LAUSR

Editor, Rare event analysis was started with the analysis of the fetal red blood cells in maternal circulation in a rare percentage.[1,2] Flow cytometry (FCM) has been used to analyze numerous rare cell populations in blood or bone marrow from circulating endothelial cells to cancer stem cells and became an important means in analysis of blood cancer.[2‐7] Initially, FCM was used only for lineage deciding in leukemia; but by years, it was developed for morphological assessment of blood and bone marrow to detect minimal residual disease (MRD), with molecular analysis used in treatment decisions of pediatric B‐acute lymphoblastic leukemia and more recently acute myeloid leukemia.[8‐13] The FCM is helpful in the detection of small tumor population to the level of 0.01% in lymph proliferative disorders.[9,10] MRD detection has been associated with time to relapse in the acute leukemia;[8,9‐11,13] there are several technical and interpretative hurdles that need to be addressed before a laboratory undertakes such an analysis. For acute myeloid leukemia (AML), there are few well‐validated protocols, and this is complicated by the fact that this disease is often composed of multiple clones at presentation and can be affected by antigen modulation during treatment.[10,11] In MRD, the time of sample processing in the rare event analysis was important because cells were very less (>0.01%) so it was important to correlate the time to get maximum number of cells and also validate staining procedure and fluorochrome. A total of four AML MRD processed samples were used in the study, previously diagnosed for AML on morphology. Immunophenotyping done at Lab Oncology, Dr. BRAICH, All India Institute of Medical Sciences, New Delhi. For flow cytometric analysis, at least 200,000 were acquired at follow‐up in all cases from each tube, and data were stored in list mode. The procedure was repeated after 15 h, and data were collected. For all the specimens, five‐color FCM was performed on Coulter FC500 instrument (Beckman Coulter [BC], Hialeah, FL, USA). Combinations of 5 antibodies in 7 different tubes,