Therapeutic Plasma Exchange: Impact on Survival in Patients With Covid-19

BACKGROUND. Five percent of cases of COVID-19 will develop acute respiratory distress syndrome and cytokine storm syndrome, which are leading causes of death in patients with SARS-CoV-2 infection. Plasma exchange therapy (PLEX) effectively removes pro-inammatory factors, modulating and restoring innate and adaptive immune responses. This clinical trial aimed to evaluate the impact of PLEX on the survival of patients with severe SARS-CoV-2 and the effect on the cytokine release syndrome. METHODS. Hospitalized patients diagnosed with SARS-CoV-2 infection and cytokine storm syndrome were selected to receive 2 sessions of PLEX or standard therapy. Primary outcome was all-cause 60-days mortality; secondary outcome was requirement of mechanical ventilation, SOFA, NEWs-2 scores modication, reduction of pro-inammatory biomarkers and hospitalization time. RESULTS. Twenty patients received PLEX were compared against 40 patients receiving standard therapy. PLEX reduced 60-days mortality (50% vs 20%; OR 0.25, 95%CI 0.071 - 0.880; p = 0.029), and this effect was independent from demographic variables and drug therapies used. PLEX signicantly decreased SOFA, NEWs-2, pro-inammatory mediators and increased lymphocyte count, accompanied with a trend to reduce affected lung volume, without effect on SatO2/FiO2 indicator or mechanical ventilation requirement. CONCLUSIONS. PLEX therapy provided signicant benets of pro-inammatory clearance and reduction of 60-days mortality in selected patients with COVID-19, without signicant adverse events. conrmation. Clinical presentation of Acute Respiratory Distress Syndrome and increased levels of interleukine-6 > 40 pg/mL, ferritin > 500 ng/mL, C reactive protein (CRP) > 60 mg/L, erythrosedimentation rate > 40 m/sec; and/or lymphopenia < 1.0 x10/L. cels Alma Medical workstation version 5.0. For determination of non-affected lung parenchyma volume (NLV), automated segmentation tool was selected, and a reference attenuation range between − 1000 and − 600 HU was designated. Vascular structures, airways, and pathologic opacities were excluded. For determination of Lung Opacities Segmentation-Lung Opacities Volume (LOV): Initial automated segmentation was attempted using thresholding-based methods. A reference attenuation range between − 500 HU and 20 HU was selected. Then, the semi-automated option was selected to perform a region-based segmentation to adjust lesion boundaries. Volumes from each side were added to calculate total NLV and LOV. Total lung volume was calculated adding NLV + LOV.


Introduction
Since December 2019, health workers and governments around the world have been ghting a virus that changed our lives. COVID-19 is an infection caused by the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). [1] Until November 2021, around ve million people had died globally as a result of COVID-19. The data recorded in America already exceeded 2.3 million deaths [2].
Coronavirus disease is characterized by a wide spectrum of manifestations, ranging from asymptomatic to acute respiratory failure, multi-organ failure, and death, the result of macrophage activation and cytokine storm. [3] According to a report with more than 44,500 con rmed cases, up to 81% were mild disease (mild or nonexistent pneumonia); 14% were moderate diseases (eg dyspnea, hypoxia, or pulmonary involvement > 50% on tomography in the rst 24 to 48 hours); and cases of severe disease (respiratory failure, shock or multi-organ dysfunction) were reported in 5% [4].
Effective antiviral treatment and measures to modulate the innate immune response and restore the adaptive immune response are imperative to break the cycle and enhance the effect of treatment. Some drug therapies would take weeks or months to remove these pro-in ammatory factors, while plasma exchange therapy (PLEX) is able to effectively remove these large molecules. [11] This technique includes the removal of large plasma volumes, which must be replaced with replacement uids (eg, albumin, fresh frozen plasma). [12] This also makes it possible to selectively eliminate substances of high molecular weight from the intravascular space or to replace a de cient circulating factor. [13] The effectiveness of PLEX depends on the plasma volume (PV) removed from the patient, on the distribution of the pathogen to be removed. One exchange (1:1) is equivalent to 65% of the initial component removed from the intravascular space. One session were the exchange is calculated at 1.5 of the total plasma volume of the patient can remove about 75% of target molecules, and about 85% is achieved with 2 exchange sessions. [14] This trial aimed to evaluate the impact of plasmapheresis therapy on the survival of patients with severe SARS-CoV-2 and cytokine release syndrome who received at least two sessions during their hospitalization compared against standard therapy.

Methods
Study design. Our trial was designed to evaluate the impact of plasmapheresis therapy in the outcome of severe COVID-19 and cytokine release syndrome at one medical center in Mexico City. The study was Study population. Hospitalized patients between April-August 2020. Patients were aged 16 to 65 years old, diagnosed with SARS-CoV-2 infection, as con rmed by typical tomographic ndings according to Radiological Society of North America and/or qRT-PCR con rmation. Clinical presentation of Acute Respiratory Distress Syndrome and increased levels of interleukine-6 > 40 pg/mL, ferritin > 500 ng/mL, C reactive protein (CRP) > 60 mg/L, erythrosedimentation rate > 40 m/sec; and/or lymphopenia < 1.0 x10/L. Patients were excluded if they had SOFA score > 11 points, active bleeding, platelet count < 50,000 cels and/or hypo brinogenemia < 80 mg/dL. Written informed consent was obtained from all the patients or from a legal representative if they were unable to provide consent. Standard therapy. It was based on the use of chloroquine, azithromycin, dexamethasone, supplementary oxygen; as well as tocilizumab and intravenous immunoglobulin. This therapy was common for the control and PLEX group, while PLEX group received tocilizumab and immunoglobulin before PLEX therapy.
Plasmapheresis. A double lumen central venous catheter was placed, either at jugular or femoral vein approaches. The plasmapheresis therapy was performed with a membrane-based system, using PrismaFlex CRRT and a TPE 1000-2000 lter according to body surface. Exchange plasma volumes of 1.5 times the estimated circulating plasma volume, according to Kapplan's formula (Plasma volume (lts) = 0.065 x Weight (kg) x (1 -Hematocrit [%])). The blood ow rate was set at range 75-150 ml/min. Replacement solution consisted of 3% albumin, at a ow rate started at 100 mL/hr, and increased up to maximum of 1500 mL/hr; while 2 fresh frozen plasma were transfused at the end of each session. Anticoagulation was performed at doses of 30-40 IU/Kg/hr of unfractionated heparin. A second plasmapheresis session was systematically performed 48 hours after the rst session. Blood samples were obtained from catheter blood before and after every session, for cytokine determination. Lung damage determination. One day before starting treatment and ten days after the last PLEX, the volume of lung involvement was calculated through tomographic volumetric assessment. Non-contrast enhanced chest CT imaging was performed using 2 CT scanners (Siemens SOMATOM drive and Siemens SOMATOM emotion scanners, Siemens Healthineers, Germany). Imaging reconstructions were performed with a 1-mm thickness slices without interstice gap. Lung segmentation was performed using the Alma Medical workstation version 5.0. For determination of non-affected lung parenchyma volume (NLV), automated segmentation tool was selected, and a reference attenuation range between − 1000 and − 600 HU was designated. Vascular structures, airways, and pathologic opacities were excluded. For determination of Lung Opacities Segmentation-Lung Opacities Volume (LOV): Initial automated segmentation was attempted using thresholding-based methods. A reference attenuation range between − 500 HU and 20 HU was selected. Then, the semi-automated option was selected to perform a regionbased segmentation to adjust lesion boundaries. Volumes from each side were added to calculate total NLV and LOV. Total lung volume was calculated adding NLV + LOV.
Outcomes. The primary outcome was all-cause mortality within 60 days after inclusion. Secondary outcomes were the free mechanical-ventilation days, changes in SOFA score, decrease of proin ammatory markers at day 7, hospital length-of-stay, and decrease of lung's volume involvement.
Statistical analysis. Data distribution was assessed by Kolmogorov-Smirnoff test. Then, qualitative and quantitative data were resumed as n(%) and mean ± SD, respectively. Inferential analyses were performed by either chi square, one-way independent T-test, or U-Mann-Whitney. Kaplan-Meyer curves were constructed. Risk estimation was evaluated through OR and CI95%. Statistical signi cance was considered at p < 0.05.

Results
Twenty patients who were eligible to receive plasmapheresis (PLEX) constituted the study population, and were compared against 40 patients who only received standard therapy.
Patients in the PLEX group were mean aged 47 years old, 85% males, with obesity (80%) as most prevalent comorbidity; and signi cantly higher ferritin, IL-6 and lower platelet count, as compared to control group. There were no differences regarding baseline SOFA and NEWs-2 scores, or other proin ammatory, pro-coagulant proteins and/or acid/base equilibrium between PLEX and control group, as shown in Table 1. Regarding time-to-PLEX, mean time to receive rst exchange was 4.9 ± 3.1 days, and mean time from the rst symptom to PLEX was 12.2 ± 5.2 days. During PLEX therapy, the mean volume exchange was 4.46 ± 0.37 liters.
Regarding the primary outcome, PLEX group reduced 60-days mortality (PLEX 20% vs 50%; OR 0.25 95%CI 0.071-0.880; p = 0.029), and survival curves are shown in Fig. 1. Moreover, effect on mortality was independent from demographic variables and drug therapies used (Fig. 2). However, PLEX did not affect the risk of mechanical ventilation and it was associated with longer hospital stay. Regarding severity scores, SOFA and NEWs2 scores tended to decrease in PLEX group, with an opposite effect in the control group (p = 0.02, Table 2). *5-7 days after treatment. # Evaluated 7 to 10 days post-PLEX.
For secondary outcomes, PLEX therapy effectively reduced pro-in ammatory mediators and increased lymphocyte count, accompanied with a trend to reduce affected lung volume, without effect on SatO2/FiO2 indicator or mechanical ventilation requirement (50% vs 55%, OR0.81, 95%CI 0.279-2.398; p = 0.78) or time to start mechanical ventilation (4.80 ± 3.94 vs 3.23 ± 2.69 days PLEX vs controls (p > 0.05); whereas the control group showed a trend to worsen most of pro-in ammatory and pulmonary indicators ( Table 2, Fig. 1).
Adverse effects related to PLEX were hypotension episodes in ve patients (5/20) that were treated with uid bolus and/or noradrenaline infusion. Four (16%) patients were diagnosed with secondary bacterial infections after 7 days since the last PLEX: 3 pneumonias, 1 empyema. Gram-negative bacteria were isolated in every case.

Discussion
In our study, plasmapheresis (PLEX) showed to improve survival from patients with severe COVID-19, as compared with control group. Consistently, several case-series have reported a reduction of 28-days mortality associated with PLEX, which ranges between 10% and 28% in patients with SARS-Cov2, ARDS and cytokine release syndrome. [15,16,17] Indeed, one study achieved zero mortality in patients with severe COVID that received 5 sessions of PLEX (0% vs 35%, PLEX vs not PLEX), without adverse events reported.
[18] This may be due to the fact that in the group that received PLEX only one patient had severe pneumonia and in the control group 50% of the patients did.
It is possible that the effect of PLEX be mediated by the clearance of pro-in ammatory mediators and toxic biological substances with molecular weights bigger than 15,000 daltons, representing a therapeutic option in patients with hyperin ammatory state secondary to SARS-CoV-2 infection. In our study PLEX was effective to clear pro-in ammatory mediators and to reduce mortality; however, it did not prevent mechanical ventilation, suggesting that additional disorders may underlie pulmonary failure.
A point to highlight is the selectivity of patients with potential bene t from PLEX therapy, which include particular features like severe course of COVID and evidence of cytokine storm syndrome. Similarly, several trials using such indications for PLEX were registered during the present pandemic. [15,16,19].
Interestingly, there is not a current accepted de nition for cytokine release syndrome, and whether this syndrome represents an appropriate in ammatory response is still controversial. [20] In the present study, cytokine release syndrome was considered as IL-6 cutoff value higher than 40 pg/mL, accompanied by elevation of other markers of in ammation and lymphopenia. Previously, Guiaro et al, found that an IL-6 cut-off value higher than 35 pg/mL was associated with increased mortality and higher risk for ICU admission. [21] According to systematic revisions, other biomarkers have been considered to select patients for potential bene ts from PLEX therapy; like CRP 132 mg/L (79-168.5), ferritin of 1332 ug/L (1125-1444) and lymphocytes 0.7x10 9 /L (0.58 -1.0). [22] Along clearance of pro-in ammatory mediators, PLEX showed to improve lymphopenia in our study, potentially representing a prognosis modi cation, since severe lymphopenia (< 500/mm 3 ) has been found as independently associated with higher mortality rate in COVID-19 (adjusted OR of 5.63). [23] Regarding adverse events, potential mechanisms related to PLEX has been described to clearance of procoagulant factors, immunoglobulins and cytokines, leading to risk of bleeding and/or immunosuppression. In our study only 4 patients developed hospital-acquired infections like pneumonia and one subject developed empyema. Finally, the length of hospital stay was signi cantly longer in the group with PLEX. Comparatively, PLEX therapy during Faqihi's trial signi cantly reduced length of ICU stay. This difference may be explained because institutional CT scan programming was performed at day 10 after the rst session of PLEX, independently from severity scores. [16] Limitations of the present study includes the heterogeneity of pharmacologic therapy, which was inherent to the available scienti c evidence during the course of pandemia; as well as the limited sample size, which may be explained by the reduce number of cases with complete information for an adequate analyses.

Conclusion
Page 13/17 PLEX therapy provided signi cant bene ts of pro-in ammatory clearance and reduction of 60-days mortality in selected patients with COVID-19, without signi cant adverse events. These results are relevant to better characterize the effect of PLEX in patients with COVID-19; which may contribute to establish more speci c therapeutic protocols, based in selection of potential candidates and expected bene ts.