Free hemoglobin concentration in severe sepsis: methods of measurement and prediction of outcome

Abstract

Introduction: Hemolysis can be induced in sepsis via various mechanisms, its pathophysiological importance has been demonstrated in experimental sepsis. However, no data on free hemoglobin concentrations in human sepsis are available. In the present study we measured free hemoglobin in patients with severe sepsis as well as in postoperative patients using four methods. It was our aim to determine the potential value of free hemoglobin as a biomarker for diagnosis and outcome of severe sepsis in critical illness.

Methods: Plasma concentration of free hemoglobin was determined in patients with severe sepsis (n = 161) and postoperative patients (n = 136) on day 1 of diagnosis and surgery. For the measurement of free hemoglobin, an enzyme linked immunosorbent assay and three spectrophotometric algorithms were used. Moreover, SAPS II- and SOFA scores as well as procalcitonin concentration and outcome were determined. Kaplan-Meier analysis was performed and odds ratios were determined after classification of free hemoglobin concentrations in a high and low concentration group according to the median. For statistical evaluation the Mann-Whitney test and logistic regression analysis were used.

Results: In non-survivors of severe sepsis, free hemoglobin concentration was twice the concentration compared to survivors. Thirty-day survival of patients, as evidenced by Kaplan-Meier analysis, was markedly lower in patients with high free hemoglobin concentration than in patients with low free hemoglobin concentration. Best discrimination of outcome was achieved with the spectrophotometric method of Harboe (51.3% vs. 86.4% survival, p < 0.001; odds ratio 6.1). Multivariate analysis including free hemoglobin, age, SAPS II- and SOFA-score and procalcitonin demonstrated that free hemoglobin, as determined by all 4 methods, was the best and an independent predictor for death in severe sepsis (p = 0.022 to p < 0.001). Free hemoglobin concentrations were not significantly different in postoperative and septic patients in three of four assays. Thus, free hemoglobin can not be used to diagnose severe sepsis in critical illness.

Conclusions: Free hemoglobin is an important new predictor of survival in severe sepsis.

Figure 1

Free hemoglobin concentrations in survivors and non-survivors of severe sepsis as obtained by ELISA and three spectrophotometric methods. Data are given in boxplots (median, quartile, minimum, maximum). For statistical evaluation the Mann-Whitney test was used. Spectrophotometric methods were the methods of Harboe [13], Noe [14] and Fairbanks [15].

Figure 2

Kaplan-Meier analyses of 30-day survival of patients with severe sepsis demonstrating the effect of high and low free hemoglobin concentrations as determined by four methods. Four sets of Kaplan-Meier curves are shown, as analysis was performed with each of the four methods used for the determination of free hemoglobin. The continuous lines show the survival of patients with free hemoglobin concentrations lower than the median, and the dotted lines show the survival when free hemoglobin concentration was higher than the median of all patients with sepsis. Significance levels for the log rank test are given in the figure. Methods used to measure free hemoglobin were ELISA and the spectrophotometric methods of Harboe [13], Noe [14] and Fairbanks [15].

Figure 3

Free hemoglobin measured in postoperative patients and patients with severe sepsis using four methods. Free hemoglobin did not differ in postoperative and septic patients using three of the four methods. Therefore, free hemoglobin cannot serve as a biomarker for the diagnosis of severe sepsis in critical illness. Data are given in boxplots (median, quartile, minimum, maximum). For statistical evaluation the Mann-Whitney test was used. Methods used to measure free hemoglobin were ELISA and the spectrophotometric methods of Harboe [13], Noe [14] and Fairbanks [15].