title: Histology verification demonstrates that biospectroscopy analysis of cervical cytology identifies underlying disease more accurately than conventional screening: removing the confounder of discordance.
abstract: BACKGROUND
  Subjective visual assessment of cervical cytology is flawed, and this can manifest itself by inter- and intra-observer variability resulting ultimately in the degree of discordance in the grading categorisation of samples in screening vs. representative histology. Biospectroscopy methods have been suggested as sensor-based tools that can deliver objective assessments of cytology. However, studies to date have been apparently flawed by a corresponding lack of diagnostic efficiency when samples have previously been classed using cytology screening. This raises the question as to whether categorisation of cervical cytology based on imperfect conventional screening reduces the diagnostic accuracy of biospectroscopy approaches; are these latter methods more accurate and diagnose underlying disease? The purpose of this study was to compare the objective accuracy of infrared (IR) spectroscopy of cervical cytology samples using conventional cytology vs. histology-based categorisation.
  
  
  METHODS
  Within a typical clinical setting, a total of n = 322 liquid-based cytology samples were collected immediately before biopsy. Of these, it was possible to acquire subsequent histology for n = 154. Cytology samples were categorised according to conventional screening methods and subsequently interrogated employing attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. IR spectra were pre-processed and analysed using linear discriminant analysis. Dunn's test was applied to identify the differences in spectra. Within the diagnostic categories, histology allowed us to determine the comparative efficiency of conventional screening vs. biospectroscopy to correctly identify either true atypia or underlying disease.
  
  
  RESULTS
  Conventional cytology-based screening results in poor sensitivity and specificity. IR spectra derived from cervical cytology do not appear to discriminate in a diagnostic fashion when categories were based on conventional screening. Scores plots of IR spectra exhibit marked crossover of spectral points between different cytological categories. Although, significant differences between spectral bands in different categories are noted, crossover samples point to the potential for poor specificity and hampers the development of biospectroscopy as a diagnostic tool. However, when histology-based categories are used to conduct analyses, the scores plot of IR spectra exhibit markedly better segregation.
  
  
  CONCLUSIONS
  Histology demonstrates that ATR-FTIR spectroscopy of liquid-based cytology identifies the presence of underlying atypia or disease missed in conventional cytology screening. This study points to an urgent need for a future biospectroscopy study where categories are based on such histology. It will allow for the validation of this approach as a screening tool.
authors.affiliation: Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Lancaster, United Kingdom ; Department of Obstetrics and Gynaecology, Lancashire Teaching Hospitals NHS Foundation Trust, Preston, United Kingdom.
authors.forename: Ketan
authors.initials: K
authors.lastname: Gajjar
authors.affiliation: Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Lancaster, United Kingdom.
authors.forename: Abdullah A
authors.initials: AA
authors.lastname: Ahmadzai
authors.affiliation: Department of Obstetrics and Gynaecology, University Hospital of Ioannina, Ioannina, Greece.
authors.forename: George
authors.initials: G
authors.lastname: Valasoulis
authors.affiliation: Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Lancaster, United Kingdom.
authors.forename: Júlio
authors.initials: J
authors.lastname: Trevisan
authors.affiliation: Department of Obstetrics and Gynaecology, Lancashire Teaching Hospitals NHS Foundation Trust, Preston, United Kingdom ; Department of Obstetrics and Gynaecology, University Hospital of Ioannina, Ioannina, Greece.
authors.forename: Christina
authors.initials: C
authors.lastname: Founta
authors.affiliation: Second Department of Obstetrics and Gynaecology, Aristotle University of Thessaloniki - Hippokration Hospital Thessaloniki, Thessaloniki, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Nasioutziki
authors.affiliation: Second Department of Obstetrics and Gynaecology, Aristotle University of Thessaloniki - Hippokration Hospital Thessaloniki, Thessaloniki, Greece.
authors.forename: Aristotelis
authors.initials: A
authors.lastname: Loufopoulos
authors.affiliation: Queen Charlotte's & Chelsea - Hammersmith Hospital, Imperial Healthcare NHS Trust, London, United Kingdom ; Department of Surgery and Cancer, Imperial College, London, United Kingdom.
authors.forename: Maria
authors.initials: M
authors.lastname: Kyrgiou
authors.affiliation: Department of Obstetrics and Gynaecology, University Hospital of Ioannina, Ioannina, Greece ; Queen Charlotte's & Chelsea - Hammersmith Hospital, Imperial Healthcare NHS Trust, London, United Kingdom.
authors.forename: Sofia Melina
authors.initials: SM
authors.lastname: Stasinou
authors.affiliation: Department of Cytopathology, "Attikon" Hospital, University of Athens, Athens, Greece.
authors.forename: Petros
authors.initials: P
authors.lastname: Karakitsos
authors.affiliation: Department of Obstetrics and Gynaecology, University Hospital of Ioannina, Ioannina, Greece.
authors.forename: Evangelos
authors.initials: E
authors.lastname: Paraskevaidis
authors.affiliation: Department of Obstetrics and Gynaecology, Lancashire Teaching Hospitals NHS Foundation Trust, Preston, United Kingdom.
authors.forename: Bianca
authors.initials: B
authors.lastname: Da Gama-Rose
authors.affiliation: Department of Obstetrics and Gynaecology, Lancashire Teaching Hospitals NHS Foundation Trust, Preston, United Kingdom.
authors.forename: Pierre L
authors.initials: PL
authors.lastname: Martin-Hirsch
authors.affiliation: Centre for Biophotonics, Lancaster Environment Centre, Lancaster University, Lancaster, United Kingdom.
authors.forename: Francis L
authors.initials: FL
authors.lastname: Martin
mesh_terms: D001706:Biopsy; D003581:Cytodiagnosis / Q000379:methods* / Q000592:standards*; D055088:Early Detection of Cancer; D005260:Female; D006801:Humans; D012680:Sensitivity and Specificity; D017550:Spectroscopy, Fourier Transform Infrared; D002583:Uterine Cervical Neoplasms / Q000175:diagnosis* / Q000473:pathology; D014626:Vaginal Smears