Clinical evaluation of presepsin considering renal function

Presepsin, a glycoprotein produced during bacterial phagocytosis, is used as a sepsis marker for bacterial infections. However, presepsin levels are affected by renal function, and the evaluation criteria according to kidney function or in chronic kidney diseases remain controversial. Furthermore, presepsin may be increased by sample stirring, but no studies have evaluated this effect.In this study, we excluded the effect of stirring by standardizing the blood collection conditions, analyzed the influence of kidney function on presepsin concentrations, and recalculated the reference range based on the findings. EDTA-whole blood from 47 healthy subjects and 85 patients with chronic kidney disease was collected to measure presepsin by PATHFAST. Presepsin was found to be significantly correlated with the levels of creatinine (r = 0.834), eGFRcreat (r = 0.837), cystatin-C (r = 0.845), and eGFRcys (r = 0.879). Furthermore, in patients with CKD, presepsin levels stratified by eGFRcys showed a significant increase in the CKD G2 patient group and with advancing glomerular filtration rate stage. The following values were obtained: Normal: 97.6 ± 27.4 pg/mL, CKD G1: 100.2 ± 27.6 pg/mL, CKD G2: 129.7 ± 40.7 pg/mL, CKD G3: 208.1 ± 70.2 pg/mL, CKD G4: 320.2 ± 170.1 pg/mL, CKD G5: 712.8 ± 336.3 pg/mL. The reference range, calculated by a nonparametric method using 67 cases of healthy volunteers and patients with chronic kidney disease G1, was found to be 59–153 pg/mL, which was notably lower than the standard reference range currently used. Presepsin concentrations were positively correlated with a few biomarkers of renal function, indicating the necessity to consider the effect of renal function in patients with renal impairment. Using the recalculated reference range considering kidney function may improve the accuracy of evaluating presepsin for diagnosis of sepsis compared to the standard reference currently in use.


Introduction
Presepsin is a protein whose blood concentrations increase specifically during sepsis.
Since its discovery in 2002 in Japan, presepsin has been widely used as a sepsis marker.
Membrane-bound CD14, a surface antigen expressed on the cell membrane of monocyte macrophages and granulocytes, is a receptor for bacterial lipopolysaccharide (LPS), which activates cells via TLR4 [1]. In addition, soluble CD14 present in the blood induces the activation of endothelial and epithelial cells without membrane-bound CD14 [2], and plays an important role in sensing invasion of bacteria in vivo. Recently, it was reported that granulocyte-mediated bacterial phagocytosis triggers elastase or cathepsin D to proteolytically cleave CD14 to produce presepsin and release it into the blood [3].
Furthermore, it was shown that the concentration of presepsin increases with infection in patients with leucopenia [4], indicating that cells other than monocytes can trigger presepsin production.
Unlike procalcitonin, which has been conventionally used for sepsis diagnosis, presepsin responds very weakly to inflammation such as trauma and burn, and is considered to be highly specific for bacterial infection [5][6][7]. Compared with conventional markers, presepsin is a good clinical indicator that responds well to changes in the disease state and thus reflects the effect of therapeutics on the condition [8][9][10].
The presepsin cut-off value for sepsis or infectious disease diagnosis is 400-700 pg/mL (500 pg/mL: Japan) [11]. However, in the blood of a patient with renal disorder such as dialysis patient, the concentration can be higher, because the presepsin is excreted from the kidney [12]. Therefore, it is unsuitable to use the general cut-off values for diagnosis of patients with chronic kidney disease (CKD), as a false high value associated with renal impairment can lead to erroneous judgment.
In the current study, presepsin concentrations were measured in patients with CKD and analyzed for their relationship with the renal function index. In addition, we established a reference range that evaluated the influence of kidney function.

Design and subjects
This study enrolled 85 outpatients with CKD who visited the Tokushima University Hospital from May 2017 to September 2017. All patients were over 18 years of age, and had no history of dialysis and infection. In addition, samples and data were also collected for 47 healthy volunteers without renal dysfunction as controls.

Blood collection and biochemical analysis
Venous blood was collected after an overnight fasting. Since presepsin concentration increases due to strong agitation, samples after complete blood count (CBC) measurement cannot be used. For this reason, collection was performed with an EDTA-2K blood collection tube exclusively for presepsin measurement. At the time of blood sampling, five gentle inversions were strictly performed. Although presepsin shows an increasing trend from the early stage due to deterioration of renal function, there was no significant difference between healthy subjects and patients in CKD G1 group. Thus, CKD G1 group might be diagnosed as normal. Meanwhile, in patients in CKD G2 group and higher stages, a significant increase in presepsin was observed even in uninfected cases. Therefore, results need to be carefully considered for effect of renal function during sepsis diagnosis in patients with renal impairment.
In addition, false high values of presepsin, arising due to vigorous agitation of the blood samples have been reported, and this could also hinder an accurate clinical assessment. A mechanical stimulus such as agitation leads to formation of a macromolecular complex, which reacts with the anti-presepsin antibody in the reagent in a larger molecular weight fraction. Although the false high value is considered to be due to cross-reaction with this polymer complex, the mechanism of complex formation is not clarified. At present, there is no efficient method other than to avoid agitation of sample at the time of blood collection/delivery, necessitating extreme caution. Although doctors and nurses collect blood on the bedside with caution, fluctuation at the pre-measurement stage can be large, affecting the reliability of the results. Therefore, it is essential to educate clinical practitioners regarding specimen collection.
In this study, recalculation of the reference range using the CKD G1 group and the healthy subject group data indicated remarkably low values that were half of the standard reference range currently in use. The sample group used for calculating the standard reference range did not take into consideration the effects of renal function or agitation, and it is highly probable that these false high values were included. Therefore, it may not be an appropriate sample group. On the other hand, the reference sample group used for calculation in this study excluded false high value cases caused by renal function and agitation, making it a more useful index.
Sepsis diagnosis is not based on only one biomarker, but is a comprehensive diagnosis based on multiple markers and clinical symptoms. The new reference range obtained in this study can be effectively used for early diagnosis of sepsis. For example, mild sepsis can be positively identified with elevated levels of presepsin in the range of 300-500 pg/mL.
In conclusion, by excluding the effect of stirring during sampling, our study showed that presepsin levels exhibit an increasing trend as GFR decreases. Currently, the false high values in the standard reference range are largely ignored. Therefore, the use of the newly recalculated reference range, which excludes the effects of renal function and agitation, is expected to improve efficiency in sepsis diagnosis, particularly at the early stage.
14. Kanda Y. Investigation of the freely available easy-to-use software '    P value was calculated using Kruskal-Wallis one-way analysis of variance. P value adjustment was calculated using the Bonferroni method.