title: Automated analysis of GPI-deficient leukocyte flow cytometric data using GemStone™.
abstract: BACKGROUND
  Flow Cytometry is the standard for the detection of glycosylphosphatidylinositol (GPI)-deficient clones in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders. Although the International Clinical Cytometry Society (ICCS) and the International PNH Interest Group (IPIG) have published guidelines for PNH assays, data analysis has not been standardized. Current analyses use manual gating to enumerate PNH cells. We evaluate an automated approach to identify GPI-deficient leukocytes using a GemStone™ (Verity Software House) probability state model (PSM).
  
  
  METHODS
  Five hundred and thirty patient samples were assayed on BD Canto II flow cytometers using a stain-lyse-wash technique. Populations were defined using CD15, CD45, CD64 and side scatter. GPI-deficient myeloid cells were recognized as FLAER-, CD24-, and dim or absent CD16. GPI-deficient monocytic cells were identified as FLAER- and CD14-. The data were not censored in any way. A PSM was designed to enumerate monocytic and myeloid cells by adjusting the peaks and line spreads of the data, and recording the normal and GPI-deficient counts. No operator adjustments were made to the automated analysis.
  
  
  RESULTS
  By human analysis, 53 of 530 samples showed GPI-deficient clones. Automated analysis identified the same 53 clones; there were 0 false positives and 0 false negatives. Sensitivity was 100% and specificity 100%. Gating and automated results (percent GPI-deficient cells) were highly correlated: r² = 0.997 for monocytic cells, and r² = 0.999 for myeloids. Mean absolute differences were 0.94% for monocytes and 0.78% for myeloid cells.
  
  
  CONCLUSIONS
  Automated analysis of GPI-deficient leukocytes produces results that agree strongly with gate-based results. The probability-based approach provides higher speed, objectivity, and reproducibility.
authors.forename: David T
authors.initials: DT
authors.lastname: Miller
authors.forename: Benjamin C
authors.initials: BC
authors.lastname: Hunsberger
authors.forename: C Bruce
authors.initials: CB
authors.lastname: Bagwell
mesh_terms: D057205:Automation, Laboratory; D003627:Data Interpretation, Statistical; D005434:Flow Cytometry / Q000379:methods; D017261:Glycosylphosphatidylinositols / Q000172:deficiency; D006457:Hemoglobinuria, Paroxysmal / Q000473:pathology*; D006801:Humans; D007962:Leukocytes / Q000378:metabolism / Q000473:pathology*; D016014:Linear Models; D015999:Multivariate Analysis; D011336:Probability; D012640:Seizures; D012984:Software
chemical_list: D017261:Glycosylphosphatidylinositols