A New Flow Cytometry-Based Single Platform for Universal and Differential Serodiagnosis of HTLV-1/2 Infection

Abstract

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.

Performance of Immunological Assays for Universal and Differential Diagnosis of HTLV-1/2 Infection in Candidates for Blood Donations from the Brazilian Amazon

“…In order to accomplish the differential diagnosis of HTLV-1 and HTLV-2 infection, the WB and the FC-Duplex assay were carried out in parallel batches. The WB results for HTLV-1 from HTLV-2 for differential diagnosis were considered according to the manufacturer instructions and the FC-Duplex results were classified using the criteria previously proposed by Pimenta de Paiva et al [25]. According to their criteria, the differential diagnosis of HTLV infection using FC-Duplex can be achieved as the ΔPPFC = (MT-2 1:32 - Mot 1:32).…”

“…Comparative analysis between the WB and FC-Duplex results (criterion 1) demonstrated 100% agreement for HTLV-1 (15/15) and HTLV-2 (7/7) ( Figure 3 ). Pimenta de Paiva et al [25] proposed two additional criteria for differential diagnosis of HTLV infection using FC-Duplex. The use of these criteria did not show outstanding agreement when compared with the WB reference diagnosis [criterion 2 (HTLV-1 (15/15), HTLV-2 (4/7) with 3/7 misclassifications); criterion 3 (HTLV-1 (15/15), HTLV-2 (5/7) with 2/7 misclassifications)] ( Supplementary Figures 1 and 2 ).…”

“…On the other hand, the FC-Simplex assay (still in the validation phase) showed better performance (agreement = 80%), and can be used to confirm the screening process, before confirming the differential diagnosis with the WB or qPCR [26]. Additionally, we observed that the FC-Duplex, the assay responsible for the viral distinction between HTLV-1 and HTLV-2 as a phase after FC-Simplex, also presented excellent results, with 100% agreement when compared to the WB for the samples analyzed [25,26]. Although these results are promising, we note that only criterion 1 (MT-2 1;32 minus MoT 1;32) was considered satisfactory for agreement with the WB results, whereby 15 (100%) reactive samples were identified for HTLV-1 and 7 (100%) for HTLV-2.…”

“…Although these results are promising, we note that only criterion 1 (MT-2 1;32 minus MoT 1;32) was considered satisfactory for agreement with the WB results, whereby 15 (100%) reactive samples were identified for HTLV-1 and 7 (100%) for HTLV-2. This highlights the ability of the FC-Duplex to distinguish other viruses from HTLV, while also being a low cost and accessible assay for blood banks in Brazil [25].…”

“…The assay aims to select positive samples, but without distinguishing between viral types, and functions as a screening step for FC-Duplex IgG1 HTLV. Subsequently, the FC-Duplex IgG1 (HTLV-1/2) assay was performed as a supporting assay for the differential diagnosis of HTLV-1/2 infection, using commercial cells infected with the viruses [25]. The assay uses a system for detecting IgG1 antibodies in a competitive immunofluorescence platform using flow cytometry, using MT-2 and MoT cell lines stained with the fluorochrome Alexa Fluor 647 (AF647) as antigenic support.…”