Intratracheal administration of autologus conditioned serum for COVID-19 associated respiratory distress syndrome.

From late 2019, China reported COVID-19 as an epidemic and in early 2020 WHO claimed it as a pandemic. Hospitalized patients may develop COVID-19 associated ARDS and cytokine storm leading to multi organ failure with a high mortality rate [1].

Although there has been progress in understanding the mechanistic basis for the initiation and propagation of COVID-19, there is unfortunately not any specific and effective treatment or vaccine for COVID-19 [2,3]. COVID-19 is associated with pulmonary inflammation and an increase in plasma concentrations of inflammatory cytokines (cytokine storm) due to dysregulated host immune respone [4]. There are many studies showing that COVID-19 is associated with immune response and hyperinflammation; so, therapeutic approaches targeting this pathway can help clinicians in the battle against this disease. Autologus conditioned serum (ACS) has been shown to be safe and effective on various disease especially osteoarthritis in previous clinical studies, all showing an excellent risk/benefit ratio [5]. ACS is enriched with anti-inflammatory cytokines like IL-1Ra, IL-10 and IL-13 and has low levels of inflammatory cytokines like tumor necrosis factor alpha (TNF-α) and IL-1β [6]. Therapy is based on the injection of signaling protein-rich serum whose efficacy is due to the synergistic effect of many of the body's own signaling proteins (cytokines and growth factors) that are present in clinically relevant concentrations in ACS. Moreover, synergistic effects of IL-1Ra and other cytokines in addition to several regenerative growth factors are responsible for the strong and long lasting efficacy of ACS therapy in previous clinical studies. It has been shown that ACS can reduce the level of inflammatory cytokines in osteoarthritis patients after intra-articular injection.

As the most important organ dysfunction in COVID-19 is related to respiratory system, we tried to administer ACS intratracheally to get better distribution in different region of lungs. We used patients own plasma as it did not need matching and was a type of an autologous transfusion with lower risk of complications. Based on the inflammatory nature of COVID-19 and strong anti-inflammatory profile of ACS, we hypothesized that intratracheal administration of ACS to critically ill COVID-19 patients would result in improvement of the inflammatory and respiratory parameters of. The primary outcome was improvement in oxygenation and the secondary outcomes were duration of mechanical ventilation, ICU length of stay and respiratory indices such as compliance and resistance.

The study protocol was approved by the Ethics Committee of Tabriz University of Medical Sciences, which is in compliance with the Declaration of Helsinki. The off-label use of the method was verbally explained to the next of kin of each patient and written consent was obtained from all of them. The consent form included all the possible advantages and disadvantages of the method and its mechanism of action. The patients' information was anonymous and analyzed in a coded format. No additional charges were paid by the patients at the end of the study, and the patients could abandon the study at any stage on personal desire. This is a preliminary data report of a clinical trial registered at http://www.irct.ir with registration number ID: IRCT20091012002582N21.

Five Patients with laboratory confirmed COVID-19, diagnosed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), were eligible to receive intratracheal ACS fulfilling the following criteria: COVID-19 associated respiratory distress syndrome being under mechanical ventilation with PaO2/FiO2 less than 150. All patients received antiviral treatment (Hydroxy chloroquine 200 mg/12 h and lopinavir/ritonavir 400 mg/12 h for 10 days), standard protocol and adjunct therapies for their disease. Severity of disease was assessed by clinical (hypoxemia, low Glascow Coma Scale, and high respiratory rate) and laboratory (high levels of D-dimer, LDH, CRP, CPK, BNP, and Ferritin) findings, and sequential organ failure assessment (SOFA) score. Thirty ml of blood was aseptically taken from each patient by EOT®II syringe. It was then incubated for 6–9 h at 37 °C and then the blood-filled syringes were centrifuged for 10 min at 5000* rpm. The acquired serum (15 ml) was filtered (0.22 μm; Millipore, Carrigtwohill, Co. Cork, Ireland), separated into 3 tubes and frozen to −20 °C till tracheal administration. The volume of injected autologous serum was based on previous researches [7] and the allowable volume which can be safely administered intratracheally without any complication. Thereafter, the plasma was intratracheally injected to each patient with co-administration of 5 ml distilled water which was followed by five manual breathing for a better distribution of plasma into different parts of the lungs. This procedure was repeated three times with the interval of three days. Before administration of ACS, tracheal suctioning was performed via closed suction system to ensure the removing of secretions from the lungs. We performed intratracheal administration under aseptic condition. To decrease the generation of aerosols, all patients received muscle relaxants before ACS injection and the ventilator circuit was blocked during the administration of ACS. Thereafter, we reconnected circuit as soon as possible and performed controlled ventilation for a better distribution of ACS to the distal parts. Meanwhile, the safety of our healthcare workers was also provided by decreasing the generation of aerosol. Demographic characteristics of patients are shown in Table 1 . The mean value of pulmonary and non-pulmonary parameters in all patients before and after ACS administration are shown in Table 2 . The most striking clinical change observed in association with the administration of ACS in this study was the rapid decline in body temperature (from 38.2 to 37.7) and serum CRP levels which can be explained by the decrease in the level of inflammation. Also, there was a corresponding radiological improvement in two patients at day 5 and in one patient at day 10. The remained two patients did not show any improvement and died. Any significant complication like hypoxemia or worsening of pulmonary indices was not reported in patients with ACS therapy and no scheduled ACS treatment was discontinued as a result of significant adverse effects. Two of five patients who received ACS died at days 8 and 14 after the first dose of ACS administration which showed lower mortality rate compared to standard treatment for patients with COVID-19 associated respiratory distress syndrome (more than 50% mortality).The most significant change after our intervention is seen for driving pressure and oxygenation index. As the driving pressure is one of the most important independent factors for mortality in critically ill patients with ARDS, it seems that this intervention may result in better outcome by improving organ function following reduction in the level of inflammation and driving pressure. Remaining three patients were discharged and their most prominent complain was decreased physical activity without any significant respiratory complication. They didn't use supplementary oxygen and only used incentive spirometry as a respiratory adjunct therapy at home. The clinical conditions of these patients including PaO2/FiO2, lung mechanic indices and chest imaging was improved. Apart from antiviral therapy, reversing of inflammation and cytokine storm can serve as an important mechanism for the restriction of virus and decreasing multi organ failure as a leading cause of this disease. The results of our study suggest the possibility of ACS in clearance of the virus as well as the improvement of symptoms due to its anti-inflammatory properties. In this study, all patients received antiviral agents during and following ACS administration, which also may have contributed to the improved symptoms. Additionally, the promising results of the study cannot be directly and exclusively contributed to ACS effects since other interventions such as personalized ventilator setting can interfere with our results. Moreover, the administration of ACS was performed after severe pulmonary involvement and it is unknown whether a different timing of administration would have been associated with different outcomes or not.

Table 1

Demographic characteristics of patients.

Variable	Mean value	Control	p-value
Age(year)	59 (38–65)	58 (35–65)	0.84
Weight(kg)	75 (95.2–60.5)	71.2 (90.5–55.7)	0.84
BMI	25.7 (23.6–31.7)	27.8 (23.7–31.0)	0.69
Sex(M/F)	3/2	2/3	1.00
SOFA	14.2 (12.3–16.4)	14.1 (13–16.6)	1.00
APACHE	27.4 (25.3–35.1)	28.1 (24.9–32.4)	1.00
Mechanical ventilation Duration(h)	14.2 (10.9–16.2)	12.3 (10.9–15.6)	0.74
ICU LOS(days)	18.1 (16.4–20.5)	17.9 (16.2–19.8)	0.86
Mortality	40%	60%	1.00
Comorbidities			1.00
Obesity	1	1
DM	2	2
CVD	2	1
Immunosuppression	1	2

BMI: body mass index SOFA: sequential organ failure assessment M/F: male/female.

APACHE: acute physiology and chronic health evaluation CVD: cardiovascular disease.

ICU LOS: intensive care unit length of stay DM: diabetes mellitus.

Table 2

Different parameters before and after of ACS therapy.

Variable		Before Ad	1st Ad	2nd Ad	3rd Ad	P⁎
PaO2 (mmHg)	Case	52 (47–59)	57 (51–64)	59 (52–64)	61 (54–68)	0.04
	Control	52 (56–58)			55 (50–62)	0.28
P		0.78			0.09
PaCO2 (mmHg)	Case	49 (44–55)	50 (46–54)	46(40–52)	47 (42–52)	0.08
	Control	49 (43–54)			49 (44–54)	0.50
P		0.92			0.54
Compliance(ml/cmH2O)	Case	38 (34–43)	39 (34–44)	42 (36–48)	46 (41–52)	0.04
	Control	38 (34–43)			40 (35–45)
P		0.92			0.04
Resistance(cmH2O/l/s)	Case	8 (7–9)	9 (7–11)	8 (7–10)	7 (6–8)	0.03
	Control	8 (7–9)			8 (7–9)
P		0.92			0.29
Plat P (cmH2O)	Case	29 (25–34)	27 (20–35)	26 (21−32)	25 (20–29)	0.04
	Control	30 (25–35)			29 (24–3)
P		0.69			0.15
DP (cmH2O)	Case	16 (14–18)	15 (14–17)	14 (12–16)	14 (11–16)	0.04
	Control	18 (16–20)			17 (15–19)
P		0.22			0.02
P/F ratio	Case	106 (91–121)	115 (44–186)	123 (107–139)	134 (122–146)	0.04
	Control	107 (94–120)			113 (98–128)
P		0.75			0.12
SPO2 (%)	Case	88 (84–93)	89 (85–94)	90 (86–95)	91 (85–97)	0.04
	Control	87 (82–92)			88 (84–92)
P		0.60			0.25
OI	Case	41 (36–47)	38 (33–42)	34 (30–37)	30 (26–34)	0.04
	Control	42 (38–46)			39 (34–43)
P		0.76			0.01
EVLWI (ml)	Case	850 (795–915)	760 (702–818)	670 (623–717)	615 (559–671)	0.04
	Control	845 (783–907)			810 (739–881)
P		0.84			0.01
PVPI	Case	2.4 (2.1–2.7)	2.1 (1.9–2.3)	1.9 (1.8–2.0)	1.5 (1.2–1.8)	0.04
	Control	2.5 (2.1–2.9)			2.1 (1.7–2.5)
P		0.56			0.02
SVR(dynes/s/cm−5)	Case	1850 (1600–2100)	1720 (1410–2030)	1630(1370–1890)	1570 (1360–1780)	0.04
	Control	1900 (1670–2130)			1780 (1490–2071)
P		0.67			0.12
Cardiac index(l/min/m2)	Case	3.1 (2.7–3.5)	3 (2.7–3.3)	3.2 (3–3.4)	3.2 (2.8–3.6)	0.02
	Control	3.1 (2.8–3.4)			3.2 (2.9–3.5)
P		0.92			1.00
Ferritin (ng/ml)	Case	621 (584–658)	601 (568–633)	587 (546–628)	554 (529–579)	0.04
	Control	626 (588–664)			605 (571–639)
P		0.60			0.03
LDH (U/L)	Case	868 (789–947)	810 (744–876)	763 (690–836)	692 (613–771)	0.04
	Control	880 (805–955)			816 (742–890)
P		0.75			0.02
BNP(pg/ml)	Case	459 (403–515)	446 (397–495)	421 (365–477)	436 (378–492)	0.04
	Control	465 (404–526)			485 (424–546)
P		0.75			0.17
CRP (mg/l)	Case	124 (99–149)	112 (101−123)	101 (84–118)	87 (72–103)	0.04
	Control	126 (104–148)			117 (101−133)
P		0.92			0.02
CPK (U/L)	Case	456 (413–499)	424 (385–463)	392 (350–434)	369 (316–422)	0.04
	Control	465 (423–507)			418 (367–469)
P		0.60			0.14
H-score	Case	259 (223–295)	215 (191–239)	182 (141–223)	153 (126–180)	0.04
	Control	264 (226–302)			218 (187–249)
P		0.60			0.01
Lactate(mmol/l)	Case	2.3 (2.0–2.6)	2.2 (1.8–2.6)	2 (1.7–2.3)	2.1 (1.7–2.5)	0.04
	Control	2.4 (2.1–2.7)			2.2 (1.9–2.5)
P		0.46			0.60
PCT (ng/ml)	Case	0.7 (0.5–0.9)	0.7 (0.6–0.8)	0.8 (0.6–1)	0.7 (0.5–0.9)	0.07
	Control	0.7 (0.4–1)			0.7 (0.4–1)
P		0.83			0.75

Ad: tracheal administration of ACS DP: driving pressure P/F ratio: Pao2/Fio2 OI: Oxygenation index EVLWI: extravascular lung water index CPK: creatinine phosphokinase CI: cardiac index (l/min/m2) PVPI: pulmonary vascular permeability index SVR: systemic vascular resistance (dynes/s/cm−5) LDH: lactate dehydrogenase BNP: brain natriuretic peptide CRP: C-reactive protein PCT: procalcitonin.

+Between groups.

Intergroup.

The absence of high-level clinical evidence to guide therapeutic interventions in such a rapidly growing pandemic let us the wide off-label use of potentially beneficial agents. While this report shows some positive effects of ACS in the management of patients with COVID-19 associated respiratory distress syndrome, it cannot lead to any absolute conclusion, and the results need confirmation in future large sample-sized randomized controlled trials.

Authors' contribution

Ata Mahmoodpoor: Conceptualization, project administration, original draft,

Seyed Kazem Shakouri: Supervision, validation of data, review and editing.

Leila Roshangar: Conceptualization, data analysis, review and editing.

All authors read and approved the final version of manuscript.

Funding

Research deputy of

Declaration of Competing Interest

The authors declare no conflict of interest.