Dried blood spots are an efficient blood sampling method for the detection of SARS-CoV-2 antibodies [version 1; peer review: awaiting peer review]

A novel coronavirus termed SARS-CoV-2 caused an outbreak in December of 2019 which has led to pandemic. Currently several serological diagnostic assays exist for the detection of SARS-CoV-2, which require the collecting of whole blood that brings about problems including the invasive nature of venepuncture, poor acceptance by patients and their storage and transportation. A more fast, efficient and less tedious method that allows mass blood sampling is necessary during a pandemic to quickly diagnose disease and obtain population serological data. Dried blood spot (DBS) sampling has been used for several decades for the accurate detection of viral specific antibodies and remains one the most convenient methods for obtaining serological data on exposed patients. Here we evaluate the use of DBS sampling on current viral serological assays including SARS-CoV-2. DBS samples were collected from six patients (five control and one positive for SARS-CoV-2 infection) and patient serum was extracted and tested blindly using commercially available antibody test kits for Coxiella burnetti, parvovirus B19 and SARS-CoV-2. The results demonstrate that antibodies recovered from DBS after elution are comparable to those found in serum, indicating that serological tests can be adapted to test DBS samples from patients using our modified protocol. Because DBS sampling is a much faster and cheaper method of sampling blood, this modification could therefore allow for potential nationwide testing for epidemiological studies.


Introduction
Coronavirus disease 2019 (COVID-19) was first reported in December, 2019 in Wuhan, China and on March 11 2020 it was declared a pandemic by the World Health Organization. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which shares up to 82% genome identity with SARS-CoV, which first caused an outbreak in 2003 1 . It is characterized by fever, cough, malaise, shortness of breath and in severe cases pneumonia 2 .
Reliable serological data are in urgent need to guide treatment, infection control, epidemiological measures and vaccination. Whilst several serological assays are now available for antibody detection in the form of anti-SARS-CoV-2 enzymelinked immunosorbent assay (ELISA) kits, most of these require the collection of serum from whole blood. Whole blood collection can pose several problems in their collection, storage and transportation. The collection of whole blood requires venepuncture, which is an invasive procedure and can be poorly accepted by patients. Equipment requirements for the isolation of serum include syringes, tubes, centrifuges, refrigerators and freezers. After collection, whole blood requires maintenance at the site of collection with refrigerated storage and transportation from distant locations 3 .
Dried blood spot (DBS) sampling offers practical, clinical, and financial advantages as compared to conventional blood testing. Compared with standard venepuncture, the fingerstick or heelpricking sampling of capillary blood is easy to perform and relatively painless. It can be carried out by patients at home without a trained health care worker or medical laboratory, which is of particular advantage in settings with limited infrastructure. Utilizing capillary blood requires less sampling volume, negates the use of centrifuges or even basic laboratory equipment, and transportation is facilitated as it does not require refrigeration or high skill preventing contamination 4 . DBS has been used for the detection of antibodies to several viral disease including rubella, HIV, measles and hepatitis and has shown good correlation to serum samples when assayed with commercially available kits [5][6][7][8][9] . Further, DBS sampling allows for the detection of different immunoglobulin isotypes IgG, IgA, IgE and IgM [5][6][7][8]10 . For this reason, we decided to evaluate the use of DBS sampling on current viral serological assays for SARS-CoV-2. DBS samples were collected from six patients (five control and one positive for SARS-CoV-2 infection) and patient serum was eluted and tested blindly using commercially available antibody test kits for Coxiella burnetti, parvovirus B19 and SARS-CoV-2.
Our results demonstrate that antibodies recovered from DBS after elution are comparable to those found in serum, indicating that commercially available serological assays can be adapted to test DBS samples from patients using our modified protocol. Adapting ELISA assays to DBS sampling may facilitate potential nationwide testing for epidemiological studies into SARS-CoV-2.

Ethical statement
According to Swiss legislation, the Human Research Act is applicable to research on human diseases and on the structure and function of the human body. As this project is a quality control on the blood samples of six participants, it does not fall under the scope of research under Article 3 HFG and an assessment by an ethics committee was therefore deemed unnecessary by the Ethics Committee Of Eastern Switzerland. All patients gave both oral and written consent to participate in this study.

DBS sampling
Potential participants were identified within our institute and selected based on their medical history. DBS samples were collected by a medical laboratory technician from six adult patients (five control and one SARS-CoV-2 positive, as confirmed by PCR and antibody testing). Blood was collected through skin puncture twice over two separate weeks. Briefly, the skin was cleaned on the palmar side of the 4th finger on the left hand with 70% isopropyl alcohol. The skin was punctured using a single-use safety BD Microtainer contact activated lancet (Becton Dickinson, Dublin, Ireland) and the first drop of blood was wiped off. Blood was collected on to a Whatman 903 Protein Saver Card (GE Healthcare, UK). DBS were allowed to dry completely overnight at room temperature. Using a single-use 6mm biopsy punch (GSK Consumer Healthcare, UK), one spot was punched out from each DBS and transferred into a well of a 96-well plate using a single-use disposable tweezer (Megro, Wesel, Germany). This process was repeated for all punched spots. To elute serum, 250 µl elution buffer (1 X PBS, 0.05% Tween 20, 0.1% BSA) was placed in each well and the 96-well plate was placed on a rotator plate overnight at RT. Eluates were then collected and stored at 4°C for ELISA.
ELISA assay DBS eluates were subjected to the following commercial immunoassay kits: Parvovirus B19 IgG (Kit number ESR122G, Serion Diagnostic, Germany), Coxiella burnetii Phase 2 IgG (Kit number ESR1312G, Serion Diagnostics), Anti-SARS-CoV-2-ELISA IgG and Anti-SARS-CoV-2-ELISA IgA (Kit numbers EI 2606-9601 G and EI 2606-9601 A, Euroimmun, Lübeck, Germany) and the EDI™ Novel Coronavirus COVID-19 IgG ELISA kit (Kit number KT-1032, Epitope Diagnostics, San Diego, USA) to detect the presence of antibodies. Because seroprevalence is high for parvovirus 11 and low for Coxiella burnetii in Europe 12,13 , the parvovirus B19 IgG and Coxiella burnetti Phase 2 IgG ELISAs acted as positive and negative controls, respectively. All the listed commercial immunoassays in our study require a minimum of 10 µl serum sample to be diluted in a 1:100 dilution ratio with sample diluent at a final volume of 1000 µl. It has been reported that a 6mm punched spot contains approximately 5 µl of serum 5 . Therefore, sample preparation for the commercial ELISA required 2 x 250 µl patient sample eluates to be pooled (500 µl), to which 500 µl of sample diluent was added to make a final volume of 1000 µl.

Elution of DBS samples
The elution buffer (1 X PBS, 0.05% Tween 20, 0.1% BSA) was found to be effective at eluting serum from DBS samples. Because each 6mm punched spot contains approximately 5 µl of serum, and elution with 250 µl represented a 1:50 dilution of the DBS sample.

ELISA with DBS samples
All six patient (A-F) DBS samples were tested for antibodies using the commercial ELISA kits parvovirus B19 (IgG), Coxiella burnetii Phase 2 (IgG), anti-SARS-CoV-2-ELISA (IgG and IgA) and the EDI ™ novel coronavirus COVID-19 (IgG) ELISA kit. All DBS samples were positive for parvovirus B19 IgG antibodies, whilst negative for Coxiella burnetti IgG as expected (Table 1). DBS samples assayed on both the SARS-CoV-2 IgG from Euroimmun and Epitope Diagnostics were negative in control patients (A-E), whilst positive for the single patient (F) with a previous history of SARS-CoV-2 infection ( Table 2, Table 3).
The ELISA for IgA antibodies from Euroimmun indicated that the DBS sample from the SARS-CoV-2 patient (F) was positive for specific IgA antibodies (Table 2). Interestingly, several DBS samples from control patients displayed borderline positive results, whilst one DBS sample from one patient (E) with no apparent history of exposure to SARS-CoV-2 showed the presence of IgA antibodies (Table 2).

Discussion
The results that we have obtained serve to confirm the effectiveness of using DBS samples for the serological detection of antibodies, whilst extending the applicability of current commercial immunoassays for the detection of SARS-CoV-2. Our results are in accordance with the expected seroprevalence of Coxiella burnetii and parvovirus in Europe [11][12][13] . Of importance is that we were able to detect both IgG and IgA antibodies in the patient with a history of SARS-CoV-2 infection using the commercially available kits. This indicates that DBS sampling may be as reliable as serum sampling via venepuncture for the detection of antibodies to SARS-CoV-2. Although IgA was detected as both borderline and positive results in asymptomatic control patients which were thought to be negative for exposure to SARS-CoV-2, this observation may be due to several reasons. Borderline cross-reactivity has been reported for the anti-SARS-CoV-2-ELISA (IgA) from Euroimmun 14 . Samples   negative for IgG and borderline or positive for IgA may also indicate prior exposure to SARS-CoV-2.
The advantages of adapting commercial immunoassays to DBS are many. Rapid diagnostic testing is necessary in limitedresource settings. DBS offers an affordable alternative to venepuncture. Because little training is required to sample blood using the DBS method, a greater number of the population can be sampled for epidemiological studies. DBS samples can be dried and stored at room temperature for several weeks, they require little storage space and can even be sent to testing sites via mail. Such sampling may be especially suitable in serological screening programs in developing countries, where SARS-CoV-2 is having its greatest impact. DBS sampling may further facilitate widespread sampling of capillary blood, enabling analysis with high-throughput laboratory-based immunoassays, which would allow rapid nationwide serological screening that is of absolute necessity for disease control during a pandemic.
The main disadvantage of DBS sampling is that most of the existing commercial immunoassays have not been validated or received the necessary regulatory approval for DBS sampling to be implemented. Despite this drawback, DBS sampling has shown promise, in that it has been successfully used to detect the presence of antibodies to several disease-causing viral pathogens including rubella, HIV, measles and hepatitis [5][6][7][8][9] . A meta-analysis on the diagnostic accuracy of HIV-antibody and HBV-surface antigen from DBS samples compared to venous blood samples was associated with excellent diagnostic accuracy 9 . However, the study found that the lack of standardization of sampling, handling, processing, storage and transportation processes limits their use. The use of DBS sampling for routine or high-throughput serological assays would therefore require a standardized method for the pre-analytical treatment of specimens and processing of the sample and subsequent validation with commercial assays.
Several commercially available automated biopsy punch instruments exist that would facilitate the processing of DBS samples for high-throughput immunoassay application. Coupled with automated immunoassays platforms, automated biopsy punch instruments could facilitate the standardization of DBS technology and bring us one step closer to adapting DBS sampling to routine immunoassays. What remains a challenge is the standardization of the sampling protocol, the storage and transportation to a facility that would combine such a set-up.

Conclusions
DBS sampling offers a promising alternative to serum sampling via venepuncture and can accurately detect antibodies to SARS-CoV-2. DBS sampling could therefore be adapted for future serological screening of large populations. Lack of standardization of sampling and testing limits the wider application of DBS.

Data availability
All data underlying the results are available as part of the article and no additional source data are required.