For patient #3 (Fig. 5b), the controls produced expected results: no dots with the negative control (anti-RUNX1 alone) and 21.2 (±7.3, n = 24) dots per nucleus with the positive control (2 different anti-RUNX1 antibodies). Interestingly, the mix of anti-RUNX1 and anti-ETV6 revealed 34.6 PLA dots (34.6 ± 10.8, n = 49) per nucleus, demonstrating a close proximity between ETV6 and RUNX1 that we allocated to the presence of the ETV6-RUNX1 fusion protein. The presence of the fusion protein was validated by the FISH result later on. Similarly, in patient #4 (Fig. 5b), the non-specific background was negative and the pair of anti-RUNX1 gave a positive result with 49.6 (±14.6, n = 20) dots per cell. PLA with the mix of anti-RUNX1 and anti-ETV6 showed an equivalent level of detected interactions, 33.1 (±13.5, n = 32) dots per nucleus that we attributed to the presence of the fusion protein ETV6-RUNX1. The FISH result was concordant with our PLA result. We conclude that the optimized PLA can also be used on non-adherent cells from patients, facilitating the study of fusion protein and protein-protein interactions and their subcellular localization.