Research

External quality assessment of SARS-CoV-2 serology in

European expert laboratories, April 2021
Ramona Mögling1,*, Francesca Colavita2,* , Johan Reimerink1 , Angeliki Melidou3 , Katrin Leitmeyer 3 , Maria Keramarou3 , Daniele
Lapa2 , Massimo Francalancia2 , Jean-Luc Murk 4 , Ann Vossen5 , Fabrizio Carletti2 , Boris Hogema6 , Adam Meijer1 , Liesbet Deprez7
, Antonino di Caro8,9 , Concetta Castille tti2,9,* , Chantal BEM Reusken1,*
1. Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, the
Netherlands
2. National Institute for Infectious Diseases ‘Lazzaro Spallanzani’ IRCCS (INMI), Rome, Italy
3. European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden
4. Microvida, location St Elisabeth-Tweesteden Hospital, Tilburg, The Netherlands
5. Leiden University Medical Center, Leiden, The Netherlands
6. Sanquin Research, Amsterdam, The Netherlands
7. European Commission, Joint Research Centre (JRC), Geel, Belgium
8. Unicamillus, International Medical University, Rome, Italy
9. IRCCS Sacro Cuore Don Calabria Hospital, Negrar di Valpolicella, Italy

* These authors contributed equally to this article.

Correspondence: Concetta Castilletti (concetta.castilletti@sacrocuore.it)

Citation style for this article:

Mögling Ramona, Colavita Francesca, Reimerink Johan, Melidou Angeliki, Leitmeyer Katrin, Keramarou Maria, Lapa Daniele, Francalancia Massimo, Murk Jean-
Luc, Vossen Ann, Carletti Fabrizio, Hogema Boris, Meijer Adam, Deprez Liesbet, di Caro Antonino, Castilletti Concetta, Reusken Chantal BEM. External quality
assessment of SARS-CoV-2 serology in European expert laboratories, April 2021. Euro Surveill. 2022;27(42):pii=2101057. https://doi.org/10.2807/1560-7917.
ES.2022.27.42.2101057

Article submitted on 11 Nov 2021 / accepted on 09 Aug 2022 / published on 20 Oct 2022

Background: Countries worldwide are focusing to miti-

Key public health message
gate the ongoing SARS-CoV-2 pandemic by employing
public health measures. Laboratories have a key role
in the control of SARS-CoV-2 transmission. Serology What did you want to address in this study?
for SARS-CoV-2 is of critical importance to support Robust serological test systems to detect antibodies
diagnosis, define the epidemiological framework and against SARS-CoV-2 in human serum samples are important
evaluate immune responses to natural infection and to establish whether past infections occurred and to assess
the extent of immunity in the population. We want to
vaccine administration. Aim: The aim of this study enable laboratories and countries employing public health
was the assessment of the actual capability among measures to assess the quality and robustness of different
laboratories involved in sero-epidemiological studies serological tests that are being used in European expert
laboratories.
on COVID-19 in EU/EEA and EU enlargement countries
to detect SARS-CoV-2 antibodies through an external What have we learnt from this study?
quality assessment (EQA) based on proficiency test-
The majority of laboratories (53 of 55) used at least one
ing. Methods: The EQA panels were composed of eight serological test that was able to characterise all the serum
different, pooled human serum samples (all collected samples correctly. The performance of serological assays
in 2020 before the vaccine roll-out), addressing sensi- varied for different types of antibodies.
tivity and specificity of detection. The panels and two What are the implications of your findings for public
EU human SARS-CoV-2 serological standards were sent health?
to 56 laboratories in 30 countries. Results: The overall
Our study showed a high capability across European
performance of laboratories within this EQA indicated reference laboratories for reliable diagnostics for SARS-
a robust ability to establish past SARS-CoV-2 infec- CoV-2 antibody responses. Aside from assessing the total
tions via detection of anti-SARS-CoV-2 antibodies, share of people with immunity in the population, reliable
serological diagnostics are also important to guide public
with 53 of 55 laboratories using at least one test that health actions by helping to estimate the proportion of
characterised all EQA samples correctly. IgM-specific asymptomatic cases.
test methods provided most incorrect sample char-
acterisations (24/208), while test methods detecting
total immunoglobulin (0/119) and neutralising anti-
bodies (2/230) performed the best. The semiquantita-
tive assays used by the EQA participants also showed
a robust performance in relation to the standards.
Conclusion: Our EQA showed a high capability across

www.eurosurveillance.org 1
European reference laboratories for reliable diagnos- Table 1
tics for SARS-CoV-2 antibody responses. Serological SARS-CoV-2 serology external quality assessment
panel composition and overall test results (n = 162) of
tests that provide robust and reliable detection of anti participating laboratories (n = 55), EU/EEA, February–
SARS-CoV-2 antibodies are available. April 2021

Introduction Anti-SARS- Correct resultsa

Countries worldwide are focusing to mitigate the ongo- Sample CoV-2 Sample
ing coronavirus disease (COVID-19) pandemic [1] by ID antibodies information % Number Total
present
employing public health measures, including increas-
Hospitalised
ing vaccination roll-out and restriction of movements. A Yes (IgA+/IgG+/ 98.8 160 162
Laboratories have a key role in the control of the trans- IgM+)
mission of severe acute respiratory syndrome corona- Mild disease
virus 2 (SARS-CoV-2) by detecting acute and previous B Yes (IgA+/IgG+/ 98.7 157 159b
IgM+)
infections with the virus in a reliable and timely fash-
ion. While the detection of acute infections, typically Negative
C No
pre-pandemic
95.0 153 161b
done by real-time reverse transcription PCR (RT-PCR)
Hospitalised
or antigen (Ag) testing, can be used to stop transmis- D Yes (IgA+/IgG+/ 99.4 161 162
sion chains through isolation and quarantine measures IgM+)
[2], the detection of immunological markers for past Acute CMV+EBV
E No
infection
96.3 155 161b
SARS-CoV-2 infections and/or vaccinations against
SARS-CoV-2 is used to estimate immunity [3] in both F No
Negative
88.8 143 161b
pre-pandemic
individuals and communities, thereby informing miti-
gation strategies. Most serological assays detect anti- Mild disease
G Yes (IgA+/IgG+/ 96.3 156 162
bodies against SARS-CoV-2 as the main immunological low IgM+c)
marker. The emergence of SARS-CoV-2 variants in the Hospitalised
spike protein represents one of the main concerns for H Yes (IgA+/IgG+/ 100.0 162 162
the potential of immunological escape from the anti- IgM+)
bodies response. EURM-
JRC standard
Yes (low IgA+c/ 94.9 149 157
017
IgG+/low IgM+c)
The vast impact of the COVID-19 pandemic on public JRC standard
health, economies and societies drives the rapid devel- EURM-
Yes (low IgA+c/ 96.8 152 157
018
opment of numerous serological assays by laborato- IgG+/low IgM+c)
ries and commercial entities [4]. These tests are not
only based on different techniques, e.g. enzyme-linked CMV: cytomegalovirus; EBV: Epstein–Barr virus; EU/EEA: European
immunosorbent assays (ELISA), chemiluminescence Union and European Economic Area; JRC: Joint Research Centre;
SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.
immunoassays (CLIA), lateral flow assays (LFA) and
virus neutralisation tests (VNT), but also use different a
Correct results at test level.
antigenic targets and are able to detect different types
of anti-SARS-CoV-2 antibodies, e.g. total immunoglob- b
Samples B, C, E, F and both standards were not tested with the
ulin (Ig), IgG, IgM, IgA and/or neutralising antibodies. maximum number (n = 162) of submitted tests. Laboratories did
not indicate the reasons for not testing these panel entries.
Besides the fact that each laboratory needs to perform
validation and evaluation studies before implementing c
Pooled serum samples contained anti-SARS-CoV-2 IgM and/or IgA
a new test [5], it is crucial to assess the capability of antibodies but in low quantities.
the laboratory to perform the test through an external
quality assessment (EQA) based on proficiency testing.
Proficiency testing enables a comparison of the accu-
racy of different tests and the performance of different human SARS-CoV-2 serological standards produced
laboratories based on the same material [6-10]. and described by the Joint Research Centre (JRC),
EURM-017 and EURM-018 [11,12], to assess the per-
Here, we describe the set-up and results of such an formance of semiquantitative assays used in different
EQA of detection of SARS-CoV-2 antibodies among laboratories in comparison with reference material.
European expert laboratories that are members of the  
European Centre for Disease Prevention and Control
(ECDC) COVID-19 and influenza laboratory networks, Methods 
laboratories involved in sero-epidemiological stud-
ies on COVID-19 in the European Union and European External quality assessment scheme
Economic Area (EU/EEA) and EU-enlargement countries organisation
and/or members of the Emerging Viral Diseases-Expert In December 2020 and January 2021, European expert
Laboratory Network (EVD-LabNet). The proficiency laboratories that are members of the ECDC COVID-
panel comprised different isotypes of SARS-CoV-2 19 and influenza laboratory networks, laboratories
antibodies. In addition, laboratories also received two involved in sero-epidemiological studies on COVID-19

2 www.eurosurveillance.org
Figure 1
Number of expert laboratories participating in external quality assessment, per country and overall laboratory (n = 55) and
test (n = 162) performance, EU/EEA, February–April 2021

A. Number of laboratories participating in SARS-CoV-2 serology EQA, by country

EU/EEA participating country

Pre-accession participating country
Non-participating country
Non-eligible country

Non-visible countries
Malta
Liechtenstein

B. Number of different tests performed by individual laboratories

n=36 n=17 n=2 laboratories

7
Number of tested methods

6
5
All EQA samples characterised
4 correctly by test method
Not all EQA samples characterised
3
correctly by test method
2

1
0

Individual laboratories (n=55)

C.

100% correct
10
results (n=133)
Number of tested samples

1 false result (n=19)

≥2 false results (n=10)
Correct
5
False-negative
False-positive
Not tested
0

5
Individual test methods (n=162)

EEA: European Economic Area; EQA: external quality assessment; EU: European Union.

www.eurosurveillance.org 3
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Table 2
Performance of assays in external quality assessment, grouped by SARS-CoV-2 antibody isotype detection, EU/EEA, February–April 2021 (n = 171)

Performance compared
with total Ig b
Number of Number of Number of false-negative tests Number of false-positive tests
Antibody
submitted different p value
type(s)
tests per assays per
detectable by Sample A Sample D Sample H Sample C Sample F Sample E
antibody antibody Sample B – mild Sample G – mild False- False-
assay – hospitalised – hospitalised – hospitalised – negative – negative – negative
typea typea negative positive
Pre- Acute results results
IgA+/IgG+/IgM+ IgA+/IgG+/IgM+ IgA+/IgG+/IgM+ IgA+/IgG+/IgM+ IgA+/IgG+/low IgM+ Pre-pandemic
pandemic CMV+EBV
Total Ig 15 3 0 0 0 0 0 0 0 0 NA NA
IgA 19 6 1 1 0 0 1 2 2 1 0.3394 0.1151
IgG 78 32 1 0 0 1 1 2 4 4 0.9741 0.3243
IgM 26 14 0 0 2 1 6 3 11 1 0.0499 0.0043
c 1 1 0 0 0 0 0 0 0 0 NA NA
IgM/IgG
c 3 1 0 0 0 0 0 0 0 0 NA NA
IgM/IgA
Neutralising
29 8 0 0 0 0 0 1 1 0 NA 0.7846
antibodies

CMV: cytomegalovirus; EBV: Epstein–Barr virus; EEA: European Economic Area; EU: European Union; NA: not applicable; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2.
a 
Assays that are able to detect multiple antibody types, and for which individual results for the respective antibody types were submitted, were analysed separately for each antibody type in this table.
b
 Performance of antibody type specific tests was compared with performance of total Ig tests using two-sided Yates’ corrected chi-squared test. Antibody-specific tests that performed significantly worse
than total immunoglobulin tests are underlined.
c 
Combinations of multiple antibody types are possible for some tests. For assays of the groups ‘IgM/IgG’ and ‘IgM/IgA’, EQA participants did not submit the individual results for the respective antibody
types.

www.eurosurveillance.org
in EU/EEA and EU-enlargement countries and/or mem- samples were provided coded Sample A to Sample H,
bers of the Emerging Viral Diseases-Expert Laboratory with no further identifying information given.
Network (EVD-LabNet) were invited to participate this The standards EURM-017 and EURM-018 were produced
EQA study. Registration was closed on 2 February 2021. and shipped by JRC. Product information on the materi-
Fifty-four laboratories received one EQA panel and two als is publicly available [11,12].
JRC standards between 22 February and 5 March 2021,
one laboratory registered later and received the pack- Samples characterisation, testing instructions
ages on 24 March 2021. The online submission form to Each sample of the EQA panel was extensively char-
submit EQA results was open until 8 April 2021. acterised by the World Health Organization (WHO)
COVID-19 reference laboratories at INMI and RIVM,
using a wide variety of serological tests (tests and
Panel composition outcomes of the panel characterisation are provided
The EQA panels were composed of eight different, in  Supplementary Table S1). Based on this characteri-
fully characterised, pooled human serum samples and sation, the final composition of the EQA panels was
addressed both sensitivity and specificity of detection. defined (Table 1). All samples of the EQA panel, includ-
Five samples contained anti-SARS-CoV-2 antibodies, ing the anti-SARS-CoV-2-negative samples (C, E and
while three samples did not (Table 1). Anti-SARS-CoV-2 F) were determined to contain antibodies against the
antibody-positive sera were collected from individuals four common human coronaviruses (HCoV-229E, HCoV-
with different severities of disease: mild, non-hospi- HKU1, HCoV-OC43, HCoV-NL63) by protein micro-array
talised cases (two samples that contained mixed sera as a quantitative multiplex immunoassay [13].
from multiple patients) and severe, hospitalised cases
(three samples that contained mixed sera from multiple The EQA panels were shipped at room temperature.
patients). The sera from individuals with SARS-CoV-2 The serological standards were shipped separately
infection were a collection of residual routine diagnos- on dry ice. Laboratories received detailed reconstitu-
tic anonymised samples and not suitable to evaluate tion, testing and storage instructions with the panels,
antibody waning. The median time from infection diag- advising to centrifugate the lyophilised samples for 1
nosis to sample collection was 17.5 days (range: 5–92 min at 3,000 rpm to sediment material which might
days). The obtained serology results for the preparation stick to the cap, reconstitute the samples in 200 µL
of the EQA were not used for the clinical management sterile water for ca 1 h, vortex the samples for ca 10 sec
of the patients. All sera from individuals with a SARS- to resuspend the material properly, and centrifugate
CoV-2 infection were collected in 2020 before COVID-19 the samples to spin down the reconstituted materials
vaccine roll-out. The sera from individuals with acute to avoid contamination. Laboratories were informed
cytomegalovirus (CMV) infection were collected before that the EQA panel samples consisted of inactivated,
the emergence of SARS-CoV-2 at the end of 2019 and non-infectious human sera, but no specific informa-
were anonymised. In addition, sera from individuals tion was provided, i.e. positivity and negativity to
with an acute Epstein-Barr virus (EBV) infection were anti-SARS-CoV-2.
anonymised. The anti-SARS-CoV-2-negative sera were
pre-pandemic residual sera from routine diagnos- The JRC standards were ready to use upon receipt. It
tics provided by the National Institute of Infectious was advised to store the material as follows: original
Diseases (INMI) in Rome, Italy and the National material of the EQA panel at room temperature (accept-
Institute for Public Health and the Environment (RIVM) able range: −20 °C to +25 °C), and at 4 °C once the
in Bilthoven, the Netherlands. All panel sera, including material was reconstituted; original material of the JRC
the pre-pandemic sera, contained antibodies directed standards frozen (acceptable range: −20 °C to −80 °C),
against the common cold coronaviruses (HCoV-OC43; and at 4 °C once the material was thawed. Laboratories
HCoV-229E; HCoV-NL63; HCoV-HKU1). In addition to the were informed that the provided standards contained
EQA panel, all participants received two human SARS- anti-SARS-CoV-2 antibodies, as they received the prod-
CoV-2 serological standards produced and described uct information sheet alongside the EQA panel.
by the JRC: EURM-017 and EURM-018 [11,12].
Evaluation of results
External quality assessment panel preparation The testing instructions provided a link to the online
The freeze-dried equivalent of 0.2 mL pooled result submission platform. In the online submission
anonymised sera was prepared for each panel sam- form, laboratories were asked to give detailed infor-
ple (Table 1). Samples were pooled to obtain sufficient mation on each of the tests that they performed on
volumes for uniform preparation of the total number of the EQA panel and JRC standards, including the type
required panels. All pooled samples were heat-inac- of detectable antibodies. Since a the majority of sero-
tivated (56 °C for 30 min) and freeze-dried in 0.2 mL logical tests only require small volumes, multiple dif-
aliquots (FreeZone Benchtop Freeze Dryer, LABCONCO, ferent tests could be performed. For each method, the
United States (US)). Successful virus inactivation of laboratories had to indicate whether they detected
panel samples was confirmed by the absence of viral anti-SARS-CoV-2 antibodies in the EQA samples, the
growth in two consecutive cell culture passages. All specific result of the test and how they interpreted
the outcome of the test. Because laboratories had to

www.eurosurveillance.org 5
6
Table 3
Performance of different SARS-CoV-2 assays that were used by three or more laboratories, external quality assessment, EU/EEA, February–April 2021 (n = 18)
Assay performance (samples characterised correctly/samples tested) EQA performance
Sample A Sample D Sample H Sample B Sample G Sample C Sample F Sample E Total correct
– hospitalised – hospitalised – hospitalised – mild – mild – negative – negative – negativ
Method Antibody (samples False- False-
Method details Unit*
type type IgA+/ characterised negative positive
IgA+/IgG+/ IgA+/IgG+/ IgA+/IgG+/ IgA+/IgG+/ Pre- Pre- correctly/ results results
IgG+/ Acute CMV+EBV
IgM+ IgM+ IgM+ low IgM+ pandemic pandemic samples
IgM+
tested)
Abbott SARS-CoV-2 IgG CLIA/CMIA IgG Index (S/C) 11/12 12/12 12/12 11/12 11/12 12/12 12/12 12/12 93/96 3 0
SARS-CoV-2 IgG II
Abbott CLIA/CMIA IgG AU/mL 9/9 9/9 9/9 9/9 9/9 9/9 9/9 9/9 72/72 0 0
Quant
Abbott SARS-CoV-2 IgM CLIA/CMIA IgM Index (S/C) 6/6 6/6 6/6 5/6 6/6 5/6 6/6 6/6 46/48 1 1
Beijing Wantai
SARS-CoV-2 Ab ELISA ELISA Total Ig Ratio 10/10 10/10 10/10 9/9 10/10 10/10 10/10 10/10 79/79 0 0
Biological
Beijing Wantai
SARS-CoV-2 IgM ELISA ELISA IgM Ratio 3/3 3/3 3/3 3/3 3/3 3/3 1/3 3/3 22/24 0 2
Biological
LIAISON SARS-CoV-2
Diasorin Inc. CLIA/CMIA IgG AU/mL 5/5 5/5 5/5 4/4 5/5 4/4 4/4 2/4 34/36 0 2
S1/S2 IgG
Anti-SARS-CoV-2
Euroimmun ELISA IgA Ratio 14/14 14/14 14/14 14/14 14/14 13/14 12/14 14/14 109/112 0 3
ELISA IgA
Anti-SARS-CoV-2
Euroimmun ELISA IgG Ratio 17/17 17/17 17/17 17/17 17/17 17/17 15/17 17/17 134/136 0 2
ELISA IgG
Anti-SARS-CoV-2 NCP
Euroimmun ELISA IgG Ratio 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 23/24 0 1
ELISA IgG
Anti-SARS-CoV-2 NCP
Euroimmun ELISA IgM Ratio 5/5 5/5 5/5 5/5 1/5 4/5 0/5 5/5 30/40 4 6
ELISA IgM
Anti-SARS-CoV-2
Euroimmun ELISA IgG RU/mL 4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4 32/32 0 0
QuantiVac ELISA IgG
cPass SARS CoV-2
Neutralising
GenScript Neutralisation sVNT % of inhibition 3/3 3/3 3/3 3/3 3/3 3/3 2/3 3/3 23/24 0 1
Ab
Antibody Detection Kit
SARS-CoV-2 Surrogate
Neutralising
GenScript Virus Neutralisation sVNT % of inhibition 5/5 5/5 5/5 5/5 5/5 5/5 5/5 5/5 40/40 0 0
Ab
Test Kit
In-house NA ELISA IgA, IgG, IgM Index 4/4 4/4 4/4 4/4 4/4 2/4 2/4 4/4 28/32 0 4
Neutralising
In-house NA PRNT Titre 4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4 32/32 0 0
Ab
Neutralising
In-house NA (s)VNT Titre 14/14 14/14 14/14 13/13 14/14 14/14 14/14 14/14 111/111 0 0
Ab
a
Other NA Various Various Various 40/41 40/41 41/41 41/41 40/41 38/41 38/41 37/41 315/328 3 10
Elecsys Anti-SARS-
Roche CLIA/CMIA Total Ig U/mL 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 24/24 0 0
CoV-2 S
Vircell COVID-19 ELISA
ELISA IgA + IgM Index 3/3 3/3 3/3 3/3 3/3 3/3 3/3 3/3 24/24 0 0
microbiologists IgM + IgA

Ab: antibody; AU: arbitrary unit; CLIA: chemiluminescence immunoassay; CMIA: chemiluminescent microparticle immunoassay; CMV: cytomegalovirus; COVID-19: coronavirus disease; EBV: Epstein–Barr virus; EQA: external quality assessment; EEA:
European Economic Area; ELISA: enzyme-linked immunosorbent assay; EU: European Union; Ig: immunoglobulin; NA: not applicable; PRNT: plaque reduction neutralisation test; RU: relative units; U: units; SARS-CoV-2: severe acute respiratory
syndrome coronavirus 2; (s)VNT: (surrogate) virus neutralisation test.

www.eurosurveillance.org
a
 The category ‘other’ comprises all tests that were used by less than three laboratories.
 A A
-h -
os Inhibition of binding (%) ho Arbitrary units/mL
D p D s
-h ita - h p it
os lis o s al is
H H
Figure 2

- h p ita ed - h p it ed

1
10

-100
1,000
10,000
100,000

100

50

-50
0
100
150
os lis A+ o s al is A+
pi ed /G p i ed /G
+
ta + ta
G lis A+ /M+ G l is A+ /M +
-m B ed /G+ -m B ed /G+
il - A /M il - A /M
C d Ig mi + / + C d Ig mi + /
- n A ld G+ - A ld G +
+ / A /M ne +/ A+ +/M

www.eurosurveillance.org
F
eg + F ga I g
- a Ig G /G + - G /G +

95.0% confidence interval.

ne t iv e + / +/M ne tiv e + / +/M
ga p Ig M + ga pr Ig M +
t iv r e - + t iv e - +
e pa l ow e pa l ow
E pr e n d E pr e n d
- e - e

respiratory syndrome coronavirus 2.

a c - pa m i a c - pa m i
ut nd c u t nd c
e e
EB emi EB emi
V c V c
EU /C M EU /C M
RM V R V
M
EU -0 EU -0
R 17

Virus NeutralizationTe st Kit (n=5)

R M 17 M

C. GenScript - SAR S-CoV- 2 Surrogate

-0 -0
18 18
A. Abbott - SARS-CoV-2 IgG II Quant (n=9)

A A
- -
ho ho
D sp D s
-h it - h pita Arbitrary uni ts/mL
Units/mL
used by at least three laboratories, EU/EEA, February–April 2021 (n = 22)

o s al is os lie
H p H
- h it ed - h pita s d

0.1
1

10
10

1,000

100
1,000

100

o s al is A+ o s lis A +
p i ed /G p i e d /G+
ta + ta
G l is A+ /M+ G l is A + /M+
-m B ed /G+ -m B ed /G+
il - A /M ild -
C d Ig mi + / + C Ig
m A+ / /M+
- A ld G+ - il G
ne +/ A+ /M ne A+ d A +/M
F g I F g /
- a g G /G +
- a IgG +/G +
ne tiv e + / +/M ne t iv e +/ +/M
ga pr Ig M + ga p Ig M +
t iv e - + t iv r e - +
e pa l ow e pa l ow
E pr e nd E pr e nd
- a -p em - e
cu a n ic a c - pa m i
ut nd c
te d e e
EB mi EB emi
V c V c
EU /C M EU /C M
RM V RM V
EU -0 EU -0
RM 1 7 RM 1 7
-0 -0
18 18
D. Roche - Elecsys Anti-SARS-CoV-2 S (n=3)

In panel B, n = 4/5 indicates that one laboratory only tested five of eight EQA samples with this test method. Dotted blue lines indicate
method specific cut-off values for positivity as indicated by EQA participants and manufacturers’ instructions. Red lines show median and
B. Diasorin Inc. - LIAISON SARS-CoV-2 S1/S2 IgG (n=4/5)
Reported numerical results for EQA and JRC standards (EURM-017 and EURM-018), semiquantitative SARS-CoV-2 assays

EEA: European Economic Area; EQA: external quality assessment; EU: European Union; JRC: Joint Research Council; SARS-CoV-2: severe acute

7
submit their results per specific test used, the EQA out- 98.7% and 100.0% correct results, samples C (nega-
comes could be analysed on individual test level, as tive pre-pandemic), E (acute CMV+EBV infection) and G
well as on laboratory level (multiple tests per labora- (mild COVID-19, A+/G+/M+(low)) showed slightly lower
tory). There was no minimum or maximum limit for how proportions for correct identification ranging from
many tests could be assessed with the EQA panels and 95.0% to 96.3% (Table 1). The sample with the least
submitted in the online submission form. correct results, 88.8%, was sample F (negative pre-
pandemic,  Table 1). The correct identification propor-
For the purposes of this EQA, the panel outcomes tion for the JRC standards was 94.9% for EURM-017 and
with an individual test submitted by a laboratory were 96.8% for EURM-018 (Table 1).
referred to as a ‘test’, i.e. in total, results of 162 tests
were submitted by 55 laboratories (for instance, two Assay performance
laboratories used seven tests to characterise the EQA Overall, false-negative results occurred less frequently
samples, 16 used three tests, 10 used one test only) than false-positive results (Figure 1C,  Table 2). Of the
(Figure 1). Tests that were used by multiple labora- 161 tests, 135 (83.9%) characterised all specificity
tories that were either the same commercial test or samples (C, E, F) correctly. Furthermore, we analysed
had the same principle (e.g. VNT) were referred to assays separately for each antibody isotype they
as ‘assays’, i.e. in total 53 commercial and in-house could detect when laboratories submitted these indi-
assays (Supplementary Table S2  lists the commercial vidual results. Assays for which no individual isotype
and in-house tests assessed by EQA participants) were results were submitted were grouped as ‘IgM/IgG’
used by 55 laboratories resulting in a submission of and ‘IgM/IgA’ (Table 2). IgM-specific tests performed
results of 162 tests. least accurately and were significantly worse at iden-
  tifying samples correctly than tests detecting total
immunoglobulin (Table 2). For each group, IgA, IgG or
Statistics IgM, multiple assays gave at least one false outcome;
Data were collected and analysed in Microsoft Excel respectively, two of six, seven of 32 and eight of 14
(Microsoft Corp., Bellingham, US) and GraphPad Prism assays had at least one false result.
9 software for Windows version 9.1.0 (GraphPad Soft-
ware, San Diego, US). Performance of specific tests In total, 41 commercial assays and 12 in-house
was analysed by comparison of the amount of correct assays were used by the EQA participants (listed
vs false results, either grouped by assay type or by iso- in  Supplementary Table S2). Overall, 3.5% (37/1,058)
types of detectable antibodies, using two-sided Yates’ of reported results using commercial assays and 2.6%
corrected chi-squared test. Results with a p value ≤ 0.05 (6/231) using in-house assays were incorrect. Of all
were considered statistically significant. Furthermore, 162 tests used by participants, 75 (46.3%) were ELISA-
Spearman’s rank correlation test was used to assess based, 43 (26.5%) were CLIA/chemiluminescent micro-
correlation of EQA performance as fraction of correct particle immunoassay (CMIA)/electrochemiluminescent
results, with sample input volume as specified by the immunoassay (ECLIA) tests and 22 (13.6%) VNTs. The
EQA participants. remaining 22 (13.6%) included, among other types of
tests, LFA, plaque reduction neutralisation test (PRNT),
Results protein micro-array and immunofluorescence assay/
enzyme-linked fluorescence assay.
External quality assessment participation and
overall laboratory and test performance Among those assays performed by three or more EQA
Fifty-six laboratories registered to participate in participants, two in-house assays (VNT, PRNT) and six
the EQA. In total, 55 laboratories from 30 countries, commercial assays (Abbott – SARS-CoV-2 IgG II Quant,
namely 27 of 30 EU/EEA countries and three of seven Beijing Wantai Biological – SARS-CoV-2 Ab ELISA,
EU pre-accession countries, reported individual panel Euroimmun – Anti-SARS-CoV-2 QuantiVac ELISA IgG,
results representing 162 tests (Figure 1A), while one Roche – Elecsys Anti-SARS-CoV-2 S, Vircell microbiolo-
laboratory did not submit any results. The number of gists – COVID-19 ELISA IgM+IgA and GenScript - SARS-
different tests assessed per laboratory varied between CoV-2 Surrogate Virus Neutralisation Test Kit) correctly
1 and 7 (Figure 1B). characterised all EQA samples in all laboratories that
assessed these assays (Table 3). None of the assays
Thirty-six of 55 laboratories characterised all EQA performed significantly better than the group ‘Other’
samples correctly with all tests they assessed, while (i.e. all tests that were used by fewer than three labo-
17 additional laboratories used at least one test that ratories) considering two-sided Yates’ corrected chi-
identified all EQA samples correctly (Figure 1B). Only squared test. One test (Euroimmun – Anti-SARS-CoV-2
two laboratories could not identify all samples cor- NCP ELISA IgM) performed significantly worse than
rectly (Figure 1B). On test level, 82.1% of the submitted ‘Other’ (p value < 0.0001) (Table 3).
tests (133/162) detected all tested samples correctly.
While the outcome for the samples of pooled sera from
hospitalised patients and for one sample of the pooled
sera from mild patients (A, D, H and B) ranged between

8 www.eurosurveillance.org
 Table 4
Results for JRC standards EURM-017 and EURM-018, semiquantitative SARS-CoV-2 assays used by three or more laboratoriesa in this EQA and previously available product
information for the reference material, EU/EEA, February–April 2021 (n = 22)

EQA participants JRC reference product informationb

Method details
EURM-017 EURM-018 EURM-017 EURM-018
Median 1,271 AU/mL 1,914 AU/mL NA NA

www.eurosurveillance.org
95.0% CI of median lower confidence limit 1,174 AU/mL 1,815 AU/mL NA NA
Abbott - SARS-CoV-2 IgG II Quant, n = 9 95.0% CI of median upper confidence limit 1,367 AU/mL 2,157 AU/mL NA NA
Mean 1,271 AU/mL 1,836 AU/mL 1,155 AU/mL 1,797 AU/mL
Standard deviation 74 AU/mL 397 AU/mL NA NA
Median 84 AU/mL 89 AU/mL NA NA
95.0% CI of median lower confidence limit 74 AU/mL 82 AU/mL NA NA
Diasorin Inc. - LIAISON SARS-CoV-2 S1/S2 IgG, n = 5c 95.0% CI of median upper confidence limit 86 AU/mL 98 AU/mL NA NA
Mean 81 AU/mL 89 AU/mL NA NA
Standard deviation 5 AU/mL 7 AU/mL NA NA
Median 73.8% inhibition 76.4% inhibition NA NA

GenScript - SARS-CoV-2 Surrogate 95.0% CI of median lower confidence limit 56.7% inhibition 66.6% inhibition NA NA
95.0% CI of median upper confidence limit 89.0% inhibition 89.0% inhibition NA NA
Virus Neutralisation Test Kit, n = 5 Mean 71.3% inhibition 77.0% inhibition 73.8% inhibitiond 76.4% inhibitiond
Standard deviation 12.6% inhibition 8.0% inhibition NA NA
Median 190 U/mL 146 U/mL NA NA
95.0% CI of median lower confidence limit 188 U/mL 141 U/mL NA NA
Roche - Elecsys Anti-SARS-CoV-2 S, n = 3 95.0% CI of median upper confidence limit 208 U/mL 151 U/mL NA NA
Mean 195 U/mL 146 U/mL 199 U/mL 154 U/mL
Standard deviation 11 U/mL 5 U/mL NA NA

AU: arbitrary unit; CI: confidence interval; EEA: European Economic Area; EQA: external quality assessment; EU: European Union; JRC: Joint Research Council; NA: not available; SARS-CoV-2: severe acute
respiratory syndrome coronavirus 2; U: Unit.
a
 One additional semiquantitative assay, namely Euroimmun (Anti-SARS-CoV-2 QuantiVac ELISA IgG, n = 4) was used by more than three laboratories. However, the results could not be included for this
analysis, as not all laboratories submitted results in units as prescribed by the manufacturer.
b
 Reference material product information published by JRC for JRC standards EURM-017 [11] and EURM-018 [12].
c
 One laboratory only tested five of eight EQA samples with this test method.
d
 Information not publicly available, direct communication by JRC.

9
Qualitative and semiquantitative results in used at least one assay that identified all samples cor-
relation to Joint Research Centre standards rectly as SARS-CoV-2 antibody positive or negative.
Four qualitative assays in this EQA had been previ- IgM-specific assays showed the most incorrect charac-
ously used to characterise the JRC standards as posi- terisations of the EQA samples. In particular, sample G
tive, both EURM-017 [11] and EURM-018 [12]: Genscript (IgA+/IgG+/IgM+(low)) was missed by four of five labo-
(cPass SARS-CoV-2 Neutralisation Antibody detection ratories that used the same commercial IgM-only assay
kit), Roche (Elecsys Anti-SARS-CoV-2), Abbott (SARS- (Euroimmun – Anti-SARS-CoV-2 NCP ELISA IgM) and by
CoV-2 IgG) and Abbott (SARS-CoV-2 IgM). In this EQA, two additional assays that should have been able to
Genscript and Roche scored all samples correctly, while detect the presence of IgM antibodies based on their
the assays of Abbott SARS-CoV-2 IgG and SARS-CoV-2 technical specifications (AAZ - COVID-PRESTO TROD
IgM each scored one standard incorrectly (EURM-018) IgG/IgM; Hangzhou Biotest Biotech - RightSign COVID-
as negative. 19 IgG/IgM Rapid Test Cassette). The remaining 20 lab-
oratories were able to identify sample G correctly using
Laboratories were asked to specify the exact (numeri- 11 different assays. Indeed, this sample was charac-
cal) results of each test they used to assess how terised by the two reference laboratories to have IgM
semiquantitative assays performed across different levels below the detection limit of some assays (find
laboratories in comparison to the reference product the detailed results listed in Supplementary Table S1).
information available for the two JRC standards. Figure Furthermore, eight of 14 IgM-specific assays resulted in
2 and Table 4 show an overview of the numerical results one or more false-positive test results. Several studies
for all semiquantitative specific assays that were used have shown that in general, IgM assays have a lower
by three or more laboratories. Notably, results of three sensitivity and specificity than IgG or total IgG assays
of the semiquantitative assays, namely by Abbott [14-16]. Hence it is recommended to perform sero-
(SARS-CoV-2 IgG II Quant), GenScript (cPass SARS- diagnostics on multiple isotypes simultaneously to
CoV-2 Surrogate Virus Neutralisation Test Kit) and improve the specifics of the overall diagnosis. In addi-
Roche (Elecsys Anti-SARS-CoV-2 S) characterised all tion, although specific IgM antibodies can be detected
EQA samples correctly; their results for the JRC stand- as early as 4 days after infection and they can help to
ards corresponded to previous JRC characterisations define the early antibody response, SARS-CoV-2 infec-
(Table 4). Absolute titres obtained with the various tion may trigger unconventional antibody responses,
virus neutralisation assays varied between laborato- with cases developing IgG before IgM or others with no
ries (range: 12- to 115-fold differences). IgM [17-19]. Indeed, the majority of laboratories (45/55)
used more than one serological test to assess the
Influence of various parameters on the samples. In case of result discrepancies between IgM-
performance of the external quality assessment specific tests and tests detecting other types of immu-
The vast majority of laboratories used the advised noglobulins, laboratories should consider the timing of
volume of 200 µL sterile water to reconstitute the EQA a potential infection, but also the higher unreliability
samples (51/55 laboratories) as well as the storage of IgM-specific tests. Overall, test methods detecting
advice for both the EQA panels (54/55 laboratories) total immunoglobulin, neutralising antibodies or IgG
and the JRC standards (50/55 laboratories). We did not performed better than test methods that are IgA- or
see any influence of these parameters on test perfor- IgM-specific. Test method performance was different
mance. Although sample input volume for the different neither for commercial and in-house assays, nor for a
tests ranged from 1 µL to 170 µL, it did not correlate specific test principle, e.g. ELISA, (s)VNT, PRNT or CLIA/
with the fraction of correct results by test (p = 0.3251; CMIA/ECLIA. This was also shown by the variety of test
Spearman correlation test). methods that characterised 100.0% of tested samples
correctly (Table 3).
Considering antibodies directed to different antigenic
proteins targeted in the serological assays, the major- Correct characterisation of the three specificity sam-
ity of the assays used (n = 27) was based on the spike ples (C, E and F) was more problematic than of the
(S) protein, including recombinant full S or its specific sensitivity samples. Variable test performance for
domains (S1, S2 or receptor-binding domain). Fifteen specificity samples has been reported before [20,21].
assays used both N and S proteins, while six targeted the A likely explanation is potential cross-reactivity with
anti-N response only. In addition, 18 assays used whole antibodies raised by previous infection with other
live virus isolates for VNT/PRNT, while for three in-house human coronaviruses causing the common cold [13],
assays, the viral antigen was not reported. No influ- as all samples in the proficiency panel of this EQA also
ence of the target protein on test results was observed. contained antibodies against all other seasonally cir-
culating human coronaviruses (Supplementary Table
S1). If this was the case, specificity test performance
Discussion would probably be further reduced in periods of high
The overall performance of laboratories within this EQA prevalence of co-circulating seasonal coronaviruses.
indicated a robust ability to establish whether past
SARS-CoV-2 infections occurred via detection of anti- As expected, the absolute titres obtained with the
SARS-CoV-2 antibodies. All except two laboratories various virus neutralisation assays varied between

10 www.eurosurveillance.org
laboratories as underlying protocols can vary exten- follow-up studies. The wide variety in tests used and
sively (e.g. whole viruses or pseudotype viruses, num- general lack of routine incorporation of international
ber of median tissue culture infectious dose units, standards in SARS-CoV-2 serology prevents compari-
incubation period and temperature, cell lines, cut-off, sons of immune responses across laboratories, thereby
read-out) and this gold standard serology method is hampering standardised cross-border sero-epidemi-
mostly used as a research tool in the virology field. ological surveillance and the wide implementation
A recent European study showed that indeed a sub- of immunoassays to identify correlates of protection
stantial heterogeneity exists in neutralising antibody against SARS-CoV-2, e.g. in the context of vaccination
testing approaches, resulting in almost 100-fold dif- policies or the EU Digital COVID Certificate Regulation
ferences in raw neutralising titres. However, a direct [25].
comparison was possible through harmonisation by
the use of a standard defined in IU/mL, which reduced Nevertheless, proficiency testing is important for indi-
the inter-laboratory variability ca 10-fold [22]. Another vidual laboratories. The results of an EQA allow labora-
study showed that the inter-laboratory variation for tories to identify potential problems and improve the
neutralisation tests was reduced more than 50-fold reliability of their diagnosis. Interestingly, one labo-
when assay outcomes were reported relative to the ratory repeated their tests upon receipt of their EQA
same (WHO) standard [23]. In our study, the results of results which included three false negative results
the VNT could not be compared through normalisation using two different assays. In this second attempt, the
as the JRC standards were defined with a titre range for results were correct. This laboratory indicated insuf-
neutralisation tests and not with a defined IU/mL as in ficient homogenisation of the samples as a potential
the WHO standard [24]. explanation for the observed discrepancies. 

The characterisation of the JRC standards with the sem- Conclusion

iquantitative binding assays by the EQA participants A general capability for reliable, harmonised and
was in range with the standards product information, standardised characterisation of the main immuno-
indicating a robust performance of these semiquantita- logical markers of a previous SARS-CoV-2 infection or
tive assays across different laboratories. The qualita- vaccination against SARS-CoV-2 is crucial to increase
tive assays that were used to define the JRC standards the overall utility of serology testing. Potential incon-
performed similarly in the EQA as well. sistent test performance and the absence of a ration-
ale to use quantitative antibody assays due to the,
Notably, all samples used in this study were obtained yet, undetermined correlates of protection of anti-
from patients with natural SARS-CoV-2 infections. We body levels, currently limits the overall usefulness of
did not include samples from vaccinated individuals. serology. Participation in EQAs may help to improve
For future sero-epidemiological surveillance stud- implementation of diagnostic tests. Our EQA showed
ies, the capacity of serological tests to differentiate a high capability across European reference laborato-
between antibodies derived from natural infections ries for reliable diagnostics for SARS-CoV-2 antibody
and antibodies induced by vaccination is desirable. responses. Serological tests that provide robust and
This can be achieved by using serological tests with reliable detection of anti-SARS-CoV-2 antibodies are
different antigenic targets, i.e. anti-N (natural infec- available. However, the use of standards is necessary
tion only) and anti-S detection, to determine the level for meaningful quantitative measurements to increase
of natural immunity in the study population and assess the overall use of serology testing, including the
the risk of vaccination not being sufficient to protect evaluation of antibody waning and possible reactivity
against a potentially increasing burden on the health- against more relevant virus variants with immunologi-
care system. EQA programmes including also sera from cal escape potential in the prospective of a more ‘per-
vaccinated people would be of great interest to expand sonalised’ approach to vaccination strategies. This is
the evaluation of the accuracy of different tests during particularly relevant given that serology is no longer
the next phases of the pandemic when vaccination cam- restricted to reference laboratories. Numerous labo-
paigns are consolidated in most countries worldwide. ratories non specialised in microbiology are perform-
ing serological tests using a wide panel of commercial
Proficiency testing in EQA schemes allows to compare assays. In order to maintain serological test quality, it
the accuracy of different tests and the performance would also be advisable to implement EQAs at national
of different laboratories based on the same material. level, including schemes to evaluate and distinguish
Although the number of study participants (n = 55) and natural immunity and vaccination response.
moreover the total number of performed tests (n = 162)
was sufficient to gain valuable insights into the qual-
ity of serological SARS-CoV-2 assays, the abundance, *Erratum
availability and variety of different SARS-CoV-2 assays A superscript “b” was included in some of the cells in the
used by the participants, limits the number of results Unit column in Table 3 in the originally published version.
obtained per specific assay by different laboratories These superscripts had no associated footnote and were re-
and therefore the statistical power and confidence in moved on 25 October 2022. We apologise for this oversight.
the results. It would be desirable to perform additional

www.eurosurveillance.org 11
Ethical statement Lithuania; Microbiology department, Laboratoire National
de Santé, Luxembourg; Virology Serology Pathology depart-
The current study was performed in accordance with the ment, Mater Dei Hospital, Malta; Laboratory for Virology
guidelines for sharing of patient data of observational sci- and Molecular Diagnostics, Institute of Public Health, North
entific research in emergency situations as issued by the Macedonia; Department of Virology, Norwegian Institute
Commission on Codes of Conduct of the Federation of Dutch of Public Health, Norway; Department of Bacteriology
Medical Scientific Societies [26]. The use of residual clini- and Biocontamination Control, National Institute of Public
cal samples and the related data on non-infringing personal Health - National Institute of Hygiene, Poland; Department
information for studies on diagnostics validation was ap- of Virology, National Institute of Public Health - National
proved by INMI Ethical Board (“Comitato Etico INMI Lazzaro Institute of Hygiene, Poland; National Influenza and Other
Spallanzani IRCCS/Comitato Etico Unico Nazionale Covid-19”, Respiratory Viruses Reference Laboratory, Infectious
issue n. 70/2018). Diseases Department, National Institute of Health Dr.
Ricardo Jorge, Portugal; Seroepidemiological laboratory,
National Institute of Public Health, Romania; Department
Funding statement for Laboratory Diagnostic, Institute of Virology, Serbia;
Laboratory for Public Health Virology, National Laboratory of
This work was supported by the European Centre for Disease Health, Environment and Food, Slovenia; Laboratory for di-
Prevention and Control (ECDC) under specific contract no. 4 agnosis of zoonoses, Faculty of Medicine, Slovenia; Serology
ECD.11186 for implementation of the framework contract laboratory, Centro Nacional de Microbiología, Spain; Klinisk
ECDC/2017/002 to CBEM and CC. Mikrobiologi, Serologi, Karolinska Universitetslaboratoriet,
Sweden; Microbiology department, The Public Health Agency
of Sweden, Sweden; Department of Medical Biochemistry
and Microbiology, Uppsala University, Sweden; Department
Acknowledgements Viroscience, Erasmus MC, The Netherlands; National Virology
We thank all EQA participants: Center for Virology, Medical Reference Laboratory, Public Health General Directorate of
University of Vienna, Austria; Clinical Reference Laboratory, Turkey;
Institute of Tropical Medicine, Belgium; Immunology depart-
ment, National Centre of Infectious and Parasitic Diseases, In addition, we thank the technicians providing laboratory
Bulgaria; Microbiology department, National Centre of support: Fion Brouwer, Marieke Hoogerwerf, Sophie van
Infectious and Parasitic Diseases, Bulgaria; Department of Tol, Gert-Jan Godeke and Sakinie Misiedjan, at RIVM; Aurora
Microbiology and Immunology, Medical University, Plovdiv, Bettini, Eliana Specchiarello, and Silvia Sarti, at INMI. We
Bulgaria; Department of Virology, Croatian Institute of Public also thank Silvia Meschi, Giulia Matusali, and Licia Bordi for
Health, Croatia; Virology department, University Hospital for the laboratory support at INMI, and Raffaella Marconi and
Infectious Diseases “Dr. Fran Mihaljević”, Croatia; Molecular Lorena Fiorentini for the administrative support at INMI.
Virology, Cyprus Institute of Neurology and Genetics, Cyprus;
Clinical Labs Immunology Department, Nicosia General
Hospital, Cyprus; NRL for Influenza and other Respiratory
Viruses, National Institute of Public Health, Czech Republic; Conflict of interest
Department of Virology, Institute of Public Health, Ostrava, None declared.
Czech Republic; VMS Serology, Virus & Mikrobiological
Specialdiagnostic, Statens Serum Institut, Copenhagen,
Denmark; Laboratory of Communicable Diseases, Health
Board, Estonia; Expert Microbiology Unit, National Institute Authors contributions
for Health and Welfare, Finland; Laboratoire Associé au CNR CR, CC, ADC, AMel, KL, MK, AMei, RM and FCo were involved
des Virus des Infections Respiratoires (France Sud), Institut with the study design and study organisation. FCo, JR, MF,
des Agents Infectieux, France; Virology department, Institut DL, FCa, CC, JLM, AV, BH and LD were involved with prepa-
Pasteur Paris, France; FG17 Influenza and Other Respiratory ration of serum pools for EQA panels and provision of sera
Viruses, Robert Koch Institute, Germany; Institute of Novel and standard material. JR, FCo and CC were involved in pre-
and Emerging Infectious Diseases, Friedrich-Loeffler- testing of the EQA panels. RM, CR, FCo and CC were involved
Institute, Germany; ZBS1 Highly Pathogenic Viruses, Robert with the data collection and analysis. RM, CR, FCo and CC
Koch Institute, Germany; Public Health Laboratory Support, co-wrote the manuscript. All authors read and approved the
ZIG 4, Robert Koch Institut, Germany; Diagnostic Department, final manuscript.
Institute of Philipps-Universität Marburg, Germany; Institute
of Virology, Charité Universitätsmedizin Berlin, Germany;
Immunology laboratory, Central Public Health Laboratory,
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