Blood purification therapy with a hemodiafilter featuring enhanced adsorptive properties for cytokine removal in patients presenting COVID-19: a pilot study

The purpose of this work was to assess the feasibility of EBP treatment with oXiris in patients with COVID-19, by means of a prospective, multicenter, and observational study on data recorded into the oXirisNet Registry (aRRT - http://​www.​arrt.​eu/​) [19], an Italian registry on patients with multiple organ dysfunction who have undergone EBP with the oXiris membrane. The study considered data of all patients with a confirmed diagnosis of COVID-19 admitted to the ICU between February and April 2020, who received treatment with the oXiris membrane for immunomodulation and/or support to renal function during AKI.

Diagnosis of SARS-CoV-2 infection was defined as a positive outcome to a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) at nasal/oral swab. AKI was defined in accordance with the KDIGO criteria. Previous treatments with other filters or further sequential treatment after treatment with oXiris represented criteria for exclusion from the study.

Finally, in order to describe the overtime variation of biochemical and clinical features among critically ill patients with SARS-CoV-2 infection but not treated with EBP, the study included data of a cohort of COVID-19 patients from the same centers.

Objectives of this preliminary study were (1) to assess the variation of IL-6 and multiorgan dysfunction scores overtime, (2) to compare observed versus predicted mortality rate in terms of disease severity scores, and (3) to assess the occurrence of procedure-related complications, clotting, and treatment duration.

Data considered for the study included main anthropometric, clinical and biochemical parameters (including inflammatory markers), comorbidities, disease-associated symptoms, and organ dysfunction severity indexes (APACHE IV score [20]; SOFA score). Measurement of IL-6 was evaluated by immunoassay analysis (Simple PlexTM, ProteinSimple, San Jose, California, USA) [21] across all centers. EBP details considered included data on prescription (e.g., flows, dose, and anticoagulation), delivery (e.g., filtration fraction or treatment effective time), and outcome (e.g., clotting and unintended discontinuation).

Clinical and technical evaluations were performed immediately before EBP initiation (T0), after 12 h (T1), and every 24 h thereafter for the first 10 days from T0. The follow-up ended either at ICU discharge or death if it occurred in the ICU.

All patients included in this prospective observational study received antimicrobials, EBP, mechanical ventilation, and any other supportive treatment in accordance with the clinical judgment of the treating center. Similarly, according to local practice, patients were placed in the prone position for at least 16 h a day when Pa = 2/FiO₂ < 150 (moderate-severe ARDS) [22].

EBP was carried out with the oXiris hemodiafilter (all patients had been prospectively recorded into the oXirisNet Registry). The choice for the specific use of the oXiris membrane was based on a patient-basis personalized approach and clinical judgment of the ICU team in consideration of the membrane’s specific technical features. The membranes were used on the PrismaFlex systems (Baxter, IL - USA).

The normality distribution of the variables was tested by the Chen-Shapiro test [23]. Descriptive statistics were performed. Frequencies and percentages were used for qualitative variables, while means and respective standard deviations were calculated for quantitative variables. Median and interquartile range (IQR) were calculated for quantitative variables with non-normal distribution.

Spearman’s and Pearson’s correlation coefficients were used to explore the relationship between quantitative variables. Kruskal-Wallis test was carried out to compare the groups for the variables with non-normal distribution. Sidak post hoc adjustments were performed for multiple comparisons.

In the regression analysis, the explanatory variables were coded as binary variables. For each of these events, we assessed by multivariate regression the combined effect of continuous and ordinal variables for which the previous univariate regression analysis had evidenced a significant effect at the time of the event. Boxplots were drawn to describe IL-6 and SOFA score variations.

No statistical comparisons were made between COVID-19 patients treated and not-treated with EBP, nor did we perform any sample calculation as this was a feasibility assessment and was åbeyond the scope of this study.

All calculations were carried out using a standard statistical package (STATA for Windows version 14.1, College Station, Texas, USA).

The present study was approved by the local Ethics Committee, “Comitato Etico di Area Vasta Centro, Regione Toscana”, Florence, Italy [rif. CEAVC 14334]. Given its observational design, the study did not involve any medical, pharmacological, or behavioral interventions in addition to the standard practice implemented by the physicians regardless of the registry. All research has been carried forth in the agreement with the principles laid out in the original Declaration of Helsinki and its later amendments, and data were handled in agreement with patient informed consent.

The median baseline IL-6 was 1230 pg/ml (IQR 895) and correlated with the severity of baseline SOFA score (rho = 0.44, p = 0.03) and in particular with the PaO₂/FiO₂ ratio (rho = − 0.47, p = 0.02).

During the first 72 h of the treatment, IL-6 significantly decreased overtime (p < 0.001 at Kruskal-Wallis test) (see Fig. 1a). In particular, the IL-6 serum concentration fell to 479 pg/ml (IQR 531) at 24 h to 320 pg/ml (IQR 259) at 48 h and to 160 pg/ml (IQR 141) at 72 h (p = 0.001 for each time point if compared to the baseline; a p = 0.05/3 = 0.016 was considered for statistical significance). Serum IL-6 reduction was statistically significant in the first 24 h of treatment (p = 0.001), but not in the second 24 h (∆ IL-6 = 197.5 pg/ml, p = 0.17, IQR 323) or third 24 h (∆ IL-6 = 118 pg/ml, IQR 100.5, p = 0.16) (a p = 0.05/3 = 0.016 was considered for statistical significance) (see Fig. 1a). Increased values of serum IL-6 were observed immediately after the EBP discontinuation (median 1615 pg/ml, IQR 1149).

The reduction in serum IL-6 concentrations correlated with improvement in organ function, as measured in a decrease of SOFA score (rho = 0.48, p = 0.0003). Such reduction observed during the first 72 h of treatment was statistically significant (p = 0.002 at Kruskal-Wallis test) (see Fig. 1b). Median values of SOFA score at main time points were 13 (IQR 6) at the baseline, 11 (IQR 6) at 24 h (p = 0.09), 8 (IQR 5) at 48 h (p = 0.001), 8.5 (IQR 4) at 72 h (p = 0.002); a p = 0.05/3 = 0.016 was considered for statistical significance (see Fig. 1b). The median SOFA score immediately after the EBP discontinuation was 7 (IQR 0).

With specific reference to organ functional recovery and mortality, the SOFA items that showed the most improvement with treatment related to the hemodynamic stability (in terms of Vasoactive Inotropic Score and vasoactive requirements in the first 72 h) and lung functions (quantified in terms of PaO₂/FiO₂ ratio).

The variation of the clinical and prescription parameters of the EBP at the various time points is described in Table 3.

Finally, eight COVID-19 patients not treated with EBT were also retrospectively observed as to overtime variation of the clinical and biochemical parameters (including the natural decay of IL-6 and SOFA score variation) (See Supplementary material Table S1).

In this cohort, every patient was treated with hydroxychloroquine and azitromycine, while no patient was treated with corticosteroids. Among them, 15 (40.5%) were also treated with tocilizumab, and 9 (24.3%) with lopinavir-ritonavir. Baseline levels and overtime variations in IL-6 and SOFA score in patients treated with tocilizumab and/or lopinavir-ritornavir are described in Table 4.

The expected mortality rate calculated based on the baseline APACHE IV score (117 ± 20) was 64.7% ± 16.2, whereas the ICU mortality observed was 56.4%, with a 8.3% mean difference. Stratifying patients based on ICU death, deceased patients had a longer time-to-EBP from the onset of COVID-19 symptoms compared to non-deceased patients (p < 0.001), independently from baseline SOFA score (p = 0.478 at multivariate logistic regression analysis adjusted for SOFA score).

The difference between expected and observed mortality was further analyzed by discriminating between early and delayed treatment first in terms of time and then of treatment needs. In the former case, patients were divided into early and delayed using cut-off as the median time-to-EBP of 14 days, which evidenced a 47.4% mortality rate for patients receiving early treatment against the 73.3% (IQR 34.3) expected mortality rate (median difference was 25.9%, IQR 33.6). Patients receiving delayed treatment had a 66.6% mortality rate compared to the 67.2% (IQR 11.3) expected APACHE IV mortality rate; (median difference was 0.1% (IQR 10.2).

Table 5 describes the treatment trend over time: 7 treatments (18.9%) prematurely clotted (running < 24 h); 2 of them were carried out without anticoagulation (50% of all treatments performed without anticoagulation), while 5 with regional citrate anticoagulation (18.6% of all treatment performed with citrate). These 7 treatments had also higher effluent dose (median 33.6 ml/kg/hr., IQR 9) and higher filtration fraction (median 31%, IQR 7.4). No electrolyte disorders were recorded during the treatment, nor catheter displacement, circuit disconnection, unexpected bleeding, air, or thromboembolisms due to venous cannulation of EBP performance. As to adverse events/complications, only one occurred related to infection of the vascular access during the extracorporeal treatment, which required replacement of the access.

With a specific reference to the inflammatory response, results from our population confirm the correlation between serum concentration of pro-inflammatory cytokines (such as IL-6) and multiorgan dysfunction. IL-6 is a leading mediator influencing systemic inflammation and has shown increased concentrations among COVID-19 patients with ARDS [27]. Higher concentrations of cytokines in COVID-19 patients are associated with organ dysfunction and worse outcome, and generally, the higher IL-6 in the blood, the higher level of SOFA score [13]. COVID-19 patients considered by the physician as “not severely inflamed” and who therefore did not undergo EBP (Table S1) had lower levels of IL-6 and SOFA score than those undergoing EBP with the oXiris membrane. Application of EBP, in this context, has been suggested by some authors for cytokine removal [14, 28] and thus as prevention or attenuation of inflammatory-related organ damage. In this context, the oXiris membrane has been proven to remove cytokines and endotoxin via unselective adsorption. Interestingly, a net decrease in IL-6 was observed in this study during EBP, along with a stable reduction of SOFA score, independently from other pharmacological therapies such as tocilizumab [27]. In accordance with other observational findings in critically ill septic patients, also in this study, the improvement in multiorgan dysfunction during EBP was mainly related to the restoring of hemodynamic stability (expressed in terms of vasoactive inotropic score) and oxygenation (measured as PaO₂/FiO₂). Although these reductions cannot be univocally advocated to EBP with oXiris in this study, our results seem to suggest a potential role of this treatment in reducing inflammatory mediators and improving multiorgan function. Table S1 shows the natural decay of IL-6 in critically ill patients with SARS-CoV-2 infection not treated with EBP admitted to the same enrolling centers. Clinical comparison with the overtime variation of IL-6 observed in COVID-19 patients treated with oXiris is particularly interesting. In particular, in the latter cohort, IL-6 serum concentrations markedly fell within the first 24 h of extracorporeal treatment, reaching asymptotically a steady state. IL-6 reduction was statistically significant in the first 24 h of treatment, becoming less important through the following 48 h and 72 h. Technically, this phenomenon might be explained considering the expected membrane fouling that occurs during continuous extracorporeal therapies (running for 24–72 h) and the expected reduction of adsorption properties due to overtime saturation of sites available for the unselective link between solutes and the membrane. For these reasons, a scheduled replacement of the membrane every 24 h might be considered in order to maximize the clearance effect over time [11]. In fact, this aspect should be carefully considered in a pandemic period that calls for careful allocation of limited resources available.

Interestingly, a slight increase in serum IL-6 can be observed at the EBP discontinuation. This effect may further suggest that EBP might have had an effect on maintaining serum IL-6 concentrations stable and low during the treatment. Nevertheless, this “rebound” in circulating inflammatory mediators (likely due to the increased IL-6 half-life related to the tocilizumab infusion [29, 30]) seems uncorrelated with the worsening of multiorgan function and naturally resolves in 2–4 days.

Notably, results on IL-6 and SOFA score overtime reduction should be carefully interpreted, considering that data might be positively influenced by the rate of drop-off of patients who died within the 72 h of observation (being those with worst values).

Although the pathophysiological rationale of the use of EBP is to attenuate systemic inflammation and prevent or mitigate multiple organ dysfunction as short-time outcomes, in this study, we also observed a slight though an interesting decrease in observed mortality vs predicted mortality rates predicted by APACHE IV score. Compared to the 64.7% expected mortality for our population, calculated based on the APACHE IV score, the observed rate markedly decreased after treatment down to 56% (in contrast with the 80% mortality reported for COVID-19 patients admitted in the ICU in China) [3, 5]. The comparison between expected and observed mortality rate was used as a surrogate outcome to better describe the potential role of EBP in critically ill patients with systemic inflammation. Interestingly, reductions in mortality rate were observed for all patients independently from baseline SOFA score and were most marked in those who underwent an early EBP both in terms of timing (time from COVID-19 symptoms and EBP initiation) and indication for extracorporeal treatment (proactive treatment for immunomodulation prior to the need of renal support due to AKI). In general, the time from symptom onset to extracorporeal therapy initiation in our population was in agreement with data from recent studies of COVID-19 patients [1‐3], with a median time of 14 days from symptom onset (vs 15 days in previous studies) [2, 4].

Overall, the treatment was administered with no specific complications, such as catheter displacement, accidental disconnection, bleeding, thromboembolisms, air embolism, or electrolyte disorders. In particular, changing the patient to a prone position did not affect the feasibility of EBP, which continued according to prescription. Such a result is extremely positive and addresses a shared concern among anesthesiologists on complications during maneuvers turning the patient with a large venous access. Despite many publications often describe extracorporeal therapies in COVID-19 as unfeasible due to hypercoagulability leading to unintended discontinuation of the treatment [29], clotting rates in our patients were similar to continuous RRT performed on other critically ill patients. Certainly, the delivery of extracorporeal therapies requires a more careful management of pharmacological and non-pharmacological strategies to reduce the occurrence of clotting. Some specific examples are the proper positioning a large vascular access—as recently recommended by an expert panel [11]—which has proven to be very effective in providing the adequate blood flow reducing filtration fraction or the optimization of anticoagulation (either with systemic heparinization or regional citrate). In our study, most patients underwent femoral venous cannulation with a vascular access over 25 cm, which guaranteed an optimal performance in terms of the blood flow (never below 150 ml/min). Similar to what was reported in other papers on treatment duration, treatments performed with regional citrate anticoagulation in our study also had a longer duration and were less affected by unintentional early interruption due to clotting. Interestingly, each RRT that had prematurely clotted (treatment < 24 h) had higher effluent doses (often prescribed to compensate the expected downtime of these patients) and higher filtration fraction (> 30%). However, beside these “classical approaches,” other newly developed concepts and technological advances that reduce membrane fouling and preserve extracorporeal clearance in patients with COVID-19 might also be considered. For instance, the use of highly biocompatible heparin-coated disposables such as the oXiris membrane might prevent local clotting activation and further prevent membrane fouling independently of the anticoagulation strategy applied.

The present study is the first to provide data on the use of this heparin-coated hemodiafilter in severely ill patients with SARS-CoV-2 infection, and it is potentially of use for conditions of hypercoagulation and hyperinflammation in COVID-19.

Given the observational nature of this study, we were unable to provide evidence of a causal relationship between this specific EBP and better patient outcomes nor establish the efficacy of EBP in improving organ dysfunction and ICU mortality. This would require further controlled studies. Also, given the few patients included, results did not point to any values or illness stage (with or with/without AKI) that might indicate a threshold or cutoff for the best moment for EBP initiation.

One aspect that was not addressed was an effluent drug and cytokine removal. Antibiotics and vasopressors are theoretically cleared by high-flux hemodiafilters (as the oXiris membrane is). Nevertheless, extracorporeal clearance of drugs specifically used for COVID-19 is theoretically not expected because of the drug’s molecular mass, surface electric charges, and protein binding. Tocilizumab, in particular, is unlikely removed by high-flux hemodiafilters considering its high molecular weight (149 KDa) [31]; furthermore, the electric charge of this complex protein likely reduces interactions with the filter membrane for the Gibbs-Donnan effects. Cytokines were not assessed nor in the effluent (to demonstrate the transmembrane clearance of this solutes across the high-flux membrane of oXiris), neither pre- and post-filter (to assess the adsorption of these solutes into the membrane). The overtime reduction of IL-6 during the treatments cannot thus be formally attributed to the EBP effect. Endotoxin removal was also not addressed due to the lack of specific measurements, but this was beyond the scope of our study.