title: Development and evaluation of a stabilized whole-blood preparation as a process control material for screening of paroxysmal nocturnal hemoglobinuria by flow cytometry.
abstract: BACKGROUND
  Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disorder in which correct diagnosis is essential for effective patient management. Demonstration of deficiency of glycosylphosphatidylinositol (GPI)-linked antigens from red cells and/or granulocytes by flow cytometry represents a highly specific diagnostic test for PNH. Currently, no external quality assessment (EQA) programme or reference material is available for whole-blood PNH testing (red cells and leucocytes) by flow cytometry.
  
  
  METHODS
  In order to address this issue, we report the development of a stabilized whole-blood PNH sample. We present the results of a detailed time course study by flow cytometry that demonstrates the stability of GPI-linked antigen expression on granulocytes and red cells in a stabilized PNH peripheral blood sample, using a previously described method.
  
  
  RESULTS
  The PNH cells, as well as the coexisting normal red cell and granulocyte populations, remained stable for up to 120 days, both in terms of immunophenotypic and light scatter characteristics. Subsequent samples were used for a PNH EQA programme and issued to 92 laboratories worldwide.
  
  
  CONCLUSIONS
  This study has highlighted that PNH testing by flow cytometry has significant problems with regard to false-positive and -negative results. In addition, the variation in GPI-linked antigen detection methods has highlighted the urgent need for standardized protocols.
authors.affiliation: Haematological Malignancy Diagnostic Service, St James's University Hospital, Leeds LS9 7TF, United Kingdom. stephen.richards2@nhs.net
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Richards
authors.forename: Liam
authors.initials: L
authors.lastname: Whitby
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Cullen
authors.forename: Anita J
authors.initials: AJ
authors.lastname: Dickinson
authors.forename: Viv
authors.initials: V
authors.lastname: Granger
authors.forename: John T
authors.initials: JT
authors.lastname: Reilly
authors.forename: Peter
authors.initials: P
authors.lastname: Hillmen
authors.forename: David
authors.initials: D
authors.lastname: Barnett
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D001793:Blood Preservation / Q000379:methods; D001800:Blood Specimen Collection / Q000592:standards*; D004912:Erythrocytes / Q000378:metabolism; D005240:Feasibility Studies; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D058851:GPI-Linked Proteins / Q000378:metabolism; D006098:Granulocytes / Q000378:metabolism; D006457:Hemoglobinuria, Paroxysmal / Q000097:blood / Q000175:diagnosis*; D006801:Humans; D008137:Longitudinal Studies; D008403:Mass Screening; D055550:Protein Stability; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D015703:Antigens, CD; D058851:GPI-Linked Proteins