Treatment of 5 Critically Ill Patients
With COVID-19 With Convalescent
Plasma

Introduction
• The epidemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
originating in Wuhan, China,has rapidly spread worldwide
• As of March, 2020, cases were reported in approximately 195 countries
• No specific therapeutic agents or vaccines for COVID-19 are available
• The use of convalescent plasma was recommended as an empirical treatment
during outbreaks of Ebolavirus in 2014
• A protocol for treatment of Middle East respiratory syndrome coronavirus with
convalescent plasma was established in 2015

Introduction
• In a study involving patients with pandemic influenza A(H1N1) 2009 virus infection,
treatment of severe infection with convalescent plasma (n=20patients) was associated
with reduced respiratory tract viral load, serum cytokine response, and mortality
• The purpose of this study was to describe the initial clinical experience with convalescent
plasma transfusion administered to critically ill patients with COVID-19.

Methodology
• This study was conducted at the infectious disease department,
Shenzhen Third People's Hospital, Shenzhen, China,
• From January 20,2020 to March 25,2020 and the final date of
follow-up was March 25, 2020.

Patient – inclusion criteria
• Patients with laboratory confirmed COVID-19, diagnosed using quantitative
reverse transcriptase–polymerase chain reaction (qRT-PCR) were eligible to
receive convalescent plasma treatment if they fulfilled the following criteria –
(1) severe pneumonia with rapid progression and continuously high viral load
despite antiviral treatment
(2) PAO2/FIO2 of <300 (PAO2 measured in mm Hg and FIO2 measured as fraction
of inspired oxygen)
(3) currently or had been supported with mechanical ventilation

Patient details
• The serum of each recipient was obtained
• ELISA and neutralizing antibody titers were tested one day prior to the convalescent
plasma transfusion
• Each received 2 consecutive transfusions of 200 to 250 mL of ABO-compatible
convalescent plasma (400 mL of convalescent plasma in total )on the same day it was
obtained from donor
• They received antiviral agents continuously until the SARS-CoV-2 viral loads became
negative

Disease Severity Classification
Patients with laboratory-confirmed COVID-19 infection who had any of the following were
considered in critical condition
• (1) respiratory failure requiring mechanical ventilation
• (2) shock, identified by the use of vasopressor therapy and elevated lactate
levels(>2mmol/L)despite adequate fluid resuscitation
• (3) failure of other organs requiring admission to the intensive care unit(ICU).

Donors
• 5 donors of convalescent plasma were between 18 and 60 years
• The donors had recovered from SARSCoV-2 infection
• The donors should be asymptomatic for at least 10 days, with a serum SARS-CoV-2–
specific ELISA antibody titer higher than 1:1000 and a neutralizing antibody titer greater
than 40.
• Following donation, 400 mL of convalescent plasma was obtained from each donor by
apheresis
• Plasma was immediately transfused to the recipients on the same day it was obtained

Data
Clinical information for the 5 patients before and after convalescent plasma
transfusion was obtained from are view of the hospital computer medical system-
• Demographic data
• days of admission from symptom onset
• presenting symptoms
• Mechanical ventilation , antiviral therapies, and steroids
• body temperature, PAO2/FIO2 ,and Sequential Organ Failure Assessment(SOFA)
score

Data
• Lab data - WBC ,lymphocyte count, LFT , KFT , cycle threshold value (Ct),
CRP procalcitonin, and IL-6, and serum antibody titer (IgG ,IgM ,and
neutralizing antibodies);
• Chest imaging studies
• Information on complications ,such as acute respiratory distress syndrome
(ARDS), bacterial pneumonia, and multiple organ dysfunction syndrome

Rt PCR
• Nasopharyngeal specimens collected during hospitalization were sent to the
laboratory in a viral transport case.
• Each RT-PCR assay provided a Ct value, which is the number of cycles required
for the fluorescent signal to cross the threshold for a positive test: a higher Ct
value is correlated with a lower viral load
• The specimens were considered positive if the Ct value was 37 or lower and
negative if the results were undetermined.
• The specimen was considered positive if the repeated results were the same as the
initial result and between 37 and 40.
• The Ct values of the 5 recipients were obtained on day−1,3,7, 12 after the
transfusion

• Microtiter plates were coated over night at 4°C with 4μg/mL
recombinant SARS-CoV-2 RBD (receptor binding domain) proteins (50 μL
per well) expressed through 293-T cells
• The plates were washed 3 times with phosphate-buffered saline (PBS)
containing 0.1% vol/vol Tween-20(PBST)
• Blocked with blocking solution (PBS containing 2%wt/ vol nonfat dry milk
) for 2 hours at 37°C
• The serum samples were diluted to 200-fold into PBS as initial
concentration
• After 3 washes, 100 μL of horseradish peroxidase–conjugated goat anti–
human IgG & IgM added & incubated at 37°C for 60 minutes.

• After 5 washes, 100 μL of tetramethylbenzidine substrate (Sangon
Biotech) was added at room temperature in the dark
• The absorbance was measured at 450nm . All samples were run in
triplicate.
• The ELISA titers were determined by end point dilution

• Vero cells were seeded in a 96-well plate
• Serum samples from patients were incubated at 56°C for 30 minutes and then diluted 2-
fold
• Aliquots (40 μL) of diluted serum samples were added to 50 μL of cell culture medium
containing TCID50 of virus
• Virus antibody mix was then added to cells in 96-well plates
• Microscopic examination for cytopathic effect done after a 5-day incubation
• The highest dilution of serum that showed inhibition activity of SARS-CoV-2was
recorded as the neutralizing antibody titer

Clinical Characteristics of SARS-CoV-2 –Infected Patients Who Received Convalescent
Plasma

.Comparison of Viral Load, Clinical Indexes before and after plasma therapy

.Comparison of Lab parameters before and after plasma therapy

Temporal changes of Cycle Threshold value, SOFA score, in patients
receiving convalescent Plasma Transfusion
A- Change in cycle threshold (Ct) value in nasopharyngeal swabs of infected patients at day 0,day 3,day 7,andday
12after the plasma transfusion . A Ct value of 40 was defined as undetectable.
B- Change in Sequential Organ Failure Assessment (SOFA) score of the patients with convalescent plasma
treatment (range 0-24

Temporal changes of PaO2/FiO2 & body temperature , in patients receiving
convalescent Plasma Transfusion

b, c- ELISA end point dilution titers (IgG and IgM antibody).The expected titer of negative control from a healthy
person <200
D Neutralization end point dilution titers.The expected titer of negative control from a healthy personis10

Changes of Receptor Binding Domain–Specific IgG and IgM ELISA and Neutralizing Antibody Titers
Before and After Convalescent Plasma Transfusion in Patients

Results
• All 5 patients were receiving mechanical ventilation at the time of transfusion ,and
3 patients (patients3,4,and5) were weaned from mechanical ventilation
• Patient2 was receiving ECMO at the time of plasma treatment but did not require
ECMO on day 5 after transfusion
• Patients 3,4,and 5 were discharged from the hospital (length of stay: 53, 51, and
55 days, respectively

Discussion
• Plasma was obtained from the donors and transfused in the recipients on the same
day, which helps preserve the natural activity of the plasma.
• Viral load decreased and became undetectable so on after convalescent plasma
treatment .As determined by ELISA , all plasma from the donors had high virus-
specific IgG and IgM ELISA titers
• the neutralizing antibody titers, vital for the restriction of viral infection of the 5
recipients ,significantly increased after plasma transfusion.

• All patients received antiviral agents ,including interferon and
lopinavir/ritonavir ,during and following convalescent plasma
treatment, which also may have contributed to the viral clearance
observed.

Limitations
• First, this was a small case series that included no controls
• It is unclear if these patients would have improved without transfusion of
convalescent plasma, although the change in Ct and PAO2/ FIO2 represent
encouraging findings
• All patients were treated with multiple other agents (including antiviral
medications),and it is not possible to determine whether the improvement observed
could have been related to therapies other than convalescent plasma.
• Plasma transfusion was administered 10 to 22 days after admission ; whether a
different timing of administration would have been associated with different
outcomes cannot be determined

• The epidemic spread rapidly worldwide within 3 mo and was
characterized as a pandemic by WHO on March 11, 2020
• Currently, there are no approved specific antiviral agents targeting
the novel virus, while some drugs are still under investigation
• Convalescent plasma (CP) therapy, a classic adaptive immunotherapy,
has been applied to the prevention and treatment of many infectious
diseases for more than one century

• A meta-analysis from 32 studies of SARS coronavirus and severe
influenza showed a statistically significant reduction in the pooled
odds of mortality following CP therapy, compared with placebo or no
therapy (odds ratio, 0.25; 95% CI- 0.14–0.45)
• Patients who have recovered from COVID-19 with a high neutralizing
antibody titer may be a valuable donor source of CP.
• the potential clinical benefit and risk of convalescent blood products
in COVID-19 remains uncertain.

• this pilot study was done in three participating hospitals to explore
the feasibility of CP treatment in 10 severe COVID-19 patients.

Neutralizing Activity of CP against SARS-CoV-2
• The neutralizing activity against SARS-CoV-2 was evaluated by
classical plaque reduction test using a recently isolated viral strain
• Among the first batch of CP samples from 40 recovered COVID-19
patients,39showedhighantibodytitersofatleast1:160,whereas only
one had an antibody titer of 1:32.
• This result laid the basis for pilot clinical trial using CP in severe
patients.

General Characteristics of Patients in the Trial
• From January 23, 2020, to February 19, 2020, 10 severe COVID-19 patients
(six males and four females) were enrolled and received CP transfusion
• The median time from onset of symptoms to hospital admission and CP
transfusion was 6 d (IQR, 2.5 d to 8.5 d) and 16.5 d (IQR, 11.0 d to 19.3 d),
respectively.
• The most common symptoms at disease onset were fever (7 / 10 s), cough
(8 ), and shortness of breath (8 ),
• Less common symptoms -sputum production (5), chest pain (2), diarrhea
(2), nausea and vomiting (2), headache (1), and sore throat (1).

Patient details
• 4 - had underlying chronic diseases- cardiovascular and/or cerebrovascular diseases and HTN
• 9 - received arbidol monotherapy or combination therapy with remdesivir, or ribavirin, or
peramivir, while one patient received ribavirin monotherapy
• 6 patients received intravenous (i.v.) methylprednisolone (20 mg every 24 h).
• All patients – in CT , bilateral ground-glass opacity and/or pulmonary parenchymal
consolidation with predominantly subpleural and bronchovascular bundles distribution in the
lungs
• Seven patients had multiple lobe involvement, and four patients had interlobular septal
thickening.

Effects of CP Transfusion- Improvement of
clinical symptoms
• All symptoms in the 10 patients, especially fever, cough, shortness of
breath, and chest pain, disappeared or largely improved within 1 d to
3 d upon CP transfusion.
• Prior to CP treatment - A fter CP
o 3 patients mechanical ventilation 2 weaned
o 3 received high-flow nasal cannula O2 1 discontinued

Reduction of pulmonary lesions on chest CT
examinations
• all patients showed different degrees of absorption of pulmonary
lesions after CP transfusion

Fig. 1. Chest CTs of two patients. (A) Chest CT of patient 9 obtained on February 9 (7 dpoi) before CP
transfusion (10 dpoi) showed ground-glass opacity with uneven density involving the multilobal segments
of both lungs. The heart shadow outline was not clear. The lesion was close to the pleura. (B) CT Image of
patient 9taken on February 15 (13 dpoi) showed the absorption of bilateral ground-glass opacity after CP
transfusion

Chest CT of patient 10 was obtained on February 8 (19 dpoi) before CP transfusion (20 dpoi). The brightness of both lungs was
diffusely decreased, and multiple shadows of high density in both lungs were observed. (D) Chest CT of patient 10 on February 18
(29 dpoi) showed those lesions improved after CP transfusion.

Lab parameter changes

Increase of neutralizing antibody titers and disappearance of
SARS-CoV-2 RNA
• The neutralizing antibody titers of five patients increased and four patients
remained at the same level after CP transfusion
• SARS-CoV-2 RNA was decreased to an undetectable level in three patients on day
2, three patients on day 3, and one patient on day 6 after CP therapy.

Outcome of patients treated with CP as compared to a recent
historic control group
• A historic control group was formed by random selection of 10 patients from the cohort
treated in the same hospitals and matched by age, gender, and severity of the diseases to
the 10 cases in our trial
• 3 cases discharged while 7 cases in much improved status and ready for discharge in CP
group
• 3 deaths, 6 cases in stabilized status, and one case in improvement in the control group (P
< 0.001);

Adverse Effects of CP Transfusions