Endotoxin and Cytokine Adsorption Properties of the Oxiris Hemofilter in Septic Shock: Evaluation of Saturation Phenomena and Inflammatory Mediators' Clearance Capacity
Conditions: Sepsis, Septic Shock, Blood Purification, Hemoadsorption
Sponsor: Pauls Stradins Clinical University Hospital

Official Study Title: 
Endotoxin and Cytokine Adsorption Properties of the Oxiris Hemofilter in Septic 
Shock:  
Evaluation of Saturation Phenomena and Inflammatory Mediators’ Clearance Capacity 
 
Sponsor: 
Pauls Stradiņš Clinical University Hospital, Riga, Latvia 
 
Study Type: 
Investigator-Initiated Study 
 
ClinicalTrials.gov Identifier: 
Pending (to be assigned) 
 
Document: 
Study Protocol  
 
Version: 
Version 1.0 
 
Date: 
[04.04.2026] 
 
 
 
 
[04.02.2026]
[23.01.2026]

 
Study Title: Endotoxin and cytokine adsorption properties of the Oxiris hemofilter in septic shock: 
evaluation of saturation phenomena and inflammatory mediators’ clearance capacity. 
Brief Title: OxiCLEAR (Oxiris Cytokines and Endotoxin Adsorption Rate) study: Evaluating Saturation 
Phenomena of Oxiris Membrane  
Hypothesis: We hypothesize that the Oxiris membrane becomes saturated with endotoxin and 
inflammatory mediators over time (24-hours period), resulting in reduced adsorptive capacity and potentially 
limiting the therapeutic efficacy of blood purification in patients with septic shock. 
Objectives:  
[O1] to assess the dynamics of the adsorption capacity of AN69-polyethylenimine membrane (Oxiris®) for 
endotoxin over a 24-hour period in septic shock patients who are admitted to the intensive care unit. 
[O2] to assess the dynamics of the adsorption capacity of AN69-polyethylenimine membrane (Oxiris®) for 
pro-and anti-inflammatory mediators over a 24-hour period in septic shock patients who are admitted to the 
intensive care unit. 
[O3] To assess the association between decreased performance of the AN69-polyethylenimine membrane 
(Oxiris®), potentially related to early saturation, and the clinical course and 28-day mortality in patients with 
septic shock. 
Primary Endpoint: 
The primary endpoint of this study is to evaluate the adsorption capacity of the AN69-polyethylenimine 
membrane (specifically the Oxiris® hemofilter) for endotoxins [O1] and inflammatory mediators [O2] in 
patients with septic shock undergoing continuous veno-venous hemofiltration (CVVH). Objective [O2] will 
be extended to include the evaluation of cytokine removal through convective transport by analyzing 
cytokine concentrations in the effluent fluid. This will allow for a more accurate assessment of the 
membrane’s net adsorption capacity by distinguishing between cytokine clearance via convection and 
adsorption. The study aims to assess the correlation between inlet endotoxin concentration and the rate of 
adsorption over a continuous 24-hour period, in order to characterize membrane saturation dynamics and 
their potential clinical implications [O3]. 
Number of Sites: Pauls Stradinš Clinical University Hospital (PSCUH), Department of Anesthesiology 
and Intensive care, Riga, Latvia. 
Study Population: 
The study population will include adult patients (≥18 years of age) of any gender who are diagnosed with 
septic shock and admitted to the intensive care unit (ICU). All enrolled patients must receive continuous 
veno-venous hemofiltration (CVVH) with a high-adsorptive AN69-polyethyleneimine membrane (Oxiris®) 
for a minimum duration of 24 hours as part of their clinical management. 
Key Inclusion Criteria: Should meet all the criteria mentioned below: 
1. Septic shock patient with a Dynamic scoring system1 of 6-8 points  
/Septic shock definition based on Sepsis-3: a vasopressor requirement to maintain a mean arterial 
pressure of 65 mmHg or greater and serum lactate greater than 2mmol/L despite optimal (30ml/kg body 
weight) bolus fluid resuscitation/  

2. CVVH with AN69-polyethylenimine membrane for at least 24 h 
3. Age >18 years 
4. Informed consent provided by the patient or by a person with decisional responsibility. 
 
1. Kogelmann, K.; Hübner, T.; Schwameis, F.; Drüner, M.; Scheller, M.; Jarczak, D. First Evaluation of a New Dynamic Scoring 
System Intended to Support Prescription of Adjuvant CytoSorb Hemoadsorption Therapy in Patients with Septic Shock. J. Clin. Med. 
2021, 10, 2939. https:// doi.org/10.3390/jcm10132939 
Key Exclusion Criteria: 
Patients will be excluded from the study if they meet any of the following conditions: age under 18 years, 
pregnancy, known contraindications to citrate anticoagulation, or a clinical judgment indicating a high 
likelihood of death within the next 24 hours due to irreversible comorbidities (e.g., end-stage cardiac, 
pulmonary, or hepatic disease; hepatorenal syndrome; or advanced, uncontrolled malignancy). 
Time of follow-up / Duration of subject participation: Each subject will be followed for a total duration 
of 28 days after the initiation of CVVH therapy with the Oxiris® hemofilter. Data collection will include 
intensive monitoring during the first 24 hours to assess adsorption dynamics, followed by ongoing clinical 
assessments up to day 28 to evaluate outcomes such as organ function, treatment response, and the 
potential impact of membrane saturation. 
Modality of CRRT: to ensure uniformity of therapy across all study participants, standardized CRRT 
settings will be applied in accordance with clinical best practices. All patients enrolled in the study and 
undergoing CRRT will be treated using continuous veno-venous hemofiltration (CVVH) with the PrisMax or 
Prismflex system and the high-adsorptive AN69-polyethyleneimine membrane (Oxiris®). CRRT parameters 
will follow the institution's local protocol for septic shock patients and include: 
● 
Anticoagulation: Continuous citrate infusion for circuit anticoagulation 
● 
Filtration dose: 25–30 mL/kg/h 
● 
Blood flow rate: 100–150 mL/min 
● 
Net ultrafiltration rate: 0 mL/h (as the standard approach for hypovolemic septic shock patients) 
● 
Replacement fluid rate: 1000–1500 mL/hour, with a 50/50 distribution between pre-dilution and 
post-dilution 
These standardized parameters are intended to minimize variability and ensure consistent treatment 
conditions for accurate comparison of adsorption dynamics and patient outcomes. 
Data collection: 
Sample Collection Time Points: blood samples for endotoxin and inflammatory mediators measurement will 
be collected at the following time points: T0 (Baseline): Immediately before initiation of CVVH with Oxiris® 
and during the next 24 hours of the treatment at the following times: 1hour (T 1), 3hours (T 3), 6hours (T 
6), 12hours (T 12) and 24 hours (T 24). 
Sample Collection Sites in the CRRT circuit: 
● 
Inlet / Arterial Line (C_in for endotoxin and cytokines 
● 
Outlet / Venous Line (C_out) for endotoxin and cytokines 
● 
Effluent Samples: Effluent fluid samples will be collected at the corresponding time points to detect 
anti- and pro-inflammatory mediators, enabling assessment of clearance via the convection 
mechanism. 
Sample Processing: immediately after collection, blood samples will be centrifuged for 10 minutes, and the 
plasma and samples from effluent will be stored at –80°C until analyses will be performed for endotoxin 
and inflammatory mediators concentration is performed. 

Endotoxin concentrations will be measured using assays based on LSBio, Gram Negative Endotoxin 
(Competitive EIA) ELISA Kit with detection range of 100-2500 pg/ml and sensitivity for endotoxin 
concentration detection of ≤0.005 EU/ml.  Method Precision (Analysis of Preliminary Data): Across all 
sample series and dilution levels, the relative standard deviation (RSD) ranged from 2.0% to 2.3%, 
indicating low variability and high precision of the method under the tested conditions. 
Inflammatory mediators’ concentrations in the plasma and effluent will be measured using the following 
methods:  
- 
Inflammatory mediator concentrations will be assessed using the Multiplex Biochip Analyzer 
Evidence Investigator platform (Randox Laboratories, EV3513 Cytokine and growth factors 
array), integrated within the RADOX system. The platform enables simultaneous quantification of 
multiple cytokines from a single sample using Cytokine Array I, ensuring high sensitivity, 
reproducibility, and broad biomarker coverage relevant to inflammatory response profiling. 
- 
Cytokines Included in the Cytokine Array I panel: EGF (Epidermal Growth Factor), IFN-γ 
(Interferon gamma), IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, MCP-1 (Monocyte Chemoattractant 
Protein-1), TNF-α (Tumor Necrosis Factor alpha), VEGF (Vascular Endothelial Growth Factor). 
Investigators will ensure strict adherence to the study protocol. All anonymized patient medical records 
will be securely stored in electronic format and retained indefinitely to allow for future review, audit, or 
verification, if required. 
To assess the efficacy of the Oxiris® hemofilter in adsorbing endotoxin [O1] and inflammatory mediators 
[O2], and to evaluate the impact of premature membrane saturation on the clinical course and outcomes 
[O3] of septic shock patients, the following data will be collected during the course of this study: 
- 
patient data (age, gender, BMI); main diagnosis prompting ICU admission, pre-existing 
comorbidities, severity as assessed using Sequential Organ Failure Assessment (SOFA) and 
Dynamic score system, source control interventions, and bacterial growth in blood. Antibacterial 
therapy (dosage and administration of a/b therapy (intermittent vs continuous) and timing of 
initiation of antibiotic therapy.  
- 
To detect endotoxin [O1] and inflammatory mediators [O2] absorption capacity of Oxiris® 
membrane, and the membrane saturation limit, endotoxin and inflammatory mediators 
concentration will be measured on both sides of the filter,in addition to inflammatory mediators 
detection in the effluent Blood and effluent samples will be obtained from each patient immediately 
before initiation of the treatment with Oxiris® hemofilter (T0), and during the next 24 hours of the 
treatment at the following times: 1h (T 1), 3h (T 3), 6h (T 6), 12h (T 12) and 24 hours (T 24). The 
adsorption rate will be calculated based on the concentration difference between inlet and outlet 
samples, combined with the blood flow rate. These values will be expressed as units per minute 
(U/min) and normalized to the membrane surface area (U/cm²/min) to enable comparison across 
patients and time points. Inflammatory mediators concentrations will be determined by measuring 
levels in both blood and effluent samples, accounting for clearance through the filter. Effluent 
clearance of each cytokine will be calculated by multiplying its concentration in the effluent by the 
effluent flow rate. 
- 
Clinical outcomes [O3] will be defined by various measures, including 28-day mortality, renal 
recovery, need for further renal support, length of ICU stay, length of in-hospital stay and 
vasopressor-free days. Secondary outcomes also include hemofilter efficacy markers (at 24h), 
such as hemodynamic improvement, a decrease in lactate levels, reduced need for vasopressors, 
and a decline in inflammatory markers like Procalcitonin (PCT) and C-reactive protein (CRP). 

- 
Laboratory parameters will be measured at baseline (T0) and at T24 (the end of the first Oxiris® 
session) to assess systemic response and organ function. The evaluated parameters will include 
full blood count (hemoglobin, leukocyte count, thrombocyte count), coagulation markers (D-dimers, 
prothrombin time (PT), international normalized ratio (INR)), inflammatory markers (procalcitonin 
(PCT), C-reactive protein (CRP)), kidney and liver function biomarkers (urea, creatinine, alanine 
aminotransferase (ALAT), aspartate aminotransferase (ASAT), bilirubin), as well as nutritional and 
electrolyte levels (albumin, phosphorus (P), magnesium (Mg)), as recommended by standard 
clinical practice for patients undergoing CRRT. 
- 
Respiratory and hemodynamic parameters, including heart rate, arterial blood pressure, central 
venous pressure, and vasopressor dosage, will be recorded in the ICU in accordance with the 
standard monitoring protocol. Arterial blood gas analysis will be performed every 8 hours during 
the first 24 hours of treatment. 
CRRT parameters, including blood flow rate, replacement fluid rates, and transmembrane pressure (TMP) 
and Δ within the CRRT circuit, will be documented to assess filter lifespan and the occurrence of 
premature filter clotting or clogging. Additionally, the rate of complications potentially related to CVVH 
therapy will be recorded 
Statistical Methods - Primary Endpoint Analysis: 
The primary endpoint of this study is the adsorption rate of endotoxins and inflammatory mediators by the 
Oxiris® hemofilter, defined as the difference between inlet and outlet concentrations (in U/mL), multiplied 
by blood flow rate (mL/min). This value reflects the amount of adsorptive molecules removed per minute 
and is further normalized to the filter’s surface area (U/cm²/min). 
To evaluate the primary objective, the statistical analysis will test the correlation between inlet concentration 
and adsorption rate across multiple time points per patient over a 24-hour treatment period. 
Analytical Approach: 
● 
Pearson correlation coefficients will be calculated between inlet concentration and the 
corresponding adsorption rate at each available time point (excluding T0 to account for system 
equilibration). 
● 
Each individual correlation coefficient will be transformed using Fisher’s Z transformation to 
stabilize variance. 
● 
The mean Fisher Z value across all patients and time points will be computed and subsequently 
back-transformed to obtain the overall correlation coefficient (ρ).  
● 
A one-sided hypothesis test will be used to test: 
o 
Null Hypothesis (H₀): ρ = 0.5 
o 
Alternative Hypothesis (H₁): ρ = 0.8 
● 
Statistical significance will be assessed at α = 0.05. 
Sample Size Justification: 
A one-tailed test is justified because the study specifically expects a decrease in the correlation between 
inlet endotoxin concentration and adsorption rate as membrane saturation occurs. Since membrane 
saturation can only reduce adsorption efficiency—not increase it—testing for a decrease (rather than any 
change) aligns with the biological mechanism and improves the study’s statistical power. Using standard 
formulas for correlation testing with Fisher Z-transformation, a minimal sample size of 24 patients is 
required to detect a correlation of 0.8 versus 0.5 with 80% power and α = 0.05 (one-tailed). These 
calculations are reproducible and may be verified using standard statistical software (e.g., G*Power, R, or 
STATA). However, a target of 29 patients was chosen to provide a buffer for attrition and variability, 
maintaining statistical power and strengthening the validity of the study outcomes. 

Software and Tools: statistical analyses will be performed using R (v4.3 or later) and validated using 
GraphPad Prism or SPSS, with full scripts and datasets available for reproducibility. 
In addition, the project will utilize MATLAB and SAS for specialized analysis needs: 
• 
MATLAB (MathWorks) will be used for advanced mathematical modeling, including time-
dependent adsorption kinetics, membrane saturation dynamics, and nonlinear regression.  
• 
SAS (Statistical Analysis System) will be employed for confirmatory statistical modeling and clinical 
data management.  
 
Statistical Methods - Secondary Endpoint(s) Analysis: 
Secondary outcomes will include hemofilter efficacy markers at 24 hours, specifically hemodynamic 
improvement (e.g., increase in mean arterial pressure, decrease in heart rate), reduction in lactate levels, 
decreased vasopressor requirements, and a decline in inflammatory markers such as procalcitonin (PCT) 
and C-reactive protein (CRP). 
• 
Continuous variables will be reported as mean ± standard deviation or median with interquartile 
range, depending on data distribution. Categorical variables will be summarized as counts and 
percentages. Between-group comparisons will be performed using the Student’s t-test or Mann-
Whitney U test for continuous variables and the chi-square or Fisher’s exact test for categorical 
variables. Changes in continuous secondary outcomes over 24 hours will be assessed using paired 
t-tests or Wilcoxon signed-rank tests, as appropriate. 
• 
Missing data will be handled using complete case analysis; if the proportion of missing data 
exceeds 5%, multiple imputation techniques will be considered. Statistical significance will be set 
at a two-sided p-value < 0.05. Based on preliminary estimates and accounting for the inclusion of 
hemofilter adsorption rate as a covariate, a total sample size of 29 patients is planned to ensure 
sufficient power to detect meaningful differences in primary outcomes. 
 
Ethics:  
The study has been approved by the Ethics Committee as part of a PhD project entitled 'Endotoxin and 
cytokine adsorption properties of the AN69-based polyethyleneimine membrane in septic shock patients.' 
The approval was granted under decision number 2-PĒK-4/429/2022 on September 29, 2022