title: Tandem dyes: Stability in cocktails and compensation considerations.
abstract: BACKGROUND
  The stability and performance of tandem-conjugated antibodies can be impaired when stored in antisera cocktails (Biancotto et al., J Immunol Methods 2011;363:245-261; Rawstron et al., Leukemia 2013;27:142-149). This, and the need for frequent re-compensation due to the possible spectral spillover variation between tandem lots, reduces the robustness of clinical flow cytometry panels that include tandems. Since tandems are required for standard 8-10 color screens, further studies of the stability of tandems in cocktails and their spillover variability are warranted.
  
  
  METHODS
  The performance of PE- and APC-tandems stored in cocktails was tested on fresh bone marrow, preserved blood and lyophilized cell samples over 1-, 6-, or 8-week periods, respectively, and their spillover matrices were compared. The observed correction factor differences were used as the basis for analyzing how the application of an incorrect compensation matrix could influence data interpretation.
  
  
  RESULTS
  Signal intensities and background fluorescence remained constant for all fluorochromes in the cocktails tested. Spillover correction factors for different PE-Cy7 mAbs did not exceed or were only marginally higher than those for non-tandem organic dye-conjugated mAb. By applying the correction factor differences observed between tandem mAb lots to clinical data, it was found that the over and under compensation would not alter the clinical interpretation.
  
  
  CONCLUSIONS
  Tandems can be safely stored and used in cocktails. However, each cocktail should be tested on relevant material prior to use. Exact compensation settings are a requirement for accurate data. Provided that careful evaluation of tandem compensation requirements is carried out, certain tandems may use a generic compensation matrix.
authors.affiliation: Haematology Oncology Diagnostics, Bristol Royal Infirmary, Bristol, BS2 8HW, United Kingdom.
authors.forename: Ulrika
authors.initials: U
authors.lastname: Johansson
authors.forename: Marion
authors.initials: M
authors.lastname: Macey
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D001854:Bone Marrow Cells / Q000166:cytology*; D002232:Carbocyanines / Q000592:standards*; D003116:Color; D015925:Cryopreservation; D005434:Flow Cytometry / Q000592:standards*; D005456:Fluorescent Dyes / Q000592:standards*; D005612:Freeze Drying; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry; D010799:Phycoerythrin / Q000592:standards*; D059629:Signal-To-Noise Ratio
chemical_list: D000911:Antibodies, Monoclonal; D002232:Carbocyanines; D005456:Fluorescent Dyes; D018796:Immunoconjugates; C501744:indotricarbocyanine; D010799:Phycoerythrin
keywords: compensation matrix; monoclonal antibody cocktails; spectral spillover; tandem conjugates