Visual semi-quantitation
Document Type
Book Chapter
Publication Date
3-13-2026
Abstract
When a biological sample (such as blood, saliva, or urine) is applied to the sample pad of a lateral flow assay (LFA), it travels via capillary action across the membrane, where the analyte of interest binds to specific antibodies or capture probes (Koczula and Gallotta, 2016). These antibodies are conjugated to colored particles, usually gold nanoparticles or colored latex beads, which provide the visual output. The mainstay of LFAs provide a simple visual binary result indicating the presence or absence of the target analyte often used for quick screening, such as for pregnancy or infectious disease screening (for, e.g., SARS-CoV-2 antigen detection tests), where only a "yes/no" answer is required on the test line and the control line confirms assay validity (Di Nardo et al., 2021; Kakkar et al., 2024; Sajid et al., 2015). Due to its rapid results, ease of use, and low cost, LFAs are well-positioned to meet the RE-ASSURED criteria for use at point-ofcare (Land et al., 2019), especially in areas with limited access to laboratory infrastructure.
While much of the progress in LFA innovation in the last few decades appears to be focused on increasing sensitivity and specificity of tests through development of novel nanomaterials (Andryukov, 2020; Kakkar et al., 2024; Liu et al., 2021), there has also been increasing recognition that there lies opportunity to expand the utility of LFAs beyond providing a binary result for early diagnostic screening. While quantitative results remain elusive without the use of an instrument (Urusov et al., 2019), there have been visual semi-quantitative (vSQ) LFAs developed and/or commercialized within the last 10–15 years that not only detect the presence of the analyte but also give an approximate measurement of analyte concentration without the use of a reader instrument (Table 12.1).
A vSQ LFA is designed to detect the presence and estimate the concentration of a target analyte, usually using a capillary-based strip that integrates both detection and visual readout mechanisms. The key components of such a test include basic components found in all LFAs such as a sample pad, conjugate pad, nitrocellulose membrane with test and control lines, and an absorbent pad (Kakkar et al., 2024; Sajid et al., 2015), but many vSQ LFAs often include a third line, which serves as a reference line or a second test line. Where supported by biological and clinical data, the added information of a result above or below a certain threshold enables the LFA test use cases to be diversified to include disease monitoring and determining disease severity in addition to simply diagnosing or ruling-out a condition. Most importantly, all vSQ LFAs are accompanied by guidelines for interpretation of the vSQ results. Such standalone tests (rather than LFAs that require an instrument) may become the norm in the next generation of LFAs, but there are no known reviews focusing on the state of vSQ LFAs to date (Di Nardo et al., 2021; Sena-Torralba et al., 2022; Omidfar et al., 2023).
This chapter explores the development and current application of vSQ LFAs by summarizing: 1) test design and mechanism, 2) interpretation, 3) clinical validation and field performance, and finally 4) challenges and opportunities that have emerged.
Publication Information
Hanafiah, Khayriyyah Mohd and Anderson, David A. (2026). "Visual semi-quantitation." Lateral Flow Assays for Rapid Point-of-care Testing: Recent Advances, Emerging Trends, and Future Direction .
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