Tandem Dyes: Stability in cocktails and compensation considerations

Background: The stability and performance of tandem conjugated antibodies can be impaired when stored in antisera cocktails (1,2). This, and the need for frequent re-compensation due to the possible spectral spill over variation between tandem lots, reduces the robustness of clinical flow cytometry panels that include tandems. Since tandems are required for standard 8-10 colour screens, further studies of the stability of tandems in cocktails and their spill over variability are warranted. Methods: The performance of PE- and APC-tandems stored in cocktails was tested on fresh bone marrow, preserved blood and lyophilised cell samples over 1-, 6- or 8-week periods respectively, and their spill over matrices were compared. The observed correction factor differences were used as the basis for analysing 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. Spill over 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. © 2013 Clinical Cytometry Society.