title: Automation for clinical CD4 T-cell enumeration, a desirable tool in the hands of skilled operators.
abstract: Automation in HIV clinical flow cytometry when appropriately applied brings considerable standardisation benefits. The Canadian Immunology Quality Assessment Program (CIQAP) detected situations where operators did not manually override automated software in the event of improper output on the Epics XL and FC500 CD4 immunophenotyping platforms. The automated gating algorithm identifies lymphocytes using a double gate strategy based on CD45 × side scatter (SS) gating and a light scatter FS × SS gate known to fail with sub optimal specimens.
  
  
  
  To generate correct interpretation and results CIQAP introduced a simple protocol modification, bypassing the light scatter gate to include all cells characterized by the CD45 gate. Seventeen problem cases were reanalysed for both absolute and relative T-cell subsets accuracy and compared to the CIQAP group mean values. Results were found to be associated with the percentage of lymphocytes excluded by the automated light scatter gate.
  
  
  
  The modified manual protocol resolved poor performance in 14 instances out of 17 problem cases. It was found to improve accuracy when the light scatter gate excluded greater than 5% of the cells. The remaining three cases had a lymphocyte recovery of greater than 94.6% in the original automated analysis.
  
  
  
  There is a risk in relying solely on automated gating procedures when using the Epics XL and FC500 CD4 immunophenotyping platforms. Laboratory managers have the responsibility to intervene when required. EQA providers are equally responsible to alert the clinical laboratories of the need to update operator training to deal with stressed specimens. © 2016 International Clinical Cytometry Society.
authors.affiliation: National HIV Immunology and Retrovirology Laboratories, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: T O
authors.initials: TO
authors.lastname: Diallo
authors.affiliation: National HIV Immunology and Retrovirology Laboratories, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Bergeron
authors.affiliation: Medical Devices Bureau, Health Canada, Ottawa, Ontario, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Seely
authors.affiliation: National HIV Immunology and Retrovirology Laboratories, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: X
authors.initials: X
authors.lastname: Yang
authors.affiliation: Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Ding
authors.affiliation: National HIV Immunology and Retrovirology Laboratories, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Plews
authors.affiliation: National HIV Immunology and Retrovirology Laboratories, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: P
authors.initials: P
authors.lastname: Sandstrom
authors.affiliation: National HIV Immunology and Retrovirology Laboratories, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.|Department of Medical Microbiology and Immunology, University of Manitoba, Winnipeg, Manitoba, Canada.|Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya.
authors.forename: T B
authors.initials: TB
authors.lastname: Ball
authors.affiliation: National HIV Immunology and Retrovirology Laboratories, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.|Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.|Department of Medical Microbiology, University of Nairobi, Nairobi, Kenya.
authors.forename: A F A
authors.initials: AFA
authors.lastname: Meyers
mesh_terms: D057205:Automation, Laboratory / Q000592:standards*; D015415:Biomarkers / Q000378:metabolism; D018791:CD4 Lymphocyte Count / Q000295:instrumentation / Q000592:standards*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D002170:Canada; D005434:Flow Cytometry / Q000295:instrumentation / Q000592:standards*; D015658:HIV Infections / Q000175:diagnosis / Q000276:immunology / Q000821:virology; D006801:Humans; D016130:Immunophenotyping / Q000295:instrumentation / Q000379:methods / Q000592:standards*; D017493:Leukocyte Common Antigens / Q000378:metabolism; D011786:Quality Control; D012984:Software; D016176:T-Lymphocyte Subsets / Q000276:immunology* / Q000821:virology
chemical_list: D015415:Biomarkers; D017493:Leukocyte Common Antigens
keywords: CD4 testing; Epics XL and FC500 software; HIV; automation; flow cytometer; immunophenotyping; quality control