Two gene probes corresponding to MMP-8 and one gene probe corresponding to elastase-2 were included in the 21 gene probes used in the class prediction modeling procedure described above. Since MMP-8 and elastase-2 protein levels can be readily measured in the serum compartment, we tested the ability of MMP-8 and elastase-2 serum protein levels to predict SSAKI in the derivation cohort. There were 150 parallel serum samples from the derivation cohort available for this analysis (132 patients without SSAKI and 18 patients with SSAKI). Figure Figure22 demonstrates that both MMP-8 and elastase-2 serum protein levels were higher in patients with SSAKI, compared with patients without kidney injury, thus corroborating the microarray data.Figure 2Serum matrix metalloproteinase-8 and elastase-2 levels in patients with and without sepsis-shock-associated acute kidney injury. Serum matrix metalloproteinase-8 (MMP-8) and elastase-2 levels are higher in patients with sepsis-shock-associated acute kidney …We next constructed ROC curves to determine the ability of serum protein levels of MMP-8 and elastase-2, respectively, to predict SSAKI in the derivation cohort (Figure (Figure3).3). From these ROC curves we empirically selected cut-off values of 11 ng/ml (MMP-8) and 235 ng/ml (elastase-2) with an a priori goal of achieving a high sensitivity at the expense of specificity. The performance characteristics of the respective cut-off values are shown in Table Table3.3. For both MMP-8 and elastase-2, the respective cut-off values were able to predict SSAKI with a relatively high sensitivity. In addition, both cut-off values had a high negative predictive value for the development of SSAKI. When we incorporated both MMP-8 and elastase-2 into a multiple regression analysis, we were unable to substantially improve the ability to predict SSAKI compared with the ability of each candidate biomarker alone (data not shown).Figure 3Matrix metalloproteinase-8 and elastase-2 as candidate biomarkers for predicting sepsis-shock-associated acute kidney injury. Receiver operating characteristic curves for matrix metalloproteinase-8 (MMP-8) and elastase-2 as candidate biomarkers for predicting …Table 3Individual performance calculations of serum protein levels for predicting SSAKI in the derivation cohortValidation of MMP-8 and elastase-2 performanceHaving demonstrated the individual performances of MMP-8 and elastase-2 as candidate biomarkers for predicting SSAKI in the derivation cohort, we next applied the same respective cut-off values to a separate validation cohort of patients. The validation cohort consisted of 59 patients without SSAKI and 11 patients with SSAKI.