title: A single tube 10-color flow cytometry assay optimizes detection of minimal residual disease in chronic lymphocytic leukemia.
abstract: Levels of residual disease (RD) are an independent predictor of progression-free survival (PFS) and overall survival (OS) in patients treated for chronic lymphocytic leukemia (CLL). We modified the international standardized approach (ISA) to RD detection using flow cytometry by developing a single tube 10 color antibody assay.
  
  
  METHODS
  A single tube incorporated the following monoclonal antibodies: CD81FITC, CD22PE, CD3ECD, CD5PercP5.5, CD20PECY7, CD79bAPC, CD38A700, CD43APC Alexa750, CD19eFluor 450, and CD45KO. A modified ISA gating strategy was developed that removed contaminating events. Sensitivity assays were performed using dilution with normal peripheral blood and bone marrow. Clinical samples were compared using the ISA and the single tube assay.
  
  
  RESULTS
  Dilution studies showed that sensitivity of 0.001% was achievable when a minimum of 1.8 × 10(6) total events were acquired. One hundred twenty-nine samples were analyzed and showed RD levels from 0.003 to 22%. In 80 samples analyzed with both assays, there was an excellent correlation between the two methods (slope = 1.0, intercept = 0.07 and R2 = 0.992) and results from Bland-Altman analysis showed a bias of 0.04 ± 0.38 with 95% confidence interval of -0.71 to 0.79. Removal of contaminating events in the single tube assay led to a significant reduction in RD values (P = 0.0014).
  
  
  CONCLUSIONS
  The single tube 10-color assay for the detection of RD in CLL provides equivalent results to the ISA but requires fewer cells, uses fewer reagents, and allows for simpler analysis. By directly removing contaminating events, it improves the accuracy of CLL RD detection and may reclassify the status of some patients following chemotherapy.
authors.affiliation: Flow Cytometry Unit, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Sydney, Australia. mary.sartor@swahs.health.nsw.gov.au
authors.forename: M M
authors.initials: MM
authors.lastname: Sartor
authors.forename: D J
authors.initials: DJ
authors.lastname: Gottlieb
mesh_terms: D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived / Q000627:therapeutic use; D015703:Antigens, CD / Q000032:analysis*; D000970:Antineoplastic Agents / Q000627:therapeutic use; D018906:Antineoplastic Agents, Alkylating / Q000627:therapeutic use; D003116:Color; D003520:Cyclophosphamide / Q000627:therapeutic use; D018572:Disease-Free Survival; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000188:drug therapy; D008297:Male; D008875:Middle Aged; D018365:Neoplasm, Residual / Q000175:diagnosis*; D000069283:Rituximab
chemical_list: D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D015703:Antigens, CD; D000970:Antineoplastic Agents; D018906:Antineoplastic Agents, Alkylating; D000069283:Rituximab; D003520:Cyclophosphamide; D016650:Fluorescein-5-isothiocyanate