Titration of monoclonal antibodies and stability of tandem dyes in cocktail preparation for flow cytometry processing

Objectives: Tandem dyes are routinely used in multicolor flow cytometry (MFC) and consist of two fluorochromes, one donor and one acceptor. Antibody cocktails for various panels are prepared by combining the pre-titrated volumes of each fluorochrome-conjugated antibody. As the performance and stability of tandem dyes may deteriorate over time, this study aimed to optimize antibody titration volumes and assess the stability of pre-titrated tandem dyes in cocktail preparations. Materials and Methods: This was a quantitative study in which titration was performed, and the prepared antibody cocktail comprised cytoplasmic myeloperoxidase-Fluorescein Isothiocyanate (FITC), cytoplasmic cluster of differentiation (cCD) 79a-Phycoerythrin (PE), CD19-energy coupling dye (ECD), CD34-phycoerythrin-cyanin (PC) 5.5, cCD3-PC7, CD45-allophycocyanin (APC) 700, CD7-APC750, sCD3-pacific blue (PB), and human leucocyte antigen - D region related (HLA-DR isotype) tagged with krome orange (KO). CytoComp TM lyophilized cells were used to evaluate tandem dye stability daily for five days and weekly for four weeks. The stain-lyse-wash procedure was used, and data acquisition and analysis were performed in the Navios TM flow cytometer. Statistical analysis: The mean fluorescent intensity and percentage of positive cells were recorded for most of the antibodies in the cocktail. A coefficient of variation (CV) value of >5% was considered statistically significant, indicating the degradation of tandem dyes. Results: Antibody titration reduced the final antibody volume by 77%, reducing the cost per test. CV values remained below 5% during all assessments, confirming cocktail stability for up to four weeks when tested with lyophilized cells. Conclusions: The pre-titrated antibody cocktail was cost-effective and stable for up to four weeks, including tandem dyes, making it suitable for routine flow cytometric applications.