**2. Soluble CD14 subtype (sCD14-ST)**–**presepsin**

Presepsin, which is also known as soluble CD14 subtype (sCD14-ST) (64 aminoacids, 13 kDa) is a glycoprotein fragment mostly synthesized and released in general circulation by macrophages or monocytes/macrophages in response to infections [19, 20]. The CD14 receptor is a pattern recognition receptor in the innate immune response that has the ability to identify different pathogen-associated molecular pattern molecules of both Gram-positive and Gram-negative bacteria such as lipopolysaccharide, peptidoglycan, lipoteichoic acid, etc. The best-studied ligand is lipopolysaccharide (LPS) of Gram-negative bacteria [21, 22].

CD14 has two forms: membrane-bound CD14 (mCD14) and soluble CD14 (sCD14). The first, mCD14 receptor (356 amino acids, 55-kDa), is a membrane glycoprotein [23], whose C-terminal leader sequence of 28–30 amino acids is replaced by a glycosyl phosphatidylinositol (GPI) anchor after translation [24]. Thus, mCD14 is anchored to the cellular membrane through GPI linkage. It does not possess an intracellular tail; it is not a transmembrane protein and is unable to transmit a signal by itself. Although CD14 is detected predominantly on the surface of myeloid-lineage cells; however, low amounts are also found in non-myeloid cells, e.g., hepatocytes, adipocytes, corneal, and intestinal epithelial cells. The circulating soluble formsCD14 is found in plasma and is produced by mCD14 fall-off or cell secretion [25].

Various stimuli induce shedding of the GPI anchored mCD14, probably mediated by serine proteases such as leucocyte elastase [26] resulting in sCD14 with a molecular mass of 48–49 kDa. Some CD14 molecules stored intracellularly escape GPI anchor attachment and keep the C-terminal leader sequence, resulting in sCD14 with a molecular weight of 55–56 kDa [27]. After that, sCD14 is cleaved by cathepsin D and

#### *Presepsin as a Diagnostic and Prognostic Biomarker in Sepsis DOI: http://dx.doi.org/10.5772/intechopen.107955*

other proteases in plasma or the phagolysosome and the truncated 13-kDa form of sCD14, called presepsin is generated and released into circulation.

After being released into circulation, lipopolysaccharide (LPS, endotoxin) of Gramnegative bacteria (the prevailing cause of severe sepsis in humans) binds to a specific plasma protein known as lipopolysaccharide-binding protein (LBP). LBP presents LPS to CD14 which is recruited to the receptor after ligand binding. The LPS/LBP complex after binding to CD14, mediated by myeloid differentiation factor 2 (MD2), finally binds to Toll-like receptor 4 (TLR4), resulting in its activation. Both membrane-bound and soluble CD14 can transfer the LPS molecule to the TLR4/MD-2 complex. TLR4 activation sequentially triggers two signaling cascades. The first one is the MyD88-signaling pathway at the plasma membrane, and the second is the TRIF-dependent signaling pathway. Dimerization of the receptor complex induces the assembly of TIRAP, MyD88, and IRAK kinases in a submembrane signaling complex called the myddosome at the TIR domain of TLR4 inducing a signaling pathway inducing expression of genes encoding pro-inflammatory cytokines (IL-6, TNF-α, etc.). The MyD88-dependent signaling is followed by internalization of TLR4 in endosomes. Simultaneously, TIRAP and MyD88 dissociate from the membrane allowing TLR4 to bind in the endosome the second set of TIR-containing adaptor proteins, TRAM and TRIF, controlling the production of type I interferons, CCL5/RANTES. Via activated NF-κB TLR4 also contributes to the activation of the cytosolic NLRP3 inflammasome [28]. Notably, while CD14 is marginally important for MyD88-dependent TNFα expression, it is essential for TRIF-mediated IFN expression because it regulates TLR4 endocytosis [29].

#### **2.1 Kinetic of presepsin release**

The kinetics of presepsin is extraordinarily rapid in blood. Presepsin levels increase in the blood within 2 h after the onset of any infection, with a maximum concentration after 3 h, and tended to decrease on day 7. The half-life of presepsin is 4–5 h at the plasma level [30]. In 2008, Nakamura et al. [31] in an experimental peritonitis model of sepsis in rabbits using cecum ligation and puncture, detected presepsin in the blood of animals 2 h after initiation of the procedure. Presepsin levels peaked at 3 h and decreased 4–8 h after initiation of the procedure. In 2016, Chenevier-Gobeaux et al. [32] measured presepsin levels after LPS stimulation in peripheral mononuclear cells (PMNC) and human cell line of mononuclear cells (THP-1), and found that In THP1 cells, presepsin levels increase after 1 h, reaching a peak after 3 h, and decreasing at 4 h after LPS exposure. In PMNC, exposure to LPS induced an increase of median presepsin levels as early as hour 1, concomitantly to IL-6 synthesis.

### **3. Measurement of soluble CD14 subtype (sCD14-ST)**–**presepsin**

Different methods have been developed for the determination of presepsin. The first, presepsin concentrations were measured using a two-step sandwich enzyme-linked immunosorbent assay (ELISA). Large recombinant CD14 antigen (S286C, approximately 40 kDa) was used as standard. The dynamic range of the two-step assay was 3–150 ng/ml. The total assay time was 4 h. This test was sufficient to evaluate presepsin as a diagnostic marker for sepsis, but it lacked the speed and accuracy that are required for routine presepsin tests in ICU. Soon after, Shirakawa et al. [33] modified a traditional two-step into one-step ELISA. The standard used in the one-step method was recombinant presepsin, the sample dilution step was eliminated and two new anti-presepsin

antibodies were used: F1106-13-3 monoclonal antibody as the capture antibody and S68 polyclonal antibody as the detection antibody. The dynamic range of the one-step assay was 0.05–3.00 ng/ml. The total assay time was 1.5 h. This assay is suitable for clinical practice [33]. However, this method has low linearity and several endogenous (rheumatoid factor, cross-reactive substances, complement) and exogenous (specimen hemolysis, bacteria, and iatrogenic tube contamination) interfering factors [34].

In 2011, Okamura and Yokoi [35] have developed and evaluated the analytical and clinical performance of the highly sensitive, fully automated PATHFAST Presepsin test. This test performed on the PATHFAST instrument (Pathfast, Mitsubishi Chemical Medicine Corporation, Tokyo, Japan) is based on a noncompetitive chemiluminescent enzyme immunoassay combined with Magtration® technology. The principle of the test is as follows. The patient sample is dispensed into the reagent cartridge. The instrument combines the patient sample, the anti-presepsin monoclonal antibody-coated magnetic particles, and the alkaline phosphatase-labeled anti-presepsin polyclonal antibody and incubates the mixture for 5 min at 37°C. During this incubation, the analyte in the patient sample binds to the antibody on the coated particles, and the alkaline phosphatase conjugate binds to the analyteantibody coated-particle. After the incubation, the instrument performs Bound/ Free (B/F) separation using Magtration® technology to remove any excess unbound reagents, chemiluminescent substrate (CDP-Star Chemiluminescent Substrate) was added. The substrate is catalyzed by the bound alkaline phosphatase, which results in the emission of photons. The photo-multiplier tube in the PATHFAST instrument detects the photons that are emitted during the reaction. The chemiluminescent count is converted to analyte concentration values by the instrument based on the master calibration curve for the reagent lot [36]. According to the manufacturer's data assay range is 20–20,000 pg/ml. The assay reveals its result within 15 min in six samples simultaneously, using a sample volume of 100 μl. The calibrator for the PATHFAST Presepsin assay is recombinant presepsin (13 kDa). The sensitivity of the PATHFAST Presepsin assay was sufficient to detect the presepsin concentrations of the healthy group. It was higher than the recently reported one-step ELISA [37]. The PATHFAST Presepsin assay correlated well with a previously reported two-step presepsin ELISA [33]. PATHFAST is applicable for use in the Emergency Department (ED), ICU, and surgical wards. No interference of presepsin was noticed for bilirubin, hemoglobin, lipids, triglyceride, or rheumatoid factors [35].

Recently, evaluation results of a new automated one-step sandwich chemiluminescent enzyme immunoassay for presepsin measurement on Sysmex HISCL-5000 (Sysmex, Japan) was published [38]. Briefly, biotinylated anti-presepsin monoclonal antibodies specifically react with presepsin in the sample. This complex binds to streptavidin-coated magnetic particles (MPs). After bound/free separation, alkaline phosphatase (ALP)-labeled anti-presepsin monoclonal antibodies specifically bind to the presepsin on an MP. Then, the ALP on the MP breaks down the CDP-Star® chemiluminescent substrate which produces a luminescent signal. Assay samples were tested undiluted, with an analytical measurement range of–30,000 pg/ml. Turn-around-time for measuring the first sample was 17 min and the total processing capacity was around 200 tests per hour. For the first time, Sysmex HISCL-5000 and PATHFAST presepsin tests were performed, and a high significant correlation was found. Also, SCD14-ST concentration picogram/milliliter (pg/ml) can be measured using CL1200i (Mindray, Shenzen, China), according to manufacturer protocol based on sandwich chemiluminescent enzyme immunoassay. The measurement range of the assay was 20–20,000 pg/ml [39].
