**4.1 Key findings**

The main findings of this study are:


*Extracorporeal Blood Purification with the Oxiris Membrane in Septic Shock DOI: http://dx.doi.org/10.5772/intechopen.106227*


AKI is a severe complication of sepsis: it's a common finding in 40–50% of septic patients and correlates with a high mortality. However, no effective therapy is currently available, and recent studies challenged the notion that AKI during sepsis depends on renal perfusion and systemic hypotension. New evidences suggest that a dysregulated immunological and inflammatory response induces microcirculatory alterations not responding to the usual intensive care treatment.

Data of our study confirm these results: IL6 is high at basal levels and probably has a direct toxic on the kidney and induce a vasoplegic response, which is also detrimental for the renal function.

Shimatsu et al. found that high quartiles of IL6 increased the risk of anuria and AKI, whereas Payen et al. found that plasma cytokine's profile differed according to AKI severity [8].

In adjunct to IL6, we also evaluated procalcitonin and endotoxin: they were elevated in all the AKIN groups. Procalcitonin increases during bacterial infection and during sepsis, may induce a toxic effect on the kidney, and is associated, too, with AKI development and mortality in critically ill patients, in line with our results. Very recently, Ronco et al. have shown that a combination of [TIMP-2] Å ~ [IGFBP7] and PCT improved the predictive ability for AKI occurrence. Unfortunately, in our study we did not measure biomarkers and we cannot confirm these data [9].

Nevertheless, the serial measurements of cytokines and procalcitonin and the changes of the renal function reinforce the Ronco's hypothesis that there is an association between inflammation and AKI development.

Interestingly more than 80% of patients, in our study, had endotoxin ≥0.6 ng/mL, a cutoff value, which correlates with a predictive positive response to BP in Euphrates trial.

In our study, as in Euphrates RCT, we enrolled patients with different infections (GRA– and GRAM+) or none (**Table 9**). Endotoxiemia, therefore, seems a global response during sepsis and AKI, not depending only on Gram-negative infections, but also on Gram + bacteria and probably by bacterial translocation, as recently pointed out by Honore' et al. [10].

The IL10 changes follow the changes of all other mediators, confirming that a proand anti-inflammatory response may coexist in the early phase of the sepsis.


#### **Table 9.**

*Pathogens isolated in the study population.*

This dysregulated immunological response translated in a multiorgan dysfunction with high basal lactate levels, the need of vasopressor therapy, and of mechanical ventilation in more than 80% of patients.

CRRT with Oxiris filter had many effects on this condition. First of all, this treatment improved the renal function: creatinine decreased and urinary output increased with improvement of SOFA renal (**Figure 1**). As expected, cardiocirculatory function improved: MAP increased with the decrease of noradrenaline infusion and SOFA cardiac (**Figure 1**).

These data are well explained in the context of the cardio-renal syndrome, in which oliguria, electrolytic disturbances, and uremia depress the cardiac function. However, the circulatory function during sepsis depends also on many inflammatory mediators. In this study, in effect, the improvement of the renal and the cardiac function is associated with the decrease of IL 6 and procalcitonin (**Table 7**).

Very recently, Zhay et al. showed a superiority of oxiris filter in confront of RRT with no adsorbing filter, with data on il6 endotoxin and cardio-renal response similar to our study. Notably, they showed also the changes of procalcitonin, as in our study. These data are recently confirmed by Xie et al., who stated that the changes of procalcitonin during the treatment may have a positive impact on the survival [9, 11, 12].

We observed also the changes of the endotoxin. This is in agreement with Kellum's in vitro study and Broman's and Zai's et al.'s clinical studies [7, 11].

Different from these studies, we used a chemiluminescence method to detect EAA changes during a longer time study. Whereas Broman and Zai evaluated EAA changes during 24- and 8-h treatment; in our study, EAA changes are evaluated at basal time and after 72 h of treatment.

Notably, endotoxin improved more in sepsis of abdominal origin then in thoracopulmonary sepsis, as previously shown by Cutuli et al. [13]. Probably in our study, as in Euphas 2, patients with abdominal infection have a more rapid decrease of endotoxin trough the source control of surgery. In this case, endotoxin declines rapidly also at extracellular and tissue level, promoting a more efficient adsorbing effect of the membrane **Figure 3**.

This important pro-inflammatory reaction, as expected, activates the coagulation response: platelets are in the lower normal range, PAI-1 is increased, and TEG analysis showed high or normal MA in more of 50% of patients, confirming that hypercogulation is a common trait during sepsis [14].

During the treatment, all coagulation parameters are stable or improve, as indicated by D/D, the stability of the platelet's numbers and decrease of PAI-1. Data of TEG, too, indicate a stability of all parameters.

We can hypothesize that some properties of the oXiris filter—heparine-coated layers, the adsorbing action of pro-inflammatory, and pro-coagulant mediators with the use of regional anticoagulation with citrate may modulate the coagulation cascade and prevent DIC.

However, heparin-coated membrane itself, as assembled on oXiris membrane, probably fails to prevent a full anticoagulation. Shetz et al., Seminars et al. reported that AN69 ST membrane and Oxiris membrane do not prolong filter survival without anticoagulation [15, 16].

Probably heparin-coated layers may be saturated very early, as the anticoagulant effect is likely localized to only areas of membranes where heparin is immobilized and exposed areas to politylenimine are not spared by thombogenicity.

*Extracorporeal Blood Purification with the Oxiris Membrane in Septic Shock DOI: http://dx.doi.org/10.5772/intechopen.106227*

#### **Figure 3.**

*Endotoxin response during CRRT with oXiris filter in different infection patients. In patients with sepsis of abdominal origin (abd) endotoxin decreases more than in patients with sepsis of thoracopulmonary (thor) origin. \*\*p < 0.01 in the group.*

Secondarily, during sepsis, activated monocite and other cells adhere to surface of extracorporeal circuit, inhibit fibrinolysis, and render clots resistant to heparin [17].

Third heparin has a weak activity on calcium concentration, whereas during sepsis, the most important pro-coagulant mechanism is induced by calcium release.

Finally, histones and other dams or pumps are strongly adsorbed by heparin layers, which may be early saturated and then inhibited to anticoagulate the membrane [18]. Thus, citrate protocol we used in this study seems a rational alternative to heparin [14].

The main advantage of citrate is that regional anticoagulation achieves optimal filter anticoagulation without affecting patient's coagulation. In this study, the coagulation parameters are stable, without induction of avert DIC.

In **Table 10**, the changes of TEG parameters during oXiris treatment are shown.

In **Figure 4**, the different changes of them during citrate and heparin anti coagulation are shown. In the heparin group MA and R were longer than citrate group, confirming that citrate achieves a better stability of the coagulation.

These data are in line with a very recent study by Osterman et al., who confirmed that coagulation profiles were stable during CRRT with citrate and no increase of prothrombotic status was found. Wiegele et al. confirmed these data [18].

We aimed, also, to control the in-filter changes of the coagulation to confirm whether citrate would be able to abolish the coagulation, as compared with heparin. Filter was fully anticoagulated by citrate, but not by heparin (**Figure 5**). This was little surprising, considering that heparin is widely used to anticoagulate all the extra corporeal circuits in many clinical contexts.

During sepsis, however, a pro-coagulant response is activated, either through calcium-dependent mechanism or inhibition of normal anticoagulant factors


#### **Table 10.**

*TEG parameters during CRRT with oXiris filter.*

#### **Figure 4.**

*TEG changes during CRRT with oXiris filter from arterial sample. In citrate group, MA was higher than in heparin group, as a more firm coagulation is achieved \*\* p < 0.01 between the groups. R and K are expressed in min, ANG in grade, and MA in mm.*

(e.g., decrease of AT III). In this way, heparin response is blunted and thrombosis of circuits may occur, with failure of the treatments and worsening of the patient's condition [19].

These data are in line with Panigada et al., who reported that citrate infusion to maintain ca++ filter <0.25 mmol/L fullly anticoagulated the ECCO2 removal circuit and had TEG changes as in our study [20].

Ostermann et al., otherwise, have shown that citrate anticoagulation does not affect intra-circuit parameters and that all patients exhibited raised thrombin generation.

At variance with this study, we utilized the Haemonetic TEG, which employs a heparinized blood sample that is mixed with dried reagents within each of the four channels of the cartridge. We cannot hypothesize whether these different changes of TEG parameters depend on a different method, or reagents or a different properties of the dialytic membranes have been used.

Failure of the circuit depends not only on coagulation factors, by also on platelets, which are activated during sepsis and CRRT.

In this study, we evaluated also platelet's function by thromboelastography: this was downregulated in the filter during treatment with citrate, but not with heparin (**Figure 4**).

*Extracorporeal Blood Purification with the Oxiris Membrane in Septic Shock DOI: http://dx.doi.org/10.5772/intechopen.106227*

#### **Figure 5.**

*In filter changes during CRRT with oXiris filter. In citrate R was higher, angle and MA lower than heparin group, as a stronger filter anticoagulation. \*\*\* p < 0.001 between the groups. R and K are expressed in min, ANG in grade, and MA in mm.*

As platelets are activated via a calcium-dependent mechanisms, citrate probably decrease platelet activation in many ways, including inhibition of PF4, extracellulare vescicles, and particle microparticle, which are strongly pro-coagulant **Figure 5**.

Heparin, itself, may enhance platelet aggregation probably by P2Y receptor: the effect of Ca++ inhibition by and the action of heparin may explain the different TEG changes and confirm that citrate has a full anti coagulation, better than heparin [21].

These data are, again, at difference with Ostermann, who observed no change in platelets function, using PFA-100 analyzer. Panigada, too, did not show any effect of citrate on platelet function evaluated by aggregometry. Also in this case we can conclude whether these divergent data stem from different methods of study or from an effect of the oXiris membrane we used.
