title: Combined accurate platelet enumeration and reticulated platelet determination by flow cytometry.
abstract: BACKGROUND
  Diagnosing the cause of thrombocytopenia often requires a bone marrow aspiration or biopsy, an invasive procedure. Reticulated platelets (RP) are immature RNA containing platelets, accurate RP enumeration has yet to be achieved, partially due to the lack of a robust reference method.
  
  
  OBJECTIVE
  To refine previous work and gating strategies distinguishing RP from mature platelets while incorporating accurate platelet enumeration into the analysis. After reviewing previously published studies on Thiazole Orange (TO) staining of RP, we systematically evaluated CD41/CD61 in combination with a commercial source of TO (BDBiosciences). Previous RP methods have not taken advantage of platelet enumeration therefore our goal was to incorporate the ICSH platelet enumeration protocol into our method.
  
  
  METHODS
  TO concentration, incubation, and fixation method were determined to be 10% of stock concentration, 30 min, and 1% formaldehyde respectively. Gating strategy to determine RP fraction used an unstained control tube to set the limit of TO staining.
  
  
  RESULTS
  Normal range (n = 51) was 9.9 ± 3.1%. Analysis of 40 patients with immune-thrombocytopenia-purpura (ITP) showed a RP range from 4.3% to 81.2%. Platelet enumeration was consistent with our previous studies in this area.
  
  
  CONCLUSIONS
  Combining CD41/CD61 platelet enumeration with TO RP percentage is possible. Accurate RP percentage requires an effective gating strategy, as background fluorescence cursor placement is important. This method for enumeration of RP percentage combined with accurate platelet enumeration, particularly in the low range, should prove useful in differentiating production from consumption issues in thrombocytopenia and monitoring response to therapy.
authors: Pathology and Laboratory Medicine, London Health Sciences Centre and St. Joseph's Health Care London, London, Ontario, N6A 4G5, Canada.
authors: Benjamin D
authors: BD
authors: Hedley
authors: Abbott Diagnostics, Santa Clara, California, 95054.
authors: Nigel
authors: N
authors: Llewellyn-Smith
authors: Liverpool Hospital, Elizabeth, NSW, 2170, Liverpool, Australia.
authors: Stephen
authors: S
authors: Lang
authors: Department of Medicine, Division of Hematology, London Health Sciences Centre, Victoria Hospital, London, Ontario, N6A 5W9, Canada.
authors: Cyrus C
authors: CC
authors: Hsia
authors: Liverpool Hospital, Elizabeth, NSW, 2170, Liverpool, Australia.
authors: Neil
authors: N
authors: MacNamara
authors: Liverpool Hospital, Elizabeth, NSW, 2170, Liverpool, Australia.
authors: David
authors: D
authors: Rosenfeld
authors: Pathology and Laboratory Medicine, London Health Sciences Centre and St. Joseph's Health Care London, London, Ontario, N6A 4G5, Canada.
authors: Michael
authors: M
authors: Keeney
mesh_terms: D052160:Benzothiazoles; D015415:Biomarkers / Q000097:blood; D001792:Blood Platelets / Q000276:immunology*; D002138:Calibration; D016022:Case-Control Studies; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D005456:Fluorescent Dyes; D006801:Humans; D039661:Integrin beta3 / Q000097:blood; D015588:Observer Variation; D010976:Platelet Count / Q000379:methods* / Q000592:standards; D040201:Platelet Membrane Glycoprotein IIb / Q000097:blood; D011237:Predictive Value of Tests; D011804:Quinolines; D012016:Reference Values; D015203:Reproducibility of Results; D013921:Thrombocytopenia / Q000097:blood / Q000175:diagnosis* / Q000209:etiology; D057188:Workflow
chemical_list: D052160:Benzothiazoles; D015415:Biomarkers; D005456:Fluorescent Dyes; C061248:ITGB3 protein, human; D039661:Integrin beta3; D040201:Platelet Membrane Glycoprotein IIb; D011804:Quinolines; C051000:thiazole orange
keywords: flow cytometry; peripheral blood; platelet