For patient #1 and patient #2 (Fig. 5a), the negative controls showed no dots, confirming the high specificity of the procedure. The positive controls showed about 16.9 dots per nucleus (±4.1, n = 50) for patient #1, and 15.8 (±11.5, n = 50) for patient #2. PLA with a mix of anti-RUNX1 and anti-ETV6 showed about 2.6 (±2.4, n = 50) for patient #1 and 2.6 (±3.5, n = 48) dots per nucleus for patient #2. Therefore, the mean number of dots per nucleus for ETV6 and RUNX1 proximity is below the cut-off level for both patients. We concluded that both patient #1 and patient #2 lymphoblasts did not express the fusion protein ETV6-RUNX1, which were concordant with FISH analysesFig. 5Proximity Ligation Assay is a reliable tool for the detection of ETV6-RUNX1 fusion protein in patients’ lymphoblasts. Quantification of PLA signals per nucleus in lymphoblasts from two patients negative (a) or positive (b) for the fusion protein ETV6-RUNX1. The cells were incubated with RUNX1 ab110035 antibodies alone (−) or with RUNX1 ab23980, CBFB ab133600 or ETV6 sc11382 antibodies. The quantification of the number of PLA dot per nucleus is presented with the mean values ± S.D. The line represents the cut-off used to determine positive PLA signals