title: ISHAGE protocol: are we doing it correctly?
abstract: BACKGROUND
  Flow cytometric CD34(+) stem cell enumeration is routinely performed to optimize timing of peripheral blood stem cell collections and assess engraftment capability of the apheresis product. While a number of different flow methodologies have been described, the highly standardized ISHAGE protocol is currently the most widely employed, with 204/255 (81%) international participants in the UK NEQAS CD34(+) stem cell enumeration program indicating their use of this method. Recently, two laboratories were identified as persistent poor performers, a fact attributed to incorrect ISHAGE protocol usage/setup. This prompted UK NEQAS to question whether other laboratories were making similar errors and, if so, how this might affect individual EQA performance.
  
  
  RESULTS
  In send out 0801, where two stabilized samples were issued, the EQA center surveyed 255 participants with flow analysis data and subsequent results collected. One hundred and ninety-six laboratories returned results with 103 returning dot plots. Eighty-three out of one hundred and three stated that they used the ISHAGE protocol gating strategy but 43% (36/83) were incorrectly set-up. Analysis of the data showed those incorrectly using single platform ISHAGE gating strategy were twice as likely to fail an EQA exercise compared to those using the protocol correctly. This failure rate increased two fold when incorrect ISHAGE protocol was used in a dual platform setting.
  
  
  CONCLUSIONS
  This study suggests a widespread fundamental lack of understanding of the ISHAGE protocol and the need to deploy it correctly, potentially having significant clinical implications and highlights the need to monitor participants rigorously in their deployment of the ISHAGE protocol. It is hoped that once these findings have been disseminated, performance can be improved.
authors.affiliation: UK NEQAS for Leucocyte Immunophenotyping, Department of Haematology, Royal Hallamshire Hospital, Sheffield S10 2QN.
authors.forename: Alison
authors.initials: A
authors.lastname: Whitby
authors.forename: Liam
authors.initials: L
authors.lastname: Whitby
authors.forename: Matthew
authors.initials: M
authors.lastname: Fletcher
authors.forename: John T
authors.initials: JT
authors.lastname: Reilly
authors.forename: D Robert
authors.initials: DR
authors.lastname: Sutherland
authors.forename: Michael
authors.initials: M
authors.lastname: Keeney
authors.forename: David
authors.initials: D
authors.lastname: Barnett
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism*; D001781:Blood Component Removal; D002470:Cell Survival; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D007937:Leukapheresis; D007958:Leukocyte Count / Q000379:methods; D011785:Quality Assurance, Health Care; D011786:Quality Control; D015203:Reproducibility of Results; D012189:Retrospective Studies
chemical_list: D018952:Antigens, CD34