title: Variables in the quantification of CD4 in normals and hairy cell leukemia patients.
abstract: BACKGROUND
  Quantitative flow cytometry (QFCM) is being applied in the clinical flow cytometry laboratory. Quantitative normal T-cell CD4 expression represents a biologic standard and quality control agent. However, low levels of CD4 expression were detected in normal T-cells in Hairy Cell Leukemia (HCL) samples.
  
  
  METHODS
  The QuantiBrite System® was used to determine the level of CD4 expression (mean antibody bound per cell, ABC) in fresh and shipped HCL blood and fresh normal donor blood (NDB). The effects of shipping, lysing reagent, cell preparation method, and antibody lot were evaluated.
  
  
  RESULTS
  Shipped HCL specimens (n = 69) had a significantly lower mean CD4 ABC of 38,788 (CV = 9.1%) compared to fresh specimens (n = 105) CD4 value of 40,330 (CV = 8.4%) (P < 0.05). In NDB, significant differences were seen for fresh versus shipped specimens using the stain/lyse method but not for lyse/stain method. Consistent differences in CD4 ABC based upon antibody lot were observed in fresh HCL and NDB samples. Stain/lyse and lyse/stain methods using NH(4) Cl lyse were compared in NDB using identical samples and antibodies. The NDB CD4 ABC values obtained with the lyse (NH(4) Cl)/stain method (45,562, 3.7% CV) were lower than those obtained with the stain/lyse (NH(4) Cl) method (49,955, 3.3% CV) with P < 0.001.
  
  
  CONCLUSIONS
  CD4 expression in HCL patient samples is not inherently different from that observed in NDB and therefore may serve as a biological control in clinical QFCM. Technical variables impact significantly on QFCM of CD4.
authors.affiliation: Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 20899-8312, USA.
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
authors.forename: Fatima
authors.initials: F
authors.lastname: Abbasi
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Jasper
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Kreitman
authors.forename: David J
authors.initials: DJ
authors.lastname: Liewehr
authors.forename: Gerald E
authors.initials: GE
authors.lastname: Marti
authors.forename: Maryalice
authors.initials: M
authors.lastname: Stetler-Stevenson
mesh_terms: D015704:CD4 Antigens / Q000097:blood*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D016022:Case-Control Studies; D006801:Humans; D016130:Immunophenotyping; D007943:Leukemia, Hairy Cell / Q000097:blood*; D012016:Reference Values
chemical_list: D015704:CD4 Antigens