title: Comparison of fluorescein and phycoerythrin conjugates for quantifying CD20 expression on normal and leukemic B-cells.
abstract: BACKGROUND
  Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes as potential disease biomarkers. CD20 expression in B-cell chronic lymphocytic leukemia (B-CLL) is one of the best examples of such a biomarker, but results from the use of different QFC methods vary considerably.
  
  
  METHODS
  We measured CD20 expression on normal and B-CLL B-cells, using FITC and PE conjugates from the same monoclonal antibody (Mab). As a biological control and calibrator, we also measured CD4 expression on T-cells with FITC and PE Mab. Calibration curves were constructed using the CLSI (formerly NCCLS) consensus guidelines for QFC. Calibration with QuantiBRITE PE-labeled microspheres and the use of unimolar PE conjugates provided direct measurement of antibody bound per cell (ABC) for CD4 and CD20. Calibration for FITC conjugates was based on molecules of equivalent soluble fluorochrome (MESF), as determined by NIST RM 8640 microsphere standards. These MESF values were then converted to ABC, using the CD4 T-cell as a biologic calibrator, to normalize FITC and PE results for CD20 expression.
  
  
  RESULTS
  On normal B cells, the mean ABC value for unimolar CD20-PE conjugate was 143,500 (CV +/- 19.1%). The mean ABC value for B-CLL B-cells stained with the same conjugate was 21,700 (CV +/- 42.0%). Using the CD4 T-cell as a biologic calibrator for FITC conjugate, the mean ABC value for CD20-FITC on normal B-cells was 199,300. CD20-FITC staining on B-CLL cells was generally too weak for accurate quantification. On normal T-cells, the mean ABC value for CD4 unimolar PE conjugate was (36,800 +/- 10.4)%, and it did not differ significantly in CLL samples.
  
  
  CONCLUSIONS
  The expression of CD20 on normal and B-CLL lymphocytes can be quantified in ABC units using unimolar CD20-PE conjugates. In addition, CD4 expression on T-cells can be used as a biological calibrator to quantify CD20-FITC ABC, with reasonable agreement between the two conjugates with different fluorochromes. Issues regarding the accuracy of MESF microsphere calibrators and effective F/P ratios for FITC conjugates will require additional laboratory studies.
authors.affiliation: Biochemical Science Division, National Institute of Standards and Technology (NIST), Gaithersburg, Maryland 20899-8312, USA. lili.wang@nist.gov
authors.forename: Lili
authors.initials: L
authors.lastname: Wang
authors.forename: Fatima
authors.initials: F
authors.lastname: Abbasi
authors.forename: Adolfas K
authors.initials: AK
authors.lastname: Gaigalas
authors.forename: Robert F
authors.initials: RF
authors.lastname: Vogt
authors.forename: Gerald E
authors.initials: GE
authors.lastname: Marti
mesh_terms: D018951:Antigens, CD20 / Q000032:analysis / Q000096:biosynthesis*; D015704:CD4 Antigens / Q000032:analysis / Q000096:biosynthesis; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D005455:Fluorescent Antibody Technique; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000276:immunology* / Q000473:pathology; D010799:Phycoerythrin / Q000737:chemistry*; D012680:Sensitivity and Specificity
chemical_list: D018951:Antigens, CD20; D015704:CD4 Antigens; D010799:Phycoerythrin; D016650:Fluorescein-5-isothiocyanate