title: Increasing the accuracy and scalability of the Immunofluorescence Assay for Epstein Barr Virus by inferring continuous titers from a single sample dilution.
abstract: High Epstein Barr Virus (EBV) titers detected by the indirect Immunofluorescence Assay (IFA) are a reliable predictor of Nasopharyngeal Carcinoma (NPC). Despite being the gold standard for serological detection of NPC, the IFA is limited by scaling bottlenecks. Specifically, 5 serial dilutions of each patient sample must be prepared and visually matched by an evaluator to one of 5 discrete titers. Here, we describe a simple method for inferring continuous EBV titers from IFA images acquired from NPC-positive patient sera using only a single sample dilution. In the first part of our study, 2 blinded evaluators used a set of reference titer standards to perform independent re-evaluations of historical samples with known titers. Besides exhibiting high inter-evaluator agreement, both evaluators were also in high concordance with historical titers, thus validating the accuracy of the reference titer standards. In the second part of the study, the reference titer standards were IFA-processed and assigned an 'EBV Score' using image analysis. A log-linear relationship between titers and EBV Score was observed. This relationship was preserved even when images were acquired and analyzed 3days post-IFA. We conclude that image analysis of IFA-processed samples can be used to infer a continuous EBV titer with just a single dilution of NPC-positive patient sera. This work opens new possibilities for improving the accuracy and scalability of IFA in the context of clinical screening.
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore.
authors.forename: Sherry Meow Peng
authors.initials: SM
authors.lastname: Goh
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore; Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Muthukaruppan
authors.initials: M
authors.lastname: Swaminathan
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore.
authors.forename: Julian U-Ming
authors.initials: JU
authors.lastname: Lai
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore.
authors.forename: Azlinda
authors.initials: A
authors.lastname: Anwar
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore; Department of Biological Sciences, National University of Singapore, Singapore. Electronic address: chansh@tll.org.sg.
authors.forename: Soh Ha
authors.initials: SH
authors.lastname: Chan
authors.affiliation: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore; Department of Biological Sciences, National University of Singapore, Singapore. Electronic address: ian@tll.org.sg.
authors.forename: Ian
authors.initials: I
authors.lastname: Cheong
mesh_terms: D000328:Adult; D000368:Aged; D000914:Antibodies, Viral / Q000097:blood*; D015415:Biomarkers / Q000097:blood; D002138:Calibration; D002277:Carcinoma / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D045744:Cell Line, Tumor; D020031:Epstein-Barr Virus Infections / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect / Q000592:standards; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D016014:Linear Models; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D000077274:Nasopharyngeal Carcinoma; D009303:Nasopharyngeal Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000821:virology; D015588:Observer Variation; D011237:Predictive Value of Tests; D012015:Reference Standards; D015203:Reproducibility of Results; D012189:Retrospective Studies; D012698:Serologic Tests; D013997:Time Factors; D057188:Workflow
chemical_list: D000914:Antibodies, Viral; D015415:Biomarkers
keywords: Epstein-Barr Virus; Image analysis; Immunofluorescence; Nasopharyngeal cancer