Papers - Wakui, Masatoshi

プロトロンビン時間（PT）の試薬特性

涌井昌俊

検査と技術 (医学書院)  50 ( 7 ) 728 - 734 2022.07

Research paper (scientific journal), Single Work

メタクロマジー核酸染色によるRNP法TMを用いたCelltac G+TM試作機.の網赤血球算定の基礎的性能

荒井智子、片桐尚子、近藤直美、谷田部陽子、横田浩充、永井豊、三ツ橋雄之、涌井昌俊、村田満

日本検査血液学会雑誌 23 ( 2 ) 265 - 273 2022.07

Research paper (scientific journal), Joint Work, Accepted

COVID-19の免疫とワクチン

涌井昌俊

The Lipid 33 ( 1 ) 14 - 22 2022.04

Research paper (scientific journal), Single Work

Response kinetics of different classes of antibodies to SARS-CoV2 infection in the Japanese population: the IgA and IgG titers increased earlier than the IgM titers

KuranoM, Morita Y, Nakano Y, Yokoyama R, Shimura T, Qian C, Xia F, He F, Zheng L, Ohmiya H, Kishi Y, Okada J, Yoshikawa N, Nakajima K, Nagura Y, Okazaki H, Jubishi D, Moriya K, Seto Y, Yasui F, Kohara M, Wakui M, Kawamura T, Kodama T, Yatomi Y

Int Immunopharmacol (International Immunopharmacology)  103   1089491 2022.02

Research paper (scientific journal), Joint Work, Accepted,  ISSN  15675769

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To better understand the immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in individuals with COVID-19, it is important to investigate the kinetics of the antibody responses and their associations with the clinical course in different populations, since there seem to be considerable differences between Western and Asian populations in the clinical features and spread of COVID-19. In this study, we serially measured the serum titers of IgM, IgG and IgA antibodies generated against the nucleocapsid protein (NCP), S1 subunit of the spike protein (S1), and receptor-binding domain in the S1 subunit (RBD) of SARS-CoV-2 in Japanese individuals with COVID-19. Among the IgM, IgG, and IgA antibodies, IgA antibodies against all of the aforementioned viral proteins were the first to appear after the infection, and IgG and/or IgA seroconversion often preceded IgM seroconversion. In regard to the timeline of the antibody responses to the different viral proteins (NCP, S1 and RBD), IgA against NCP appeared than IgA against S1 or RBD, while IgM and IgG against S1 appeared earlier than IgM/IgG against NCP or RBD. The IgG responses to all three viral proteins and responses of all three antibody classes to S1 and RBD were sustained for longer durations than the IgA/IgM responses to all three viral proteins and responses of all three antibody classes to NCP, respectively. The seroconversion of IgA against NCP occurred later and less frequently in patients with mild COVID-19. These results suggest possible differences in the antibody responses to SARS-CoV-2 antigens between the Japanese and Western populations.

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Young age, female sex, and presence of systemic adverse reactions are associated with high post-vaccination antibody titer after two doses of BNT162b2 mRNA SARS-CoV-2 vaccination: An observational study of 646 Japanese healthcare workers and university staff

Uwamino Y, Kurafuji T, Sato Y, Tomita Y, Shibata A, Tanabe A, Yatabe Y, Noguchi M, Sakai A, Noguchi M, Arai T, Ohno A, Yokota H, Yamasawa W, Uno S, Nishimura T, Hasegawa N, Saya H, Wakui M*, Murata M

Vaccine (Vaccine)  40 ( 7 ) 1019 - 1025 2022.02

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0264410X

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Background: SARS-CoV-2 vaccination has started worldwide, including Japan. Although high rates of vaccine response and adverse reactions of BNT162b2 vaccine have been reported, knowledge about the relationship between sex differences and antibody response is limited. Furthermore, it is uncertain whether adverse reactions are associated with the vaccine response. Methods: This prospective observational study included 673 Japanese participants working in a medical school and its affiliated hospital in Tokyo, Japan (UMIN000043340). Serum samples were collected before the first dose and three weeks after the second dose of BNT162b2 vaccine, and antibody titers against the receptor-binding domain of the spike protein of SARS-CoV-2 were measured. Answers to questionnaires about background characteristics and adverse reactions were obtained at the time of sample collection, and the relationship between antibody titers was analyzed. Results: After excluding participants who did not complete receiving two doses of vaccination or two series of serum sample collection, 646 participants were analyzed. Although all participants became sero-positive after vaccination, antibody titers were highly variable among individuals (260.9–57,399.7A U/mL), with a median titer of 13478.0AU/mL. Mean titer was higher in females than in males and higher in young (≤45 years old) participants than in aged (>45 years old) participants. Participants who experienced adverse reactions demonstrated a higher antibody titer after vaccination than those without adverse reactions. Multivariable analysis demonstrated that young age, female sex, and adverse reactions after the second dose were independently related to higher antibody titers after the second dose. Discussion: A favorable antibody response was observed after two doses of BNT162b2 vaccination among mostly healthy Japanese participants, especially among female and young participants. Although further investigation is essential, our results imply that the systemic adverse reactions (i.e., fever and general fatigue) are associated with a higher antibody response that indicates the acquisition of humoral immunity.

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凝固波形解析

涌井昌俊

血栓止血誌 33 ( 1 ) 69 - 74 2022.02

Research paper (scientific journal), Single Work

Dynamics of antibody response and cellular immunity after two doses of BNT162b2 mRNA vaccine over 6 months among Japanese healthcare providers and breakthrough infections (preprint)

Uwamino Y*, Kurafuji T, Takato K, Sakai A, Tanabe A, Noguchi M, Yatabe Y, Arai T, Ohno A, Tomita Y, Shibata A, Yokota H, Yamasawa W, Namkoong H, Sato Y, Hasegawa N, Wakui M, Murata M

medRxi preprint 2022.01

Research paper (scientific journal), Joint Work

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The effect of the E484K mutation of SARS-CoV-2 on the neutralizing activity of antibodies from BNT162b2 vaccinated individuals

Uwamino Y, Yokoyama T#, ShimamuraT, Nishimura T, Sato Y, Wakui M, Kosaki K, Hasegawa N, Murata M

Vaccine (Vaccine)  40 ( 13 ) 1928 - 1931 2022

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0264410X

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The reduced vaccine efficacy against the SARS-CoV-2 variant lineage B. 1.351 (beta variant) containing the E484K and N501Y mutations is well known. The E484K mutation in SARS-CoV-2 is thought to be responsible for weakened humoral immunity. Vaccine efficacy against the R.1 lineage, which contains the E484K mutation but not the N501Y mutation, is uncertain. Serum samples were collected from 100 healthy Japanese participants three weeks after receiving the second dose of the BNT162b2 vaccine, and serum neutralization antibody titers were measured against five SARS-CoV-2 variants. The geometric mean neutralization titers measured for the original and R.1 lineages were equivalent (91.90 ± 2.40 and 102.67 ± 2.28, respectively), whereas a low titer was measured for the beta variant (18.03 ± 1.92). Although further investigations with other variant strains and serum samples are essential, our results imply that the weakened humoral response is not caused solely by the E484K mutation. (UMIN000043340).

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Dynamics of antibody titers and cellular immunity among Japanese healthcare workers during the 6 months after receiving two doses of BNT162b2 mRNA vaccine

Uwamino Y, Kurafuji T, Takato K, Sakai A, Tanabe A, Noguchi M, Yatabe Y, Arai T, Ohno A, Tomita Y, Shibata A, Yokota H, Yamasawa W, Namkoong H, Sato Y, Hasegawa N, Wakui M, Murata M

Vaccine (Vaccine)  40 ( 32 ) 4538 - 4543 2022

Research paper (scientific journal), Joint Work, Accepted,  ISSN  0264410X

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Background: The antibody titer is known to wane within months after receiving two doses of the Pfizer-BioNTech BNT162b2 mRNA SARS-CoV-2 vaccine. However, knowledge of the cellular immune response dynamics following vaccination is limited. This study to aimed to determine antibody and cellular immune responses following vaccination, and the incidence and determinants of breakthrough infection. Methods: This prospective cohort study a 6-month follow-up period was conducted among Japanese healthcare workers. All participants received two doses of BNT162b2 vaccine. Anti-SARS-CoV-2 antibody titers and T-cell immune responses were measured in serum samples collected at several timepoints before and after vaccination. Results: A total of 608 participants were included in the analysis. Antibody titers were elevated 3 weeks after vaccination and waned over the remainder of the study period. T-cell immune responses showed similar dynamics. Six participants without predisposing medical conditions seroconverted from negative to positive on the IgG assay for nucleocapsid proteins, indicating breakthrough SARS-CoV-2 infection. Five of the six breakthrough infections were asymptomatic. Conclusions: Both humoral and cellular immunity waned within 6 months after BNT162b2 vaccination. The incidence of asymptomatic breakthrough infection within 6 months after vaccination was approximately one percent. UMIN Clinical Trials Registry ID: UMIN000043340.

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Performance evaluation of a novel reticulocyte identification method using metachromatic nucleic acid staining based on a crossover analysis of emission DNA/RNA light (RNP DeterminationTM) in hematology analyzer Celltac G+

Yahagi K, Arai T, Katagiri H, Yatabe Y, Yokota H, Nagai Y, Mitsuhashi T, Wakui M, Murata M

Int J Lab Hematol (International Journal of Laboratory Hematology)  44 ( 6 ) 1050 - 1059 2022

Research paper (scientific journal), Joint Work, Accepted,  ISSN  17515521

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Introduction: Assessing the percentage of reticulocytes (%Retic) is useful for diagnosing and treating blood diseases that present with anaemia. The Celltac G+™ hematology analyzer (HA) uses a novel reticulocyte identification method that involves metachromatic nucleic acid staining with acridine orange and crossover analysis of emission light of DNA/RNA (determination of red cells, nucleic acid-containing cells, and platelets, RNP Determination™). The red and green fluorescence generated by stained single-stranded RNA and double-stranded DNA express immaturity and morphological abnormality of erythrocytes by detecting erythrocyte RNA and DNA content. Methods: The basic performance of the test automated analyzer (TAA) Celltac G+ was evaluated and compared with the flow cytometry reference method and the comparative automated analyzer (CAA) XN-1000/2000™. In addition, its precision, limit of quantity (LoQ), linearity, analytical measurement interval (AMI), accuracy, and comparability and the effects of interfering substances were evaluated. Results: Evaluation of %Retic by the TAA demonstrated good precision and linearity. The AMI was confirmed from 0.02 to 8.23, and the LoQ in %Retic as the coefficient of variation within an 11% limit (SD, within a 0.01 limit) was 0.14. TAA correlated well with the reference method and routine HA (CAA). Some deviations were found between TAA and CAA in DNA measurements of erythrocytes from abnormal samples. Conclusion: Celltac G+ uses a novel measurement principle and can assess erythrocyte immaturity independent of DNA contents. It represents a new HA that provides novel, useful information on immaturity and morphological abnormality of erythrocytes.

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Time course of adverse reactions after BNT162b2 vaccination in healthy and allergic disease individuals aged 5–11 years: an observational and historical cohort study (preprint)

Yoshida M, Kobashi Y, Shimazu Y, Saito H, Yamamoto C, Kawamura T, Wakui M, Takahashi K, Ito N, Nishikawa Y, Zhao T, Tsubokura M

Research Square  2022

Research paper (scientific journal), Joint Work

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What proportion of population is induced cellular immunity against SARS-CoV-2 after booster vaccination: a cross-sectional study (preprint)

Tani Y, Takita M, Kobashi Y, Wakui M, Tianchen Z, Yamamoto C, Saito H, Kawashima M, Sugiura S, Nishikawa Y, Omata F, Shimazu Y, Kawamura T, Sugiyama A, Nakayama A, Kaneko Y, Kodama T, Kami M, Tsubokura M

Research Square  2022

Research paper (scientific journal), Joint Work

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Anti-SARS-CoV-2 cellular immunity in 571 vaccinees assessed using an interferon-gamma release assay (preprint)

Uwamino Y, Wakui M, Yatabe Y, Nakagawa T, Sakai A, Kurafuji T, Shibata A, Tomita Y, Noguchi M, Tanabe A, Arai T, Ohno A, Yokota H, Uno S, Yamasawa W, Sato Y, Ikeda M, Yoshimura A, Hasegawa N, Saya H, Murata M

medRxiv  2021.12

Research paper (other academic), Joint Work

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How clinical laboratory physicians should stand and go on in near future: Their individuality and cooperativity expected for medical practice and science— Chairpersons’ remarks —

Wakui M, Tabe Y

Rinsho Byori (日本臨床検査医学会)  69 ( 12 ) 943 - 944 2021.12

Research paper (scientific journal)

Alinity hq platelet results are equivalent with the international reference method in thrombocytopenic samples

Lifson MA, Wakui M, Arai T, Mitsuhashi T, Lakos G, Murata M

Int J Lab Hematol (International Journal of Laboratory Hematology)  43 ( 6 ) 1357 - 1362 2021.12

Research paper (scientific journal), Joint Work, Accepted,  ISSN  17515521

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Introduction: Accurate and precise platelet (PLT) count is critical for the appropriate management of patients with thrombocytopenia. This study evaluated the performance of PLT counting with the Abbott Alinity hq hematology analyzer, which utilizes multi-dimensional optical technology. Methods: Imprecision, linearity, and accuracy were assessed per CLSI guidelines. Alinity hq PLT results were compared to the international flow cytometry reference method (IRM) in the concentration range of 6.3 to 103.0 × 109/L. Additional comparisons were made with Sysmex XN-3000 PLT counts: impedance (PLT-I), optical (PLT-O), and optical fluorescent (PLT-F) methods. Results: The average within-run %CV was 4.7% on patient samples with PLT concentrations ranging from 13.1 to 41.7 × 109/L, and the within-laboratory %CV was 3.6% at the level of 68.2 × 109/L. Linearity evaluation indicated a maximum deviation of 3.1% from the linear fit in the range of 0.1 to 316.8 × 109/L. Comparison between Alinity hq and the IRM PLT counts yielded a correlation coefficient of 0.99 and predicted bias of 0.0 and −0.5 × 109/L at 10.0 and 20.0 × 109/L transfusion thresholds, respectively. Alinity hq PLT counts also correlated well with Sysmex PLT counts, with strongest correlation obtained with PLT-F and PLT-O (r =.99) methods. Conclusion: This study demonstrated excellent analytical performance of Alinity hq PLT counting in thrombocytopenic samples, equivalency with the IRM and strong agreement with Sysmex PLT-F and PLT-O methods. The Alinity hq multi-dimensional optical PLT count is available with every CBC without additional reagents and may help promote efficiency in clinical laboratories.

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コロナ抗体検査の現況　～ワクチン接種後のコロナ抗体価の動態、データの見方～

涌井昌俊

臨床検査支援協会機関紙ASCL LETTER (臨床検査支援協会)   ( 2 ) 11 - 11 2021.12

Research paper (other academic), Single Work

抗体検査と免疫学

涌井昌俊

診断と治療 (診断と治療社)  109 ( 11 ) 1521 - 1525 2021.11

Research paper (scientific journal), Single Work

Characterization of antithrombin-dependent anticoagulants through clot waveform analysis to potentially distinguish them from antithrombin-independent inhibitors targeting activated coagulation factors

Wakui M, Fujimori Y, Nakamura S, Oka S, Ozaki Y, Kondo Y, Nakagawa T, Katagiri H, Murata M

Journal of Clinical Pathology (Journal of Clinical Pathology)  74 ( 4 ) 251 - 256 2021

Research paper (scientific journal), Joint Work, Accepted,  ISSN  00219746

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© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ. Aims: While antithrombin (AT)-independent inhibitors targeting thrombin or activated factor X have been assessed through clot waveform (CWA), there are no reports on assessment with respect to AT-dependent anticoagulants. The present study aims to characterise AT-dependent anticoagulants through CWA to distinguish them from AT-independent inhibitors. Methods: CWA was applied to the activated partial thromboplastin time (APTT) assay of plasma samples spiked with each of AT-dependent drugs (unfractionated heparin, enoxaparin and fondaparinux) and AT-independent drugs (rivaroxaban, apixaban, edoxaban, dabigatran, argatroban, hirudin and bivalirudin), which was performed using the CS-5100 or CN-6000 (Sysmex). The APTT-CWA data were automatically gained by the analyser program. The positive mode of clotting reaction curves was defined as the direction towards fibrin generation. Results: Regarding dose-response curves in AT-dependent anticoagulants, the maximum positive values of the first and secondary derivatives (Max1 and Maxp2, respectively) and the maximum negative values of the secondary derivative (Maxn2) seemed to drop to zero without making an asymptotic line, consistent with the irreversibility. Such a feature was observed also in hirudin, as reported previously. Notably, the symmetric property of Max1 peaks in the waveforms was distorted dose dependently in AT independent but not AT-dependent drugs. A plot of Maxp2 logarithm versus Maxn2 logarithm was linear. The slope was about 1 in AT-dependent drugs while that was more than 1 in AT-independent drugs. These features made it possible to distinguish AT-dependent and AT-independent drugs. Conclusions: The results aid in further understanding of the pharmacological aspects of anticoagulation and in screening of candidates for novel anticoagulants.

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Evaluation of usability of various rapid antibody tests in diagnostic application for COVID-19

Uwamino Y, Wakui M*#, Aoki W, Kurafuji T, Yanagita E, Morita M, Nagata M, nose R, Noguchi M, Yokota H, Hasegawa N, Saya H, Murata M and for the Keio Donner Project Team

Annal Clin Biochem ({SAGE} Publications)  58 ( 3 ) 174 - 180 2021

Research paper (scientific journal), Joint Work, Accepted,  ISSN  00045632

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© The Author(s) 2021. Background: The usability of laboratory tests related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critically important for the world undergoing the COVID-19 pandemic. The present study aimed to assess the diagnostic usability of rapid tests for the detection of antibody against SARS-CoV-2 through comparison of their results with the results of reverse transcription polymerase chain reaction (RT-PCR) test for the detection of SARS-CoV-2 genomic RNA and with the results of a quantitative test for antibody detection. Methods: Serum samples were collected from 18 patients undergoing RT-PCR testing for SARS-CoV-2. Twelve patients were RT-PCR positive while six were negative. A quantitative test based on chemiluminescent immunoassay and three rapid tests based on immunochromatography were performed to detect anti-SARS-CoV-2 IgG and IgM. Results: All the antibody tests exhibited poor sensitivity at the timing of initial RT-PCR diagnosis. IgG responses occurring prior to or simultaneously with IgM responses were observed through not only the quantitative test but also the three rapid tests. Based on concordance with the quantitative test results, the large variance among the three rapid tests was revealed. Conclusions: All antibody tests were unsatisfactory to replace RT-PCR for the early diagnosis of COVID-19. Rapid antibody tests as well as a quantitative antibody test were useful in the assessment of immune responses in COVID-19. The obvious variance among the three rapid tests suggested limited accuracy and difficult standardization. Diagnostic usability of rapid antibody tests for COVID-19 should be investigated rigorously.

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Assessment of humoral responses in COVID-19 using various quantitative antibody tests

Wakui M, Uwamino Y, Kurafuji T, Noguchi M, Ohno A, Yokota H, Kikuchi H, Hasegawa N, Saya H, Murata M and for the Keio Donner Project Team

Ann Clin Biochem ({SAGE} Publications)  58 ( 4 ) 368 - 376 2021

Research paper (scientific journal), Joint Work, Accepted,  ISSN  00045632

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Background: Quantitative antibody tests are expected to be useful in diagnostics of COVID-19 and investigation of herd immunity against SARS-CoV-2. To make it proper to perform them, understanding of the immunological aspects is critically important. The present study aimed to assess humoral responses in COVID-19 using various quantitative antibody tests. Methods: Four quantitative antibody tests that are different in targeted antigens, detectable immunoglobulin classes and avidity were used. Diagnosis was confirmed by RT-PCR for SARS-CoV-2 detection. Antibody titres of 117 samples collected from 24 COVID-19 patients and 23 non-COVID-19 patients were measured to evaluate correlations between different tests. For 24 COVID-19 patients, antibody titres measured at various time points after the onset or the RT-PCR diagnosis were subjected to assessment of humoral responses. Results: Correlations between tests were observed to some degree, although there were discrepancies putatively due to differences in measurement principle. Seronegative COVID-19 was diagnosed for some patients, in whom antibody titres were less than the cut-off value in each test throughout the time courses. IgG seroconversion without prior IgM seroconversion most frequently occurred, while predominance of IgM responses over IgG responses was observed in some severe cases. Viral burdens estimated according to threshold cycle values at the RT-PCR seemed to impact antibody responses. Conclusions: The results provide insights into the nature of humoral responses to SARS-CoV-2 and diagnostic performance of antibody tests.

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