               
               
                   
                 3 
                 8 
                 4 
                 12 
               
               
                   
                   
                 10.00 
                 5.00 
                 15.00 
               
               
                   
                   
                 50.00 
                 6.25 
               
               
                   
                   
                 66.67 
                 33.33 
               
               
                   
                 4 
                 5 
                 16 
                 21 
               
               
                   
                   
                 6.25 
                 20.00 
                 26.25 
               
               
                   
                   
                 31.25 
                 25.00 
               
               
                   
                   
                 23.81 
                 76.19 
               
               
                   
                 5 
                 1 
                 35 
                 36 
               
               
                   
                   
                 1.25 
                 43.75 
                 45.00 
               
               
                   
                   
                 6.25 
                 54.69 
               
               
                   
                   
                 2.78 
                 97.22 
               
               
                   
                 6 
                 0 
                 9 
                 9 
               
               
                   
                   
                 0.00 
                 11.25 
                 11.25 
               
               
                   
                   
                 0.00 
                 14.06 
               
               
                   
                   
                 0.00 
                 100.00 
               
               
                   
                   
                 16 
                 64 
                 80 
               
               
                   
                   
                 20.00 
                 80.00 
               
               
                   
                   
               
             
          
         
       
     
       Tests 
       [0279]      
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 N 
                 DF 
                 -LogLike 
                 RSquare (U) 
               
               
                   
                   
               
             
             
               
                   
                 80 
                 4 
                 16.298162 
                 0.4071 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Test 
                 ChiSquare 
                 Prob &gt; ChiSq 
               
               
                   
                   
               
             
             
               
                   
                 Likelihood 
                 32.596 
                 &lt;.0001 
               
               
                   
                 Ratio 
               
               
                   
                 Pearson 
                 33.447 
                 &lt;.0001 
               
               
                   
                   
               
             
          
         
       
     
         [0280]    The invention is not limited to the embodiments hereinbefore described which may be varied in construction and detail without departing from the spirit of the invention.