LAUSR

Dear Editors, Flow cytometry (FC) is essential for the quantification of residual disease in patients with acute leukemia.1,2 However, hemodilution of the sample (ie, mixing of peripheral blood [PB] with bone marrow [BM]) may potentially lead to an underestimation of disease burden.1 Despite this concern, assessment of hemodilution is not standardized. This study set out to describe the distribution of basic BM cell subsets and assess the frequency and degree of hemodilution in a cohort of patients with acute leukemia after treatment. From November 2018 until February 2020, an 8-color “cell subset distribution” tube has been used in our unit to characterize and quantify basic BM populations in samples from patients with acute myeloblastic and lymphoblastic leukemia (AML and ALL, respectively) sent for quantification of residual disease by FC. For this study, patients with a residual disease by FC > 0.5% were excluded, as we considered that greater disease burdens could alter cell subset distribution. EDTA-preserved BM samples were generally analyzed within 3 hours after collection. The “cell subset distribution” tube was processed with the TQ-Prep Workstation (Beckman Coulter). Briefly, after mixing 5 μL of each antibody with 50 μL of BM and 50 μL of fetal bovine serum (EuroClone, Cat.No.ESC0180L), the sample was placed in the TQ-prep workstation for 10 minutes of incubation in the dark. It was then lyzed and fixed with immunoprep reagent. The sample was washed before acquisition. Whenever possible, at least 20 000 events were acquired. A Navios flow cytometer (Beckman Coulter) was used. The “cell subset distribution” tube used in this study was adapted from the one proposed by Jacob et al3 and Pont et al4 for the same purpose and is shown in Table 1a. Kaluza software was used for all analyses. At least 10 events were required to define a population. Table 1a shows the populations and the antibody combinations used to define them, and Figure S1 shows the gating strategy. In addition, whenever CD117 was included in the strategy to quantify residual disease (ie, tubes aiming to acquire 500 000 leukocytes), the % of mastocytes was also recorded. To assess hemodilution, we used several proposed methods: