A novel approach for evaluating antibodies against SARS-CoV-2 using menstrual blood collected from sanitary napkins before and after vaccination and evaluation of the visual napkin score

A novel approach for evaluating antibodies against SARS-CoV-2 using menstrual blood collected from sanitary napkins before and after vaccination and evaluation of the visual napkin score | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A novel approach for evaluating antibodies against SARS-CoV-2 using menstrual blood collected from sanitary napkins before and after vaccination and evaluation of the visual napkin score Hiromitsu Shirasawa, Yukiyo Kumazawa, Shiori Kushima, Ayaka Fujishima, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-737828/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Background Although saliva, whole blood, serum, plasma, urine, and feces have been used as specimens for SARS-CoV-2 antigen and antibody tests, menstrual blood has not been reported to date. Unlike invasive blood collection methods, menstrual blood collected non-invasively from participants can be used to evaluate the presence of antibodies against SARS-CoV-2. The purpose of our report is to show an association between menstrual blood and the presence of neutralizing antibodies acquired via mRNA vaccination and the usefulness of menstrual blood as a sample type for detecting SARS-CoV-2 antibodies, considering the volume of blood in sanitary napkins as visual napkin score (VNS). Methods In this study, we collected one napkin each from 40 participants visiting the outpatient gynecology clinic of our university hospital with no symptoms related to COVID-19 and attempted to collect their menstrual blood from the napkins. In 5 of 40 participants, menstrual blood was collected after at least one dose of mRNA vaccination. For this study, the maximum volume of menstrual blood collected was set as 980 μl. In addition, the classification of napkins based on the VNS was set, with level 1 being the lowest percentage of blood on the napkin (0–20%) and level 5 being the highest category (80–100%), according to the consensus of two researchers. We have evaluated used four different antibody testing kits using menstrual blood for detecting IgG and IgM. Findings The mean amount of menstrual blood collected from the 40 participants' sanitary napkins was 364 ± 372 μl; higher VNS indicated more menstrual blood collected. Statistically, VNS 3 or higher resulted in significantly higher menstrual blood collection than VNS 1 (p<0.01). With VNS 1, the collection of menstrual blood was complicated, and antibody test kits could not be tested for all eight participants. On the other hand, 31 of 32 participants (96.9%) with VNS 2 or higher could be tested with one or more antibody test kits. For all testing kits, 100% of tests with menstrual blood had a positive control line, and all participants who tested positive for IgG and IgM had received a COVID-19 mRNA vaccine. In the five participants after mRNA vaccination, only two of the four testing kits were all positive for IgG. Interpretation We have, for the first time, evaluated antibodies against SARS-CoV-2 in menstrual blood collected from sanitary napkins with several antibody test kits. We found that if more than 20% of the napkin area has menstrual blood on it, sufficient menstrual blood can be collected for antibody testing. We also confirmed that menstrual blood collected from a sanitary napkin could be used to detect antibody after mRNA COVID-19 vaccination. We believe that our results are a pioneering effort that has not been reported previously and will lead to better public health and development of wearable devices. Funding There are no conflicts of interest to disclose for this study. Biotechnology and Bioengineering Translational Medicine Nanoscience Applied & Industrial Microbiology Virology Infectious Diseases menstrual blood COVID-19 SARS-CoV-2 antibody sanitary napkin Figures Figure 1 Figure 2 Introduction Various medical tests have been conducted for coronavirus disease 2019 (COVID-19), which has been raging worldwide since the end of 2019. These tests include antigen tests 1 , reverse-transcriptase polymerase chain reaction (RT-PCR) tests 2 , and antibody tests 3 . The materials used are different for each test and include nasopharyngeal swabs, oropharyngeal swabs, saliva samples, and blood samples taken from the fingertips. From a public health perspective, these tests are chosen based on the outbreak situation in each region and the purpose of the test 4 . In general, antigen and RT-PCR tests are used to diagnose infection in patients. IgG, IgM, and IgA antibody tests are used to confirm a history of infection. The principal antibodies against COVID-19 are the nucleoside capsid (N) and spike (S) proteins, which are structural proteins encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene. The structural S protein is used as an antigen 5,6 . In addition, enzyme-linked immunosorbent assay and chemiluminescent immunoassay are generally used for quantitative antibody tests, and immunochromatography is used for simple tests such as qualitative antibody tests 7,8 . The immunochromatographic method is used as a simple test rather than to confirm the diagnosis. There are many commercial anti–SARS-CoV-2 immunochromatographic test kits available in different countries 9 . The immune response after SARS-CoV-2 infection is associated with elevated IgA, IgG, and IgM antibodies for both N and S proteins 10 . There have been reports of post-onset tests using these simple kits to evaluate regional antibody prevalence and for rapid diagnostics 11 . On the other hand, there have been only a few reports on detecting neutralizing antibodies in response to messenger RNA (mRNA) vaccination 12 , which has been rapidly introduced in many countries since 2021, using simple test kits from various manufacturers targeting N and S proteins as antigens. Although saliva, whole blood, serum, plasma, urine, and feces have been used as specimens for SARS-CoV-2 antigen and antibody tests, menstrual blood has not been reported to date. Menstrual blood is a bloody secretion originating from the endometrium. It contains blood, endometrial epithelial cells, and bacterial components of the uterus 13 . The endometrial tissue is associated with natural killer (NK) cells, T cells, and macrophages, making it an immunologically distinctive tissue 13 . Menstrual blood is also thought to be the origin of endometriosis, which is caused by the reflux of menstrual blood into the fallopian tubes. From the immunological and inflammatory perspectives, menstrual blood is unique 14 . There have been several reports on the use of menstrual blood to evaluate infectious diseases such as chlamydia and human papillomavirus as well as correlations between levels of reproductive hormones between peripheral blood and menstrual blood 15-17 . This study involved antibody testing of menstrual blood collected from sanitary napkins with multiple immunochromatographic kits. We have been evaluating sanitary napkins from asymptomatic non-COVID-19 women since before mRNA vaccination began. We also performed antibody testing on menstrual blood from women who had received mRNA vaccination and compared results from several antibody test kits. Unlike invasive blood collection methods, menstrual blood collected non-invasively from participants can be used to evaluate the presence of antibodies against SARS-CoV-2. Our report is the first to show an association between menstrual blood and the presence of neutralizing antibodies acquired via mRNA vaccination and the usefulness of menstrual blood as a sample type for detecting SARS-CoV-2 antibodies, considering the volume of blood in sanitary napkins. Methods A) Menstrual blood collection In this study, a total of 40 participants visiting the outpatient gynecology clinic of our university hospital with no symptoms related to COVID-19 provided us with one sanitary napkin containing menstrual blood. The mean age was 35.5 ± 5.4 years. The napkin they were wearing when they visited the outpatient clinic, which was on an average day 2.8 ± 0.86 of their menstrual period, was used for the study. In 5 of 40 participants, menstrual blood was collected after at least one dose of mRNA vaccination (Comirnaty; Pfizer, New York, NY, USA). In this study, we used commercially available sanitary napkins that the patient usually used. Once collected from the participants, the napkins were promptly brought to the laboratory. A 5 ml syringe was used to collect menstrual blood (Figure 1A). The blood-stained area on the napkin was aspirated with a syringe to the greatest extent possible. The blood collected in the syringe was transferred into 300-μl tubes (Figure 1B), and 20 μl were used for each manufacturer's antibody testing. Excess blood was frozen and stored. In this study, antibody test kits from four companies were used. For this study, the maximum volume of menstrual blood collected was set as 980 μl, consisting of three 300-μl tubes and 80 μl for four antibody test kits. B) Visual napkin score evaluation In this study, we used the area of blood that had adhered to the napkin as a guide for evaluating the possibility of blood collection from the napkin. Specifically, referring to the previous report by Magnay et al., the percentage of blood on the napkin was assessed in 20% increments in this study 18 . Figure 2 shows the classification of napkins based on the visual napkin score (VNS), with level 1 being the lowest percentage of blood on the napkin (0–20%) and level 5 being the highest category (80–100%). VNS was based on the percentage of the sanitary napkin with blood according to the consensus of two researchers. C) Antibody testing with multiple immunochromatographic kits We used four different antibody testing kits A–D. Kit A is the One Step Novel Coronavirus (COVID-19) IgM/IgG Test Kit (Artron Laboratories, Burnaby, Canada) 19 . Kit B is the 2019-nCov IgG/IgM Detection Kit (Shionogi & Co., Osaka, Japan; Vazyme Medical Technology, China) 20 . Kit C is the Lepu Medical SARS-CoV-2 Antibody Test (Lepu Medical Technology, Beijing, China) 21 . Kit D is the Cellspect COVID-19 IgG LF/RCGLF011 kit (Cellspect, Iwate, Japan) 22 . Kits A–C detect both IgM and IgG against SARS-CoV-2, while Kit D detects only IgG. For all kits, 20 μl of menstrual blood were used (Figure 1C). At 15 minutes after the addition of the buffer from each kit, the control line was checked and the IgG and IgM lines were visually checked to determine whether the result was positive or negative (Figure 1D). The datasheet from each manufacturer was used as a reference to determine whether each antibody test kit recognizes N protein or S protein. The antigens, sensitivity, and specificity of each kit are summarized in supplemental Table 1, based on datasheets on the manufacturer’s website. The antigens for Kits A and B were not disclosed. Initially, three different antibody test kits (Kits A, B, and C) were used, but starting from, the twelfth participant Kit D was also added. When the amount of collected menstrual blood was insufficient for testing with all kits, Kit A was preferentially used. D) Statistical analysis The two-sample t -test or the multiple comparisons test was performed with SPSS version 25.0 (IBM Corp, Armonk, NY, USA). The Kruskal-Wallis test was used as a nonparametric test. The Dann-Bonferroni test was used for subsequent multiple comparisons. P<0.05 was determined to be statistically significant. E) Ethical considerations After obtaining approval from our institutional review board (approval number 2412, March 2020), written informed consent was obtained from all participants who participated in the study. Results In this study, we collected one napkin from each of the 40 patients and attempted to collect their menstrual blood. The classification of the 40 patients by VNS, shown in Figure 2. As shown in Table 1, the larger the VNS, the significantly more significant the amount of menstrual blood collected. Statistically, a VNS of level 3 or higher resulted in significantly higher menstrual blood collection than a VNS of level 1 ( p <0.01). In addition, VNS level 5 tended to have a higher menstrual blood collection than VNS level 2, but there was no significant difference after adjustment by Bonferroni correction ( p =0.070). In cases with a VNS of level 1, collection of menstrual blood was extremely difficult, and antibody kits could not be tested in all 8 cases. On the other hand, 31 out of 32 patients (96.9%) with VNS of level two or higher could be tested with one or more antibody kits. In 5 of 40 patients, menstrual blood was collected after at least one dose of mRNA vaccination (Comirnaty). The overall antibody testing rate, defined as the percentage of patients who could be measured with at least one antibody kit, was 77.5%. We summarized the test results of the antibody kit using menstrual blood in Table 2. In this study, Kit A was the most commonly tested, with 31 cases performed. Kit D, which was added to the test midway through the study, had the lowest number of tests with 21 participants. From Kit A to Kit D, the percentage of the control line that was positive by menstrual blood was 100%. On the other hand, there was a difference in the positive IgG line and IgM line among the kits, as shown in Table 2. All patients who tested positive for the IgG and IgM lines had received the mRNA vaccine against COVID-19. The results of the antibody testing kit for five participants who received at least one dose of mRNA vaccine are summarized in Table 3. The patient tested 13 days after the second vaccination was IgG line positive for Kit A and IgG/IgM lines positive for Kit C. The patient tested 16 days after the first vaccination were positive for IgG/IgM lines in Kit A and Kit C. The patient tested 15 days after the first vaccine was positive for IgG line for kit A, IgG/IgM lines for kit C, and weakly IgG line positive for kit D. The two patients who received the second vaccination were tested 21 and 48 days later, respectively. Only IgG line was positive in Kit A, and Kit C in these patients. Kit B did not show any positive IgG or IgM lines in patients who received one or two doses of the vaccine or did not receive the vaccine. Three of the participants who tested positive for IgM were within 20 days of vaccination. Discussion This study has several significant findings. First, it was possible to collect enough menstrual blood from sanitary napkins to perform antibody testing in 77.5% of participants. The previous study by Magnay et al. evaluated menstrual blood on sanitary napkins as a pictogram and reported a relationship with menstrual blood loss and a pictogram 18 . Our study found for the first time that napkins with VNS 2 or higher can yield sufficient menstrual blood for multiple antibody tests with syringe aspiration. A sanitary napkin has been reported to be a useful specimen for PCR-based detection of Chlamydia trachomatis 15 . Alary et al. showed that a sanitary napkin worn for 4 hours without menstrual blood could be a PCR specimen for Chlamydia trachomatis , with the sensitivity of 93.1% and specificity of 98.9%, suggesting that sanitary napkins are useful as self-collection devices. Our study showed that VNS 3 or above was associated with the significantly higher volume of menstrual blood collected than VNS 1. We think VNS will be a good indicator of whether or not antibody testing is possible. A recent report used menstrual cups to collect menstrual effluents. It examined CD45− and CD45+ cell populations using flow cytometry 23 . One of the novelties of this study is that menstrual blood absorbed by sanitary napkins was recollected and used as a sample to detect SARS-CoV-2 antibodies. This is also the first time that multiple antibody test kits have detected IgG and IgM against SARS-CoV-2 in menstrual blood collected from sanitary napkins. In this study, the upper limit was set to 980 μl of menstrual blood. The upper limit was reached in three participants, each with VNS 4 or 5. However, even with this upper limit, an average of 364 μl of menstrual blood can be collected. Thus, menstrual blood may be an attractive non-invasive specimen for SARS-CoV-2 antibody evaluation. For antibody confirmation after mRNA vaccination, a study using saliva has recently been reported 24 . Ketas et al. showed that IgG and IgA against the S protein could be detected in saliva after vaccination. Several attempts to identify SARS-CoV-2 RNA in stool have also been reported 25,26 . Although various specimens have been used, the current gold standard for evaluating antibodies against SARS-CoV-2 remains serum-based evaluation. However, very recently, a trial on remote early detection of SARS-CoV-2 infection using a wearable device has been reported 27 . We believe that this study demonstrates that menstrual blood collected from sanitary napkins can be helpful as a wearable device to determine the presence of SARS-CoV-2 antibodies in public health. In this study, the results of antibody tests differed significantly between participants by vaccination status. Non-vaccinated participants were asymptomatic at the time of testing, and a total of 92 kits were tested on 26 non-vaccinated participants. All were positive for the control line only, with no positives for IgG or IgM. On the other hand, all five asymptomatic participants who received mRNA vaccination were positive for IgG with least 1 kit. Three participants were also positive for IgM in antibody tests using menstrual blood. Thus, 11 out of 20 kits were positive for IgG or IgM in the vaccinated group, which was significantly higher than in the non-vaccinated group. In the non-vaccinated group, all 92 kits were negative for IgG and IgM ( p <0.001). There have been no reports on the concordance rate between antibody test kits after mRNA vaccination by using the menstrual blood. In this study, neutralizing antibodies against the S protein were detected in menstrual blood. These results were helpful in determining which antibody test kit to use for evaluation the retention of neutralizing antibodies in future. Since saliva is a non-invasive specimen for vaccination or antibody evaluation after SARS-CoV-2 infection, studies have been conducted on its usefulness as described above 24,28 . Like saliva, menstrual blood in a napkin can be collected non-invasively, but the napkin may have the advantage of being a wearable device. Non-invasively obtained menstrual blood has also been used as a biomarker for screening and monitoring for human papillomavirus DNA in cervical lesions 16 . In addition, menstrual blood has attracted attention for its immunological specificity, such as the differential expression of NK cells from peripheral blood 29 . It is very important to investigate the immunological effects of menstrual blood on SARS-CoV-2. In this study, both IgG and IgM were detected in antibody test kits targeting the S protein in participants who have received the mRNA vaccine. In the future, it is necessary to study the response of menstrual blood after vaccination with peptide vaccines that target both the S and N proteins 30 . Naturally, there is debate about the usefulness of SARS-CoV-2 antibody testing after vaccination. The United States Food and Drug Administration (FDA) did not recommend antibody testing to assess immunity after COVID-19 vaccination as of July 2021 31 . To date, no test kit has been approved to confirm the presence of antibodies after vaccination. It is also unclear whether qualitative assessment of IgG antibodies after vaccination can indicate immunity to SARS-CoV-2. While antigen and RT-PCR testing are certainly important in determining whether a person is infected with SARS-CoV-2, we believe that qualitative antibody testing after vaccination will become increasingly important in public health. In our study, since the antibody responses differed across kits, it may be possible to differentiate between antibodies obtained from vaccines and antibodies from SARS-CoV-2 infection based on differences in antigens from different kits used to test menstrual blood. In the future, it may be possible to embed immunochromatographic antibody test kits for the S and N proteins in the sanitary napkin itself so that the presence of antibodies can be determined by looking at the lines on a sanitary napkin. A trial for a SARS-CoV-2 detection sensor to be embedded in a face mask as a wearable device have recently been reported 32 . In the future, it is expected that electrical signals from wearable devices such as sanitary napkins will be linked to applications to obtain biometric information. One of the limitations of this study is that the sample size was small. It is necessary to include more participants considering that there will be several types of COVID-19 vaccinations in the future. It should also be noted that the participants in this study were from a region with a relatively low incidence of COVID-19. This study was conducted in a province with about 900,000 people, and there were only approximately 970 cumulative cases of COVID-19 cases as of July 2021, suggesting that the risk of infection was low even in asymptomatic participants before vaccination. In addition, there are many types of sanitary napkins, and the effect of differences in absorbent polymers in different products on the composition of the menstrual blood collected needs to be further studied in the future. In conclusion, we have, for the first time, evaluated antibodies against SARS-CoV-2 in menstrual blood collected from sanitary napkins with several antibody test kits. We found that if more than 20% of the napkin area has menstrual blood on it, sufficient menstrual blood can be collected for antibody testing. We also confirmed that menstrual blood collected from a sanitary napkin could be used to detect IgG and IgM after mRNA COVID-19 vaccination. We believe that our results are a pioneering effort that has not been reported previously and will lead to better public health and development of wearable devices. Declarations Author contributions H.S. conceived the concept of this study, conducted most of the research, wrote the manuscript, created the figures and tables, and oversaw this study. Y.K., A.F., W.S., and K.T. were responsible for the collection and processing of research materials and analysis of experimental data. S.K. provided advice on research concepts, collected research materials, analyzed data, and created figures. E.S., M.G., and K.T. analyzed the research materials and assisted in preparing figures and tables. Y.T. made overall corrections to the manuscript and figures. Competing interests All authors have no conflicts of interest to disclose for this study. Acknowledgements We want to thank Dr. Hidehiro Hayashi, Dr. Keita Saito, Chihiro Kato, and Yuki Wakamatsu of Cellspect Co., Ltd. for their collaboration and advice. A scholarship donation to our department funded this study. In addition, scholarship support from Shionogi & Co. in 2020 and a portion of the scholarship donation from Cellspect Co., Ltd. in 2020 to our department were also used. References 1) Korenkov M, Poopalasingam N, Madler M, et al. Evaluation of a rapid antigen test to detect SARS-CoV-2 infection and identify potentially infectious individuals. 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Summary of 40 participants based on visual napkin score VNS1 VNS2 VNS3 VNS4 VNS5 Total number of patients (n) 8 9 8 8 7 40 average collected menstural blood volume (μl) 0 110±77 a 412±245 a 610±359 a 774±310 a 364±372 antibody testing rate 0 88.9 100 100 100 77.5 a indicates significant difference vs. VNS 1. VNS, visual napkin score; Antibody testing rate, percentage of participants whose menstrual blood could be with at least one antibody test kit. Antibody kit Number of participants tested Control positive (%) IgG positive (%) IgM positive (%) Kit A 31 31(100) 5 (16.1) 1 (3.2) Kit B 30 30 (100) 0 (0) 0 (0) Kit C 30 30 (100) 5 (16.7) 3 (10.0) Kit D 21 21 (100) 1 (4.8) - Vaccination status Unvaccinated 26 26 (100) 0 (0) 0 Vaccinated 5 5 (100) 5 (100) 3 (60.0) Table 2. Summary of antibody test results with menstrual blood Kit A, Antron One Step Novel Coronavirus (COVID-19) IgM/IgG Test Kit; Kit B, Shionogi IgG/IgM Antibody-test Kit for COVID-19; Kit C, Lepu Medical SARS-CoV-2 Antibody Test; Kit D, Cellspect COVID-19 IgG LF/RCGLF011. Case of vaccinated participants 1 2 3 4 5 Kit A, IgG/IgM +/- +/+ +/- +/- +/- Kit B, IgG/IgM -/- -/- -/- -/- -/- Kit C, IgG/IgM +/+ +/+ +/+ +/- +/- Kit D, IgG - - weak + - - Days since vaccination 13 16 9 48 21 mRNA vaccine dose number 2 1 1 2 2 Table 3. Antibody test results from each kit in vaccinated patients Kit A, Antron One Step Novel Coronavirus (COVID-19) IgM/IgG Test Kit; Kit B, Shionogi IgG/IgM Antibody-test Kit for COVID-19; Kit C, Lepu Medical SARS-CoV-2 Antibody Test; Kit D, Cellspect COVID-19 IgG LF/RCGLF011; +, positive; -, negative Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-737828","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":41258515,"identity":"e0bbc750-a92b-424b-ad39-72b4b103e48b","order_by":0,"name":"Hiromitsu Shirasawa","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-5300-0037","institution":"Department of Obstetrics and Gynecology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Hiromitsu","middleName":"","lastName":"Shirasawa","suffix":""},{"id":41258516,"identity":"d86bbefd-9968-41e7-b6a1-fb1c2267beb6","order_by":1,"name":"Yukiyo Kumazawa","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yukiyo","middleName":"","lastName":"Kumazawa","suffix":""},{"id":41258517,"identity":"459cf3b0-a828-4a78-9803-c75770fc0be4","order_by":2,"name":"Shiori Kushima","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shiori","middleName":"","lastName":"Kushima","suffix":""},{"id":41258518,"identity":"dce91597-3453-4295-a250-7b6a43ee916e","order_by":3,"name":"Ayaka Fujishima","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ayaka","middleName":"","lastName":"Fujishima","suffix":""},{"id":41258519,"identity":"b3c97dfd-af53-46e1-b152-958c82eca364","order_by":4,"name":"Wataru Sato","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Wataru","middleName":"","lastName":"Sato","suffix":""},{"id":41258520,"identity":"a292db7d-5659-4cd6-8ecb-18cb7434f7be","order_by":5,"name":"Kazue Togashi","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kazue","middleName":"","lastName":"Togashi","suffix":""},{"id":41258521,"identity":"3808fe69-bb33-45fa-a226-cc3649457be9","order_by":6,"name":"Mayumi Goto","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mayumi","middleName":"","lastName":"Goto","suffix":""},{"id":41258522,"identity":"dd5e1b0d-b8d2-4463-a7e2-5c6528a65f96","order_by":7,"name":"Kazumasa Takahashi","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kazumasa","middleName":"","lastName":"Takahashi","suffix":""},{"id":41258523,"identity":"d27683da-8362-43d4-a5db-de2d3df0283d","order_by":8,"name":"Emiko Sato","email":"","orcid":"","institution":"Department of Obstetrics and Gynecology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Emiko","middleName":"","lastName":"Sato","suffix":""},{"id":41258524,"identity":"12bb8645-0d09-4cab-88b4-1313c5fd36bd","order_by":9,"name":"Yukihiro Terada","email":"","orcid":"","institution":"Akita Industrial Technology Center","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yukihiro","middleName":"","lastName":"Terada","suffix":""}],"badges":[],"createdAt":"2021-07-21 09:31:30","currentVersionCode":2,"declarations":"","doi":"10.21203/rs.3.rs-737828/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-737828/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":12116962,"identity":"b40dfbf7-1e00-48b1-97e3-0adebd33c5bb","added_by":"auto","created_at":"2021-08-04 19:34:29","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80703,"visible":true,"origin":"","legend":"The visual napkin score was based on percentage of the area on the sanitary napkin with blood. Menstrual blood area percentage of approximately 0–20% was classified as level 1, approximately 20–40% as level 2, approximately 40–60% as level 3, approximately 60–80% as level 4, and approximately 80–100% as level 5.","description":"","filename":"f1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-737828/v2/58629da05708ee62ffe16b1c.jpg"},{"id":12116961,"identity":"631de5ba-40de-4032-b80e-5aeba8656f47","added_by":"auto","created_at":"2021-08-04 19:34:28","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46822,"visible":true,"origin":"","legend":"A) Aspiration of menstrual blood from a napkin with a 5-ml syringe. B) Menstrual blood collected in 300-μl tubes. C) Use of an antibody testing kit with menstrual blood. D) Results of the four antibody testing kits in a participant who has received mRNA vaccination.","description":"","filename":"f2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-737828/v2/d61a04a8b263ec0906404a0c.jpg"},{"id":13708231,"identity":"62ea3c48-ed84-4303-ab09-5bc9a0d701a6","added_by":"auto","created_at":"2021-09-17 14:06:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":446522,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-737828/v2/249c9003-be4a-49cd-bcf9-59a808f7dd07.pdf"},{"id":12116963,"identity":"fdaebba5-4079-43dc-b5a6-e17eaafcd353","added_by":"auto","created_at":"2021-08-04 19:34:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15435,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-737828/v2/b26024f87f7809f530da4c24.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"A novel approach for evaluating antibodies against SARS-CoV-2 using menstrual blood collected from sanitary napkins before and after vaccination and evaluation of the visual napkin score","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVarious medical tests have been conducted for coronavirus disease 2019 (COVID-19), which has been raging worldwide since the end of 2019. These tests include antigen tests \u003csup\u003e1\u003c/sup\u003e, reverse-transcriptase polymerase chain reaction (RT-PCR) tests \u003csup\u003e2\u003c/sup\u003e, and antibody tests \u003csup\u003e3\u003c/sup\u003e. The materials used are different for each test and include nasopharyngeal swabs, oropharyngeal swabs, saliva samples, and blood samples taken from the fingertips. From a public health perspective, these tests are chosen based on the outbreak situation in each region and the purpose of the test \u003csup\u003e4\u003c/sup\u003e. In general, antigen and RT-PCR tests are used to diagnose infection in patients. IgG, IgM, and IgA antibody tests are used to confirm a history of infection. The principal antibodies against COVID-19 are the nucleoside capsid (N) and spike (S) proteins, which are structural proteins encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene. The structural S protein is used as an antigen \u003csup\u003e5,6\u003c/sup\u003e. In addition, enzyme-linked immunosorbent assay and chemiluminescent immunoassay are generally used for quantitative antibody tests, and immunochromatography is used for simple tests such as qualitative antibody tests \u003csup\u003e7,8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe immunochromatographic method is used as a simple test rather than to confirm the diagnosis. There are many commercial anti\u0026ndash;SARS-CoV-2 immunochromatographic test kits available in different countries \u003csup\u003e9\u003c/sup\u003e. The immune response after SARS-CoV-2 infection is associated with elevated IgA, IgG, and IgM antibodies for both N and S proteins \u003csup\u003e10\u003c/sup\u003e. There have been reports of post-onset tests using these simple kits to evaluate regional antibody prevalence and for rapid diagnostics \u003csup\u003e11\u003c/sup\u003e. On the other hand, there have been only a few reports on detecting neutralizing antibodies in response to messenger RNA (mRNA) vaccination \u003csup\u003e12\u003c/sup\u003e, which has been rapidly introduced in many countries since 2021, using simple test kits from various manufacturers targeting N and S proteins as antigens.\u003c/p\u003e\n\u003cp\u003eAlthough saliva, whole blood, serum, plasma, urine, and feces have been used as specimens for SARS-CoV-2 antigen and antibody tests, menstrual blood has not been reported to date. Menstrual blood is a bloody secretion originating from the endometrium. It contains blood, endometrial epithelial cells, and bacterial components of the uterus \u003csup\u003e13\u003c/sup\u003e. The endometrial tissue is associated with natural killer (NK) cells, T cells, and macrophages, making it an immunologically distinctive tissue \u003csup\u003e13\u003c/sup\u003e. Menstrual blood is also thought to be the origin of endometriosis, which is caused by the reflux of menstrual blood into the fallopian tubes. From the immunological and inflammatory perspectives, menstrual blood is unique \u003csup\u003e14\u003c/sup\u003e. There have been several reports on the use of menstrual blood to evaluate infectious diseases such as chlamydia and human papillomavirus as well as correlations between levels of reproductive hormones between peripheral blood and menstrual blood \u003csup\u003e15-17\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThis study involved antibody testing of menstrual blood collected from sanitary napkins with multiple immunochromatographic kits. We have been evaluating sanitary napkins from asymptomatic non-COVID-19 women since before mRNA vaccination began. We also performed antibody testing on menstrual blood from women who had received mRNA vaccination and compared results from several antibody test kits. Unlike invasive blood collection methods, menstrual blood collected non-invasively from participants can be used to evaluate the presence of antibodies against SARS-CoV-2. Our report is the first to show an association between menstrual blood and the presence of neutralizing antibodies acquired via mRNA vaccination and the usefulness of menstrual blood as a sample type for detecting SARS-CoV-2 antibodies, considering the volume of blood in sanitary napkins.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eA) Menstrual blood collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, a total of 40 participants visiting the outpatient gynecology clinic of our university hospital with no symptoms related to COVID-19 provided us with one sanitary napkin containing menstrual blood. The mean age was 35.5 \u0026plusmn; 5.4 years. The napkin they were wearing when they visited the outpatient clinic, which was on an average day 2.8 \u0026plusmn; 0.86 of their menstrual period, was used for the study. In 5 of 40 participants, menstrual blood was collected after at least one dose of mRNA vaccination (Comirnaty; Pfizer, New York, NY, USA). In this study, we used commercially available sanitary napkins that the patient usually used. Once collected from the participants, the napkins were promptly brought to the laboratory. A 5 ml syringe was used to collect menstrual blood (Figure 1A). The blood-stained area on the napkin was aspirated with a syringe to the greatest extent possible. The blood collected in the syringe was transferred into 300-\u0026mu;l tubes (Figure 1B), and 20 \u0026mu;l were used for each manufacturer\u0026apos;s antibody testing. Excess blood was frozen and stored. In this study, antibody test kits from four companies were used. For this study, the maximum volume of menstrual blood collected was set as 980 \u0026mu;l, consisting of three 300-\u0026mu;l tubes and 80 \u0026mu;l for four antibody test kits.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB) Visual napkin score evaluation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we used the area of blood that had adhered to the napkin as a guide for evaluating the possibility of blood collection from the napkin. Specifically, referring to the previous report by Magnay et al., the percentage of blood on the napkin was assessed in 20% increments in this study \u003csup\u003e18\u003c/sup\u003e. Figure 2 shows the classification of napkins based on the visual napkin score (VNS), with level 1 being the lowest percentage of blood on the napkin (0\u0026ndash;20%) and level 5 being the highest category (80\u0026ndash;100%). VNS was based on the percentage of the sanitary napkin with blood according to the consensus of two researchers.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC) Antibody testing with multiple immunochromatographic kits\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used four different antibody testing kits A\u0026ndash;D. Kit A is the One Step Novel Coronavirus (COVID-19) IgM/IgG Test Kit (Artron Laboratories, Burnaby, Canada) \u003csup\u003e19\u003c/sup\u003e. Kit B is the 2019-nCov IgG/IgM Detection Kit (Shionogi \u0026amp; Co., Osaka, Japan; Vazyme Medical Technology, China) \u003csup\u003e20\u003c/sup\u003e. Kit C is the Lepu Medical SARS-CoV-2 Antibody Test (Lepu Medical Technology, Beijing, China) \u003csup\u003e21\u003c/sup\u003e. Kit D is the Cellspect COVID-19 IgG LF/RCGLF011 kit (Cellspect, Iwate, Japan) \u003csup\u003e22\u003c/sup\u003e. Kits A\u0026ndash;C detect both IgM and IgG against SARS-CoV-2, while Kit D detects only IgG. For all kits, 20 \u0026mu;l of menstrual blood were used (Figure 1C). At 15 minutes after the addition of the buffer from each kit, the control line was checked and the IgG and IgM lines were visually checked to determine whether the result was positive or negative (Figure 1D). The datasheet from each manufacturer was used as a reference to determine whether each antibody test kit recognizes N protein or S protein. The antigens, sensitivity, and specificity of each kit are summarized in supplemental Table 1, based on datasheets on the manufacturer\u0026rsquo;s website. The antigens for Kits A and B were not disclosed. Initially, three different antibody test kits (Kits A, B, and C) were used, but starting from, the twelfth participant Kit D was also added. When the amount of collected menstrual blood was insufficient for testing with all kits, Kit A was preferentially used.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD) Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe two-sample \u003cem\u003et\u003c/em\u003e-test or the multiple comparisons test was performed with SPSS version 25.0 (IBM Corp, Armonk, NY, USA). The Kruskal-Wallis test was used as a nonparametric test. The Dann-Bonferroni test was used for subsequent multiple comparisons. P\u0026lt;0.05 was determined to be statistically significant.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE) Ethical considerations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter obtaining approval from our institutional review board (approval number 2412, March 2020), written informed consent was obtained from all participants who participated in the study.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn this study, we collected one napkin from each of the 40 patients and attempted to collect their menstrual blood. The classification of the 40 patients by VNS, shown in Figure 2. As shown in Table 1, the larger the VNS, the significantly more significant the amount of menstrual blood collected. Statistically, a VNS of level 3 or higher resulted in significantly higher menstrual blood collection than a VNS of level 1 (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01). In addition, VNS level 5 tended to have a higher menstrual blood collection than VNS level 2, but there was no significant difference after adjustment by Bonferroni correction (\u003cem\u003ep\u003c/em\u003e=0.070). In cases with a VNS of level 1, collection of menstrual blood was extremely difficult, and antibody kits could not be tested in all 8 cases. On the other hand, 31 out of 32 patients (96.9%) with VNS of level two or higher could be tested with one or more antibody kits. In 5 of 40 patients, menstrual blood was collected after at least one dose of mRNA vaccination (Comirnaty). The overall antibody testing rate, defined as the percentage of patients who could be measured with at least one antibody kit, was 77.5%.\u003c/p\u003e\n\u003cp\u003eWe summarized the test results of the antibody kit using menstrual blood in Table 2. In this study, Kit A was the most commonly tested, with 31 cases performed. Kit D, which was added to the test midway through the study, had the lowest number of tests with 21 participants. From Kit A to Kit D, the percentage of the control line that was positive by menstrual blood was 100%. On the other hand, there was a difference in the positive IgG line and IgM line among the kits, as shown in Table 2. All patients who tested positive for the IgG and IgM lines had received the mRNA vaccine against COVID-19. The results of the antibody testing kit for five participants who received at least one dose of mRNA vaccine are summarized in Table 3. The patient tested 13 days after the second vaccination was IgG line positive for Kit A and IgG/IgM lines positive for Kit C. The patient tested 16 days after the first vaccination were positive for IgG/IgM lines in Kit A and Kit C. The patient tested 15 days after the first vaccine was positive for IgG line for kit A, IgG/IgM lines for kit C, and weakly IgG line positive for kit D. The two patients who received the second vaccination were tested 21 and 48 days later, respectively. Only IgG line was positive in Kit A, and Kit C in these patients. Kit B did not show any positive IgG or IgM lines in patients who received one or two doses of the vaccine or did not receive the vaccine. Three of the participants who tested positive for IgM were within 20 days of vaccination.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study has several significant findings. First, it was possible to collect enough menstrual blood from sanitary napkins to perform antibody testing in 77.5% of participants. The previous study by Magnay et al. evaluated menstrual blood on sanitary napkins as a pictogram and reported a relationship with menstrual blood loss and a pictogram\u0026nbsp;\u003csup\u003e18\u003c/sup\u003e. Our study found for the first time that napkins with VNS 2 or higher can yield sufficient menstrual blood for multiple antibody tests with syringe aspiration. A sanitary napkin has been reported to be a useful specimen for PCR-based detection of \u003cem\u003eChlamydia trachomatis\u003c/em\u003e \u003csup\u003e15\u003c/sup\u003e. Alary et al. showed that a sanitary napkin worn for 4 hours without menstrual blood could be a PCR specimen for \u003cem\u003eChlamydia trachomatis\u003c/em\u003e, with the sensitivity of 93.1% and specificity of 98.9%, suggesting that sanitary napkins are useful as self-collection devices. Our study showed that VNS 3 or above was associated with the significantly higher volume of menstrual blood collected than VNS 1. We think VNS will be a good indicator of whether or not antibody testing is possible.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;A recent report used menstrual cups to collect menstrual effluents. It examined CD45\u0026minus; and CD45+ cell populations using flow cytometry\u003csup\u003e\u0026nbsp;23\u003c/sup\u003e. One of the novelties of this study is that menstrual blood absorbed by sanitary napkins was recollected and used as a sample to detect SARS-CoV-2 antibodies. This is also the first time that multiple antibody test kits have detected IgG and IgM against SARS-CoV-2 in menstrual blood collected from sanitary napkins. In this study, the upper limit was set to 980\u0026nbsp;\u0026mu;l\u0026nbsp;of menstrual blood. The upper limit was reached in three participants, each with VNS 4 or 5. However, even with this upper limit, an average of 364\u0026nbsp;\u0026mu;l\u0026nbsp;of menstrual blood can be collected. Thus, menstrual blood may be an attractive non-invasive specimen for SARS-CoV-2 antibody evaluation. For antibody confirmation after mRNA vaccination, a study using saliva has recently been reported \u003csup\u003e24\u003c/sup\u003e. Ketas et al. showed that IgG and IgA against the S protein could be detected in saliva after vaccination. Several attempts to identify SARS-CoV-2 RNA in stool have also been reported \u003csup\u003e25,26\u003c/sup\u003e. Although various specimens have been used, the current gold standard for evaluating antibodies against SARS-CoV-2 remains serum-based evaluation. However, very recently, a trial on remote early detection of SARS-CoV-2 infection using a wearable device has been reported \u003csup\u003e27\u003c/sup\u003e. We believe that this study demonstrates that menstrual blood collected from sanitary napkins can be helpful as a wearable device to determine the presence of SARS-CoV-2 antibodies in public health.\u003c/p\u003e\n\u003cp\u003eIn this study, the results of antibody tests differed significantly between participants by vaccination status. Non-vaccinated participants were asymptomatic at the time of testing, and a total of 92 kits were tested on 26 non-vaccinated participants. All were positive for the control line only, with no positives for IgG or IgM. On the other hand, all five asymptomatic participants who received mRNA vaccination were positive for IgG with least 1 kit. Three participants were also positive for IgM in antibody tests using menstrual blood. Thus, 11 out of 20 kits were positive for IgG or IgM in the vaccinated group, which was significantly higher than in the non-vaccinated group. In the non-vaccinated group, all 92 kits were negative for IgG and IgM (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001). There have been no reports on the concordance rate between antibody test kits after mRNA vaccination by using the menstrual blood. In this study, neutralizing antibodies against the S protein were detected in menstrual blood. These results were helpful in determining which antibody test kit to use for evaluation the retention of neutralizing antibodies in future.\u003c/p\u003e\n\u003cp\u003eSince saliva is a non-invasive specimen for vaccination or antibody evaluation after SARS-CoV-2 infection, studies have been conducted on its usefulness as described above \u003csup\u003e24,28\u003c/sup\u003e. Like saliva, menstrual blood in a napkin can be collected non-invasively, but the napkin may have the advantage of being a wearable device. Non-invasively obtained menstrual blood has also been used as a biomarker for screening and monitoring for human papillomavirus DNA in cervical lesions \u003csup\u003e16\u003c/sup\u003e. In addition, menstrual blood has attracted attention for its immunological specificity, such as the differential expression of NK cells from peripheral blood \u003csup\u003e29\u003c/sup\u003e. It is very important to investigate the immunological effects of menstrual blood on SARS-CoV-2. In this study, both IgG and IgM were detected in antibody test kits targeting the S protein in participants who have received the mRNA vaccine. In the future, it is necessary to study the response of menstrual blood after vaccination with peptide vaccines that target both the S and N proteins \u003csup\u003e30\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eNaturally, there is debate about the usefulness of SARS-CoV-2 antibody testing after vaccination. The United States Food and Drug Administration (FDA) did not recommend antibody testing to assess immunity after COVID-19 vaccination as of July 2021 \u003csup\u003e31\u003c/sup\u003e. To date, no test kit has been approved to confirm the presence of antibodies after vaccination. It is also unclear whether qualitative assessment of IgG antibodies after vaccination can indicate immunity to SARS-CoV-2. While antigen and RT-PCR testing are certainly important in determining whether a person is infected with SARS-CoV-2, we believe that qualitative antibody testing after vaccination will become increasingly important in public health. In our study, since the antibody responses differed across kits, it may be possible to differentiate between antibodies obtained from vaccines and antibodies from SARS-CoV-2 infection based on differences in antigens from different kits used to test menstrual blood. In the future, it may be possible to embed immunochromatographic antibody test kits for the S and N proteins in the sanitary napkin itself so that the presence of antibodies can be determined by looking at the lines on a sanitary napkin. A trial for a SARS-CoV-2 detection sensor to be embedded in a face mask as a wearable device have recently been reported \u003csup\u003e32\u003c/sup\u003e. In the future, it is expected that electrical signals from wearable devices such as sanitary napkins will be linked to applications to obtain biometric information.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;One of the limitations of this study is that the sample size was small. It is necessary to include more participants considering that there will be several types of COVID-19 vaccinations in the future. It should also be noted that the participants in this study were from a region with a relatively low incidence of COVID-19. This study was conducted in a province with about 900,000 people, and there were only approximately 970 cumulative cases of COVID-19 cases as of July 2021, suggesting that the risk of infection was low even in asymptomatic participants before vaccination. In addition, there are many types of sanitary napkins, and the effect of differences in absorbent polymers in different products on the composition of the menstrual blood collected needs to be further studied in the future.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; In conclusion, we have, for the first time, evaluated antibodies against SARS-CoV-2 in menstrual blood collected from sanitary napkins with several antibody test kits. We found that if more than 20% of the napkin area has menstrual blood on it, sufficient menstrual blood can be collected for antibody testing. We also confirmed that menstrual blood collected from a sanitary napkin could be used to detect IgG and IgM after mRNA COVID-19 vaccination. We believe that our results are a pioneering effort that has not been reported previously and will lead to better public health and development of wearable devices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.S. conceived the concept of this study, conducted most of the research, wrote the manuscript, created the figures and tables, and oversaw this study. Y.K., A.F., W.S., and K.T. were responsible for the collection and processing of research materials and analysis of experimental data. S.K. provided advice on research concepts, collected research materials, analyzed data, and created figures. E.S., M.G., and K.T. analyzed the research materials and assisted in preparing figures and tables. Y.T. made overall corrections to the manuscript and figures.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;All authors have no conflicts of interest to disclose for this study.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to thank Dr. Hidehiro Hayashi, Dr. Keita Saito, Chihiro Kato, and Yuki Wakamatsu of Cellspect Co., Ltd. for their collaboration and advice. A scholarship donation to our department funded this study. In addition, scholarship support from Shionogi \u0026amp; Co. in 2020 and a portion of the scholarship donation from Cellspect Co., Ltd. in 2020 to our department were also used.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e1) Korenkov M, Poopalasingam N, Madler M, et al. Evaluation of a rapid antigen test to detect SARS-CoV-2 infection and identify potentially infectious individuals. J Clin Microbiol 2021: Jcm0089621.\u003c/p\u003e\n\u003cp\u003e2) Stessel B, Callebaut I, Polus F, et al. Evaluation of a comprehensive pre-procedural screening protocol for COVID-19 in times of a high SARS CoV-2 prevalence: a prospective cross-sectional study. Ann Med 2021; 53(1): 337-44.\u003c/p\u003e\n\u003cp\u003e3) Sharma A, Ahmad Farouk I, Lal SK. COVID-19: A Review on the Novel Coronavirus Disease Evolution, Transmission, Detection, Control and Prevention. Viruses 2021; 13(2).\u003c/p\u003e\n\u003cp\u003e4) Lee J, Kim SY, Huh HJ, et al. Clinical Performance of the Standard Q COVID-19 Rapid Antigen Test and Simulation of its Real-World Application in Korea. Ann Lab Med 2021; 41(6): 588-92.\u003c/p\u003e\n\u003cp\u003e5) Chandel V, Sharma PP, Raj S, Choudhari R, Rathi B, Kumar D. Structure-based drug repurposing for targeting Nsp9 replicase and spike proteins of severe acute respiratory syndrome coronavirus 2. J Biomol Struct Dyn 2020: 1-14.\u003c/p\u003e\n\u003cp\u003e6) Guo L, Ren L, Yang S, et al. Profiling Early Humoral Response to Diagnose Novel Coronavirus Disease (COVID-19). Clin Infect Dis 2020; 71(15): 778-85.\u003c/p\u003e\n\u003cp\u003e7) Grandjean L, Saso A, Ortiz AT, et al. Long-Term Persistence of Spike Antibody and Predictive Modeling of Antibody Dynamics Following Infection with SARS-CoV-2. Clin Infect Dis 2021.\u003c/p\u003e\n\u003cp\u003e8) Pellini R, Venuti A, Pimpinelli F, et al. Initial observations on age, gender, BMI and hypertension in antibody responses to SARS-CoV-2 BNT162b2 vaccine. EClinicalMedicine 2021; 36: 100928.\u003c/p\u003e\n\u003cp\u003e9) de Almeida SM, Spalanzani RN, Nogueira MB, et al. Rapid Serological Tests for Sars-Cov-2: Diagnostic Performance of Four Commercial Assays. Med Princ Pract 2021.\u003c/p\u003e\n\u003cp\u003e10) Sun B, Feng Y, Mo X, et al. Kinetics of SARS-CoV-2 specific IgM and IgG responses in COVID-19 patients. Emerg Microbes Infect 2020; 9(1): 940-8.\u003c/p\u003e\n\u003cp\u003e11) Jacobs J, K\u0026uuml;hne V, Lunguya O, Affolabi D, Hardy L, Vandenberg O. Implementing COVID-19 (SARS-CoV-2) Rapid Diagnostic Tests in Sub-Saharan Africa: A Review. Front Med (Lausanne) 2020; 7: 557797.\u003c/p\u003e\n\u003cp\u003e12) Ebinger JE, Fert-Bober J, Printsev I, et al. Antibody responses to the BNT162b2 mRNA vaccine in individuals previously infected with SARS-CoV-2. Nat Med 2021; 27(6): 981-4.\u003c/p\u003e\n\u003cp\u003e13) Yang H, Zhou B, Prinz M, Siegel D. Proteomic analysis of menstrual blood. Mol Cell Proteomics 2012; 11(10): 1024-35.\u003c/p\u003e\n\u003cp\u003e14) Yovich JL, Rowlands PK, Lingham S, Sillender M, Srinivasan S. Pathogenesis of endometriosis: Look no further than John Sampson. Reprod Biomed Online 2020; 40(1): 7-11.\u003c/p\u003e\n\u003cp\u003e15) Alary M, Poulin C, Bouchard C, et al. Evaluation of a modified sanitary napkin as a sample self-collection device for the detection of genital chlamydial infection in women. J Clin Microbiol 2001; 39(7): 2508-12.\u003c/p\u003e\n\u003cp\u003e16) Wong SCC, Au TCC, Chan SCS, Ng LPW, Tsang HF. Menstrual Blood Human Papillomavirus DNA and TAP1 Gene Polymorphisms as Potential Biomarkers for Screening and Monitoring of Cervical Squamous Intraepithelial Lesion. J Infect Dis 2018; 218(11): 1739-45.\u003c/p\u003e\n\u003cp\u003e17) Zhou JP, Fraser IS, Caterson I, et al. Reproductive hormones in menstrual blood. J Clin Endocrinol Metab 1989; 69(2): 338-42.\u003c/p\u003e\n\u003cp\u003e18) Magnay JL, Nevatte TM, O\u0026apos;Brien S, Gerlinger C, Seitz C. Validation of a new menstrual pictogram (superabsorbent polymer-c version) for use with ultraslim towels that contain superabsorbent polymers. Fertil Steril 2014; 101(2): 515-22.\u003c/p\u003e\n\u003cp\u003e19) http://www.artronlab.com/products/IFU/A03-51-322%20COVID-19AbIFU.pdf (accessed 1 July 2021).\u003c/p\u003e\n\u003cp\u003e20) https://www.accessdata.fda.gov/cdrh_docs/presentations/maf/maf3278-a001.pdf (accessed 1 July 2021).\u003c/p\u003e\n\u003cp\u003e21) https://labchem-wako.fujifilm.com/jp/category/docs/01890_pamphlet.pdf (accessed 1 July 2021).\u003c/p\u003e\n\u003cp\u003e22) https://9ed3a4c3-7463-4090-8d0a-9691c6a1c873.filesusr.com/ugd/ab41e7_c02b2d0a28f648bc9e76ab1cb5f525\u003c/p\u003e\n\u003cp\u003ecb.pdf (accessed 1 July 2021).\u003c/p\u003e\n\u003cp\u003e23) Warren LA, Shih A, Renteira SM, et al. Analysis of menstrual effluent: diagnostic potential for endometriosis. Mol Med 2018; 24(1): 1.\u003c/p\u003e\n\u003cp\u003e24) Ketas TJ, Chaturbhuj D, Portillo VMC, et al. Antibody Responses to SARS-CoV-2 mRNA Vaccines Are Detectable in Saliva. Pathog Immun 2021; 6(1): 116-34.\u003c/p\u003e\n\u003cp\u003e25) Yang Z, Yu M, Li G, et al. A Convalescent of COVID-19 with RT-PCR Test Continues Positive in Stool. Clin Lab 2020; 66(12).\u003c/p\u003e\n\u003cp\u003e26) Abe T, Ikeda T, Tokuda Y, et al. A patient infected with SARS-CoV-2 over 100 days. Qjm 2021; 114(1): 47-9.\u003c/p\u003e\n\u003cp\u003e27) Brakenhoff TB, Franks B, Goodale BM, et al. A prospective, randomized, single-blinded, crossover trial to investigate the effect of a wearable device in addition to a daily symptom diary for the remote early detection of SARS-CoV-2 infections (COVID-RED): a structured summary of a study protocol for a randomized controlled trial. Trials 2021; 22(1): 412.\u003c/p\u003e\n\u003cp\u003e28) Chiang SH, Tu M, Cheng J, et al. Development and validation of a quantitative, non-invasive, highly sensitive and specific, electrochemical assay for anti-SARS-CoV-2 IgG antibodies in saliva. PLoS One 2021; 16(7): e0251342.\u003c/p\u003e\n\u003cp\u003e29) Tong X, Gao M, Du X, et al. Analysis of uterine CD49a(+) NK cell subsets in menstrual blood reflects endometrial status and association with recurrent spontaneous abortion. Cell Mol Immunol 2021; 18(7): 1838-40.\u003c/p\u003e\n\u003cp\u003e30) Ahmed SF, Quadeer AA, McKay MR. Preliminary Identification of Potential Vaccine Targets for the COVID-19 Coronavirus (SARS-CoV-2) Based on SARS-CoV Immunological Studies. Viruses 2020; 12(3).\u003c/p\u003e\n\u003cp\u003e31) https://www.fda.gov/medical-devices/safety-communications/antibody-testing-not-currently-recommended-assess-immunity-after-covid-19-vaccination-fda-safety (accessed 1 July 2021).\u003c/p\u003e\n\u003cp\u003e32) Nguyen PQ, Soenksen LR, Donghia NM, et al. Wearable materials with embedded synthetic biology sensors for biomolecule detection. Nat Biotechnol 2021.\u003c/p\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1. Summary of 40 participants based on visual napkin score\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.666151468315302%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003eVNS1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003eVNS2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003eVNS3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003eVNS4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003eVNS5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.666151468315302%\"\u003e\n \u003cp\u003enumber of patients (n)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.666151468315302%\"\u003e\n \u003cp\u003eaverage collected menstural blood volume (\u0026mu;l)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e110\u0026plusmn;77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e412\u0026plusmn;245\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e610\u0026plusmn;359\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e774\u0026plusmn;310\u003csup\u003e\u0026nbsp;a\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e364\u0026plusmn;372\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.666151468315302%\"\u003e\n \u003cp\u003eantibody testing rate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e88.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.055641421947449%\"\u003e\n \u003cp\u003e77.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e indicates significant difference vs. VNS 1.\u003c/p\u003e\n\u003cp\u003eVNS, visual napkin score; Antibody testing rate, percentage of participants whose menstrual blood could be with at least one antibody test kit.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eAntibody kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eNumber of participants tested\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eControl positive (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eIgG positive (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eIgM positive (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eKit A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e31(100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e5 (16.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e1 (3.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eKit B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e30 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eKit C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e30 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e5 (16.7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e3 (10.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eKit D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e21 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e1 (4.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eVaccination status\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eUnvaccinated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e26 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003eVaccinated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e5 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e5 (100)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20%\"\u003e\n \u003cp\u003e3 (60.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Summary of antibody test results with menstrual blood\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKit A, Antron One Step Novel Coronavirus (COVID-19) IgM/IgG Test Kit; Kit B, Shionogi IgG/IgM Antibody-test Kit for COVID-19; Kit C, Lepu Medical SARS-CoV-2 Antibody Test; Kit D, Cellspect COVID-19 IgG LF/RCGLF011.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.53941267387945%\"\u003e\n \u003cp\u003eCase of vaccinated participants\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.53941267387945%\"\u003e\n \u003cp\u003eKit A, IgG/IgM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e+/-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e+/+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e+/-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e+/-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e+/-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.53941267387945%\"\u003e\n \u003cp\u003eKit B, IgG/IgM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e-/-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.53941267387945%\"\u003e\n \u003cp\u003eKit C, IgG/IgM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e+/+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e+/+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e+/+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e+/-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e+/-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.53941267387945%\"\u003e\n \u003cp\u003eKit D, IgG\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003eweak +\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.53941267387945%\"\u003e\n \u003cp\u003eDays since vaccination\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.53941267387945%\"\u003e\n \u003cp\u003emRNA vaccine dose number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.292117465224111%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Antibody test results from each kit in vaccinated patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKit A, Antron One Step Novel Coronavirus (COVID-19) IgM/IgG Test Kit; Kit B, Shionogi IgG/IgM Antibody-test Kit for COVID-19; Kit C, Lepu Medical SARS-CoV-2 Antibody Test; Kit D, Cellspect COVID-19 IgG LF/RCGLF011; +, positive; -, negative\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"menstrual blood, COVID-19, SARS-CoV-2, antibody, sanitary napkin","lastPublishedDoi":"10.21203/rs.3.rs-737828/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-737828/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eAlthough saliva, whole blood, serum, plasma, urine, and feces have been used as specimens for SARS-CoV-2 antigen and antibody tests, menstrual blood has not been reported to date. Unlike invasive blood collection methods, menstrual blood collected non-invasively from participants can be used to evaluate the presence of antibodies against SARS-CoV-2. The purpose of our report is to show an association between menstrual blood and the presence of neutralizing antibodies acquired via mRNA vaccination and the usefulness of menstrual blood as a sample type for detecting SARS-CoV-2 antibodies, considering the volume of blood in sanitary napkins as visual napkin score (VNS).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eIn this study, we collected one napkin each from 40 participants visiting the outpatient gynecology clinic of our university hospital with no symptoms related to COVID-19 and attempted to collect their menstrual blood from the napkins. In 5 of 40 participants, menstrual blood was collected after at least one dose of mRNA vaccination. For this study, the maximum volume of menstrual blood collected was set as 980 μl. In addition, the classification of napkins based on the VNS was set, with level 1 being the lowest percentage of blood on the napkin (0–20%) and level 5 being the highest category (80–100%), according to the consensus of two researchers. We have evaluated used four different antibody testing kits using menstrual blood for detecting IgG and IgM.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFindings\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe mean amount of menstrual blood collected from the 40 participants' sanitary napkins was 364 ± 372 μl; higher VNS indicated more menstrual blood collected. Statistically, VNS 3 or higher resulted in significantly higher menstrual blood collection than VNS 1 (p\u0026lt;0.01). With VNS 1, the collection of menstrual blood was complicated, and antibody test kits could not be tested for all eight participants. On the other hand, 31 of 32 participants (96.9%) with VNS 2 or higher could be tested with one or more antibody test kits. For all testing kits, 100% of tests with menstrual blood had a positive control line, and all participants who tested positive for IgG and IgM had received a COVID-19 mRNA vaccine. In the five participants after mRNA vaccination, only two of the four testing kits were all positive for IgG.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eInterpretation\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eWe have, for the first time, evaluated antibodies against SARS-CoV-2 in menstrual blood collected from sanitary napkins with several antibody test kits. We found that if more than 20% of the napkin area has menstrual blood on it, sufficient menstrual blood can be collected for antibody testing. We also confirmed that menstrual blood collected from a sanitary napkin could be used to detect antibody after mRNA COVID-19 vaccination. We believe that our results are a pioneering effort that has not been reported previously and will lead to better public health and development of wearable devices.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThere are no conflicts of interest to disclose for this study.\u003c/p\u003e","manuscriptTitle":"A novel approach for evaluating antibodies against SARS-CoV-2 using menstrual blood collected from sanitary napkins before and after vaccination and evaluation of the visual napkin score","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2021-08-04 19:34:27","doi":"10.21203/rs.3.rs-737828/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2021-07-22 15:30:31","doi":"10.21203/rs.3.rs-737828/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"596aa8a1-1628-47b6-8180-09df1a7b1187","owner":[],"postedDate":"August 4th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":6225051,"name":"Biotechnology and Bioengineering"},{"id":6225052,"name":"Translational Medicine"},{"id":6225053,"name":"Nanoscience"},{"id":6225054,"name":"Applied \u0026 Industrial Microbiology"},{"id":6225055,"name":"Virology"},{"id":6225056,"name":"Infectious Diseases"}],"tags":[],"updatedAt":"2021-07-22T20:10:50+00:00","versionOfRecord":[],"versionCreatedAt":"2021-08-04 19:34:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-737828","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-737828","identity":"rs-737828","version":["v2"]},"buildId":"ehx78VzkSd0WSzXnipQa-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}