pathogenic cytokines, has also been hypothesized to have an additive role in managing early sepsis having onset less than 12 hours 5 . Our study was aimed to demonstrate that efficient control of the cytokine storm during early phase might be beneficial to selective patients, however, no prospective study on TPE has been conducted so far in patients with COVID-CRS.
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It was an interventional retrospective Propensity score matched (PSM) single centre based cohort study in Pak Emirates Military Hospital Rawalpindi (PEMH), Pakistan from 1st April to 30th June 2020. This study was carried out at the Department of Pulmonology and Critical care. PEMH is the largest Covid-19 designated hospital in the country.
Data of all hospitalized patients is maintained by PEMH Covid- 19 Research and evaluation cell.
The study was approved by Institutional Review Board.
Definitions of study groups: Data was extracted for patients with COVID-19 admitted with or developing Cytokine release syndrome (CRS) during their admission. CRS was defined as per National guidelines for COVID-19 6 given as: Fever of equal to or more than 100 F persisting > 48 hours in absence of documented bacterial infection and ANY of the following in the presence of moderate, severe or critical COVID- 19 (1) Ferritin >1000 mcg/L and rising in last 24 hours (2) Ferritin >2000 mcg/L in patient requiring high flow oxygen or ventilation (3) Lymphopenia < 800 cells/ul or lymphocyte percentage <20% and two of the following (a) Ferritin >700 mcg/mL and rising in the last 24 hours (b) LDH > 300 IU (reference 140-250 IU/L) and rising in the last 24 hours (c) D-Dimer >1000ng/mL (or >1mcg/ml) and rising in the last 24 hours (d) CRP >70 mg/L (or >10 hsCRP) and rising in the last 24 hours, in absence of bacterial infection (e) If any 3 of above are present on admission, a rising trend was not required.
Severity of disease was defined as per criteria designed by WHO 7 . Moderate disease was defined as COVID-19 positive case with lung infiltrates < 50% of total lung fields on CXR / peripheral ground glass opacities (GGOs) on HRCT chest but no evidence of hypoxemia. Severe disease was operationally defined as COVID-19 pneumonia with evidence of hypoxemia (RR > 30/minute or PaO2 on ABGs < 80mmHg or PF ratio < 300 or lung infiltrates > 50% of the lung field). Critical disease was defined if there was COVID-19 pneumonia with evidence of either respiratory failure (PaO2 < 60mmHg) or multiorgan dysfunction syndrome (MODS) measured by SOFA score > 10 or septic shock (Systolic BP less than 90 or less than 40mm Hg of baseline in hypertensive or Urine output < 0.5 ml/kg/hour). As per Institutional COVID-19 Management Guidelines all   patients of moderate, severe and critical COVID-19 received standard protocol of aspirin, anticoagulation, ulcer prophylaxis, awake Proning (if PaO2 < 80mmHg) and corticosteroids. All patients of CRS received Methylprednisolone 1 mg/kg irrespective of disease severity. In addition to standard care TPE was offered as a trial investigational therapy to willing patients with CRS. All patients were explained the investigational role of Therapeutic plasma exchange (TPE) in treatment of COVID-19. Written consent was taken from those who agreed for this treatment. TPE was performed once daily using COBE Spectra Apheresis machine (Manufacturer TERUMO BCT, INC) having continuous flow centrifugation. Venous access was All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint this version posted July 29, 2020. . https://doi.org/10.1101/2020.07.23.20160796 doi: medRxiv preprint achieved using an ultrasound guided double lumen catheter (Arrow -12 FR) via femoral vein.
Patient's total blood volume was calculated as per Nadler's formula. Anticoagulant acid dextrose ratio was 1:10 and flow rate 30-40 ml/minutes (Adjusted as per hemodynamic status). Patients' blood pressure, pulse, oxygen saturation was monitored throughout procedure. Duration of procedure varied from 2-4 hours and 1-1.5 times total plasma volume was removed during each procedure. Replacement fluid was fresh frozen plasma (FFP) and normal saline in 2:1 respectively. All procedures were performed in intensive care or high dependency unit by Apheresis Department of PEMH. TPE was continued till recovery.
Recovery was defined by de-escalation of patients condition from severe to moderate, or from moderate to mild, plus at least 2 of the following; serum Ferritin < 1000 ug/ml (and decreasing trend on two consecutive days), serum LDH normalization, C-reactive protein > 50% fold reduction (and decreasing trend in on two consecutive days),), ALC > 1000 and PT/APTT normalization. TPE related complications were also documented. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 29, 2020. . https://doi.org/10.1101/2020.07.23.20160796 doi: medRxiv preprint Statistical analysis: Retrospective observational studies involving therapeutic interventions are often confounded by either measured or unmeasured baseline characteristics. As a result, baseline characteristics of treated subjects differ from untreated ones. In order to account for these systematic differences, we conducted propensity score matched (PSM) analysis of patients of COVId-19 associated CRS treated with or without TPE. PS-matching was performed on cohort of patients meeting above inclusion and exclusion criteria.
For estimation of propensity score we used a logistic regression model (data matching Greedy) on NCCS statistical software v20.0.2. Number of controls was matched with TPE treatment group in 1:1 matching. Distance calculation method used was Mahalanobis distance including the propensity score, order for matching was random 1:1 and Caliper radius was set at 1*Sigma.
For ensuring comparable groups, forced matching was done for disease severity, standard care and advanced treatment at disease escalation. Co-variate matching was done for age, duration of illness, symptoms at presentation, co-morbidities, serum Ferritin, lactate dehydrogenase, ddimers, C-reactive protein levels, absolute lymphocyte count (ALC), platelet count and oxygen requirement at time of CRS diagnosis.
Median and range was used for continuous variables while frequency and percentage were used to express categorical statistics. The Chi-square test was used to evaluate differences in categorical variables while Students t-test or Mann-Whitney U test was used to evaluate continuous variables. Kaplan-Meier test was used for survival analysis and log rank was used to compare difference in two groups. Cox-proportional hazards were used to generate hazard ratio (HRs) and 95% confidence interval (CIs) for outcome. P-value <0.05 was considered significant.
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Patient selection procedure is shown in figure-1. On initial screening of data for COVID-19 patients, 315 cases of CRS were found. After applying exclusion criteria, 280 eligible patients were included in PSM analysis. Using 1:1 matching, 45 pairs of patients were formed, treated with or without TPE.
Baseline characteristics of patients before and after matching are shown in table 1. Before matching there were significant differences in TPE treated and untreated groups, which were addressed after matching. The overall eligible cohort was 245/280 (87.5%) males and 35/280 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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Time of resolution of CRS was significantly reduced in TPE group. From time of admission, cumulative incidence for normalization of CRS at day 15 was 90% in TPE group vs. 50% for PSmatched controls. Gray's test was applied to cater for completing risk, difference was statistically significant (p < 0.001) (Figure-4) .
Overall, 108/280 (38.6%) patients remained PCR positive on day 7 and 29/280 (10.35%) on day 14 with or without TPE. Median duration of hospitalization was significantly reduced in TPE treated group as compared to non-TPE controls (10 days vs. 15 days (p< 0.01).
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This study demonstrates that addition of TPE (as a single novel therapy) to the standard treatment (inclusive of steroids) for moderate, severe and critical COVID-19 with CRS is associated with significant survival benefit especially in critical disease and it remarkably decreases duration of hospitalization and resolution of CRS. However, PCR positivity at day 7 and 14 remained unchanged with addition of TPE. This is to be expected since evidence suggests presence of non-replicable viral nucleic acid material only, after day 10 of onset of illness, being picked up by the PCR 8, 9 .
To neutralize more comprehensively for biases associated with the selection of a particular mode of treatment, we stringently matched TPE and standard treatment care in both groups using a PSM analysis. The strength of conclusion stems from the fact that 15 variables in both arms had to be matched before analysis.
TPE appears promising as an investigational therapy for several convincing reasons. First, TPE 10 has been used in secondary HLH (III,2C), thrombotic microangiopathy secondary to various causes (various categories and strengths of evidence) and septic shock (III,2B) and any/all of these pathologies may be present in severe to critical COVID-19. For instance, not only Cytokine profile of severe Covid-19 closely resemble secondary hemophagocytic lymphohistiocytosis (sHLH) 11 but also it is associated with venous and arterial thromboembolic complications 12 and septic shock 13 , Therefore, it was hypothesized that TPE will be similarly beneficial if used in COVID-19 triggered CRS. Second, TPE has been used previously for the management of severe (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint this version posted July 29, 2020. suggested that TPE had an immediate effect on the treatment of the cytokine storm and improvement of PF ratio. Our study also endorses the same fact as most of our patients showed remarkable reduction in Oxygen demand after 1-2 sessions of TPE.
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The copyright holder for this preprint this version posted July 29, 2020. . https://doi.org/10.1101/2020.07.23.20160796 doi: medRxiv preprint Median duration of hospitalization in patients treated with TPE was 10 day in our study as compared to symptom onset to recovery time of 18 to 25 days in Zhang et al reported cases. This disparity might be because of the fact that our discharge criteria did not include PCR negativity.
Even in PS-matched control group, our study showed a mortality of 38.5% which is significantly lesser as compared with a large retrospective study carried out by Yang Nonetheless, our study had few limitations. Firstly, it was retrospective study which in itself has weaker evidence as compared to prospective trials. Secondly, although a strict PSM analysis was done, but still all biases cannot be neutralized. Thirdly, we did not follow up patients after discharge, due to lack of resources. Fourthly, in TPE procedure, we used centrifugation TPE machine rather than continuous hemofiltration (CHF) which is known to remove IL-6 and similar cytokines molecular mass of 21 to 54.1 kDa 24 , due to lack of availability of filters.
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Nevertheless, even after considering such limitations, using TPE early, in addition to standard treatment in patients with COVID-19 can mitigate the cytokine storm. TPE shows promise, and we propose that large, multi-centric, randomized trials be designed to further investigate its role.
In conclusion, TPE might be a lifesaving modality, with a statistically significant survival benefit, a decreased hospitalization time and an almost halved CRS resolution time, if started earlier at the onset of CRS in treatment of severe and critical COVID-19.
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Clinical management of severe acute respiratory infection ( SARI)   w  h  e  n  C  O  V  I  D   -1  9   d  i  s  e  a  s  e  i  s  s  u  s  p  e  c  t  e  d  :  i  n  t  e  r  i  m  g  u  i  d  a  n  c  e  , 1  3  M  a  r  c  h  2  0  2 All rights reserved. No reuse allowed without permission.
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