A New Flow Cytometry-Based Single Platform for Universal and Differential Serodiagnosis of HTLV-1/2 Infection Luciene Pimenta de Paiva 1† , Jordana Grazziela Alves Coelho-dos-Reis 1,2 * † , Bruno Caetano Trindade 1† , Vanessa Peruhype-Magalhães 1 , Ma ´ rcio Sobreira Silva Arau ´ jo 1 , Juan Jonathan Gonc ¸ alves 1 , Ana Caroline Nogueira-Souza 1 , Ju ´ lia Pereira Martins 1 , A ´ gata Lopes Ribeiro 1,2 , Ana Lucia Starling 3 , Luiz Carlos Ju ´ nior Alca ˆ ntara 4 , Maı ´sa Aparecida Ribeiro 5 , Anna Ba ´ rbara de Freitas Carneiro-Proietti 5 , Ester Cerdeira Sabino 6 , Kelly Alves Bicalho 1 , Andre ´ a Teixeira-Carvalho 1 and Olindo Assis Martins-Filho 1 1 Instituto Rene ´ Rachou, Fundação Oswaldo Cruz (FIOCRUZ)-Minas, Belo Horizonte, Brazil, 2 Instituto de Cie ˆ ncias Biolo ´ gicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 3 Faculdade de Medicina, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil, 4 Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (FIOCRUZ)/RJ, Rio de Janeiro, Brazil, 5 Fundac ¸ ão Centro de Hematologia e Hemoterapia do Estado de Minas Gerais- Hemominas (HEMOMINAS), Belo Horizonte, Brazil, 6 Universidade de São Paulo, Ribeirão Preto, Brazil In the present work, we developed and evaluated the performance of a new flow cytometry- based single platform, referred to as “FC-Duplex IgG1 (HTLV-1/2)”, for universal and differential serodiagnosis of HTLV-1/2 infection. The proposed technology employs a system for detection of IgG1 antibodies in a single competitive immunofluorescence platform by flow cytometry using fluorescently labeled MT-2/MoT cell line mix coupled to a highly sensitive development system (Biotin/Streptavidin/Phycoerythrin). The stability of fluorescent labeling and the antigenicity of MT-2 and MoT cell lines were confirmed upon storage at -20°C for 2, 6, and 12 months. The anti-HTLV-1/2 IgG1 reactivity, expressed as percentage of positive fluorescent cells (PPFC), was evaluated for each target antigen along the titration curve of test serum samples (1:32 to 1:4,096). Upon selection of target cell line and serum dilutions with higher segregation score between groups, the performance of “FIX” and “FIX & PERM” protocols was evaluated. The “FIX” protocol presented excellent performance indices (Se = 92%/Sp = 94%/AUC = 0.96; Se = 96%/Sp = 100%/AUC = 0.99) for the universal (HTLV-1/2 vs. NI) and differential (HTLV-1 vs. HTLV-2) diagnosis of HTLV-1 infection, respectively. Optimization of the “FIX” protocol using the principle of synchronous and asynchronous pairwise analysis further improved the performance of “FC-Duplex IgG1 (HTLV-1/2)”, using the “FIX” protocol for differential diagnosis of HTLV-1 and HTLV-2 infections (Se = 100%/Sp = 100%/AUC = 1.00). In conclusion, the “FC-Duplex IgG1 (HTLV-1/2)” method represents an innovation in the biotechnology segment with the potential to compose a serological kit for differential diagnosis of HTLV-1/2 infection for reference laboratories and blood centers. Keywords: universal and differential diagnosis, HTLV-1/2, flow cytometry, competitive assay, HTLV Frontiers in Immunology | www.frontiersin.org April 2022 | Volume 13 | Article 795815 1 Edited by: Jeffrey R. Dorfman, Stellenbosch University, South Africa Reviewed by: Claudio Fenizia, University of Milan, Italy Kerstin Malm, Örebro University, Sweden Sandrine Alais, Institut National de la Sante ´ et de la Recherche Me ´ dicale (INSERM), France *Correspondence: Jordana Grazziela Alves Coelho-dos-Reis jreis@icb.ufmg.br † These authors have contributed equally to this work Specialty section: This article was submitted to Viral Immunology, a section of the journal Frontiers in Immunology Received: 09 November 2021 Accepted: 18 March 2022 Published: 14 April 2022 Citation: Pimenta de Paiva L, Coelho-dos-Reis JGA, Trindade BC, Peruhype-Magalhães V, Silva Arau ´ jo MS, Gonc ¸ alves JJ, Nogueira-Souza AC, Pereira Martins J, Lopes Ribeiro A ´ , Starling AL, Alca ˆ ntara LCJ, Ribeiro MA, Carneiro-Proietti ABdF, Sabino EC, Alves Bicalho K, Teixeira-Carvalho A and Martins-Filho OA (2022) A New Flow Cytometry-Based Single Platform for Universal and Differential Serodiagnosis of HTLV-1/2 Infection. Front. Immunol. 13:795815. doi: 10.3389/fimmu.2022.795815 METHODS published: 14 April 2022 doi: 10.3389/fimmu.2022.795815