title: Quantification of histochemical stains using whole slide imaging: development of a method and demonstration of its usefulness in laboratory quality control.
abstract: OBJECTIVE
  Histochemical staining of tissue is a fundamental technique in tissue diagnosis and research, but it suffers from significant variability. Efforts to address this include laboratory quality controls and quality assurance schemes, but these rely on subjective interpretation of stain quality, are laborious and have low reproducibility. We aimed (1) to develop a method for histochemical stain quantification using whole slide imaging and image analysis and (2) to demonstrate its usefulness in measuring staining variation.
  
  
  METHODS
  A method to quantify the individual stain components of histochemical stains on virtual slides was developed. It was evaluated for repeatability and reproducibility, then applied to control sections of an appendix to quantify H&E staining (H/E intensities and H:E ratio) between automated staining machines and to measure differences between six regional diagnostic laboratories.
  
  
  RESULTS
  The method was validated with <0.5% variation in H:E ratio measurement when using the same scanner for a batch of slides (ie, it was repeatable) but was not highly reproducible between scanners or over time, where variation of 7% was found. Application of the method showed H:E ratios between three staining machines varied from 0.69 to 0.93, H:E ratio variation over time was observed. Interlaboratory comparison demonstrated differences in H:E ratio between regional laboratories from 0.57 to 0.89.
  
  
  CONCLUSIONS
  A simple method using whole slide imaging can be used to quantify and compare histochemical staining. This method could be deployed in routine quality assurance and quality control. Work is needed on whole slide imaging devices to improve reproducibility.
authors.affiliation: Leeds Teaching Hospitals NHS Trust, Leeds, UK.
authors.forename: Allan
authors.initials: A
authors.lastname: Gray
authors.affiliation: University of Leeds, Leeds, UK.
authors.forename: Alex
authors.initials: A
authors.lastname: Wright
authors.affiliation: Leeds Teaching Hospitals NHS Trust, Leeds, UK.
authors.forename: Pete
authors.initials: P
authors.lastname: Jackson
authors.affiliation: University of Leeds, Leeds, UK.
authors.forename: Mike
authors.initials: M
authors.lastname: Hale
authors.affiliation: Leeds Teaching Hospitals NHS Trust, Leeds, UK University of Leeds, Leeds, UK.
authors.forename: Darren
authors.initials: D
authors.lastname: Treanor
mesh_terms: D001065:Appendix / Q000473:pathology*; D057205:Automation, Laboratory; D019411:Clinical Laboratory Techniques / Q000592:standards*; D004396:Coloring Agents / Q000592:standards; D004801:Eosine Yellowish-(YS) / Q000592:standards; D006416:Hematoxylin / Q000592:standards; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000592:standards*; D007753:Laboratories / Q000592:standards*; D059021:Laboratory Proficiency Testing / Q000592:standards*; D015588:Observer Variation; D011237:Predictive Value of Tests; D011786:Quality Control; D019984:Quality Indicators, Health Care / Q000592:standards*; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000592:standards; D013194:Staining and Labeling / Q000592:standards*
chemical_list: D004396:Coloring Agents; D004801:Eosine Yellowish-(YS); D006416:Hematoxylin
keywords: DIGITAL PATHOLOGY; HISTOPATHOLOGY; IMAGE ANALYSIS; QUALITY ASSURANCE; QUALITY CONTROL