Flow cell sorting followed by PCR‐based clonality testing may assist in questionable diagnosis and monitoring of acute lymphoblastic leukemia

Introduction Multicolor flow cytometry (MFC) has highly reliable and flexible algorithms for diagnosis and monitoring of acute lymphoblastic leukemia (ALL). However, MFC analysis can be affected by poor sample quality or novel therapeutic options (e.g., targeted therapies and immunotherapy). Therefore, an additional confirmation of MFC data may be needed. We propose a simple approach for validation of MFC findings in ALL by sorting questionable cells and analyzing immunoglobulin/T‐cell receptor (IG/TR) gene rearrangements via EuroClonality‐based multiplex PCR. Patients and Methods We obtained questionable MFC results for 38 biological samples from 37 patients. In total, 42 cell populations were isolated by flow cell sorting for downstream multiplex PCR. Most of the patients (n = 29) had B‐cell precursor ALL and were investigated for measurable residual disease (MRD); 79% of them received CD19‐directed therapy (blinatumomab or CAR‐T). Results We established the clonal nature of 40 cell populations (95.2%). By using this technique, we confirmed very low MRD levels (