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  Table of Contents    
Year : 2017  |  Volume : 60  |  Issue : 3  |  Page : 449-451
Effect of processing time on analysis of rare event in minimal residual disease study

1 Department of Laboratory Oncology Unit, Dr. B.R.A. Institute Rotary Cancer Hospital, All India Institute of Medical Sciences, New Delhi, India
2 Departement of Biotechnology, Maharaj Vinayak Global University, Jaipur, Rajasthan, India

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Date of Web Publication22-Sep-2017

How to cite this article:
Rai S, Bhardwaj U, Singh S, Misra A. Effect of processing time on analysis of rare event in minimal residual disease study. Indian J Pathol Microbiol 2017;60:449-51

How to cite this URL:
Rai S, Bhardwaj U, Singh S, Misra A. Effect of processing time on analysis of rare event in minimal residual disease study. Indian J Pathol Microbiol [serial online] 2017 [cited 2021 Jul 23];60:449-51. Available from: https://www.ijpmonline.org/text.asp?2017/60/3/449/215374


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],[3],[4],[5],[6],[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],[9],[10],[11],[12],[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],[10],[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, with CD34 and CD45 as backbone markers was used for analysis. The antibody panel used is shown in [Table 1]. The instrument setup was done by Flow Check fluorospheres (BC, Hialeah, FL, USA) for alignment, Flow Set beads (BC, Hialeah, FL, USA) for voltage standardization, and compensation was also done. Alignment was done on a daily basis to ensure precise flow of cells through the laser beam intersection and the fluid stream so that each detector gave maximal and reproducible signals from the standard particles or cells. Electronic standardization (voltage and compensation) also ensured that results were reproducible on a daily basis. The same instrument settings were used for samples.
Table 1: Loss in percentage and mean fluorescence intensity by the time duration in analysis of rare events in minimal residual disease

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For data analysis, Boolean logic gating was used to identify the leukemia-associated immunophenotypes (LAIPs) at the time of diagnosis and at the time of MRD quantification in the postinduction marrow. Gating strategy includes the debris exclusion by time gate (for taking continuous sample stream) and then the SYTO 41 was used to exclude the dead cells from nucleated live cells, on which singlet gate was formed to include only single cells not doublets. These singlet cells were further used for analysis. On CD45/SS plot, at intermediate/low side scatter region, a gate was formed, followed by back gating on the CD34+ population and removal of CD19+ hematogones from analysis. These cells with different parameter were used to identify LAIP, and in the study, the percentage variation of these cells was correlated. Cleaning of the instrument is crucial step to reduce background level of noise below the threshold that would interfere the rare events. For identification of rare event accuracy and eliminating background noise, staining buffer was acquired before sample acquisition and number of events noted down, and then test sample stained with AML panel was is acquired for events that are detectable in the region of interest. This exercise should be performed periodically or any time when there is an unexpected result. It is important to acquire a saline or sheath fluid before MRD collection to ensure no carryover is there. The rare event analysis was done on the processed sample for AML MRD after 15 h to check the reduction in the percentage of the rare events noted earlier and also the change in mean fluorescence intensity [MFI] [Figure 1].
Figure 1: The difference in the events with effect of time duration. Row A shows the initial analysis and row B shows analysis after 15 h. Gating strategy includes exclusion of dead cells and doublets by SYTO 41 and singlet gate

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Our result is shown in [Table 1], shows reduction in MFI and percent loss of cells when samples were processed within 4 hours and after 15 hours. As noted, there was a significant loss of cells and reduction in MFI with time in all plots of CD56/34, CD7/34 and CD14/34. So to conclude, we could advise the feasibility and applicability of MRD estimation by flow cytometry on the fresh sample within 4 hours of reciept as compared to the long standing diagnostic samples to ensure accurate and true results.

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Conflicts of interest

There are no conflicts of interest.

   References Top

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Kraan J, Strijbos MH, Sieuwerts AM, Foekens JA, den Bakker MA, Verhoef C, et al. A new approach for rapid and reliable enumeration of circulating endothelial cells in patients. J Thromb Haemost 2012;10:931-9.  Back to cited text no. 4
Coumans FA, Ligthart ST, Uhr JW, Terstappen LW. Challenges in the enumeration and phenotyping of CTC. Clin Cancer Res 2012;18:5711-8.  Back to cited text no. 5
van de Stolpe A, Pantel K, Sleijfer S, Terstappen LW, den Toonder JM. Circulating tumor cell isolation and diagnostics: Toward routine clinical use. Cancer Res 2011;71:5955-60.  Back to cited text no. 6
Zimmerlin L, Donnenberg VS, Donnenberg AD. Rare event detection and analysis in flow cytometry: Bone marrow mesenchymal stem cells, breast cancer stem/progenitor cells in malignant effusions, and pericytes in disaggregated adipose tissue. Methods Mol Biol 2011;699:251-73.  Back to cited text no. 7
Campana D, Coustan-Smith E. Detection of minimal residual disease in acute leukemia by flow cytometry. Cytometry 1999;38:139-52.  Back to cited text no. 8
Borowitz MJ, Pullen DJ, Winick N, Martin PL, Bowman WP, Camitta B. Comparison of diagnostic and relapse flow cytometry phenotypes in childhood acute lymphoblastic leukemia: Implications for residual disease detection: A report from the children's oncology group. Cytometry B Clin Cytom 2005;68:18-24.  Back to cited text no. 9
Buccisano F, Maurillo L, Del Principe MI, Del Poeta G, Sconocchia G, Lo-Coco F, et al. Prognostic and therapeutic implications of minimal residual disease detection in acute myeloid leukemia. Blood 2012;119:332-41.  Back to cited text no. 10
Inaba H, Coustan-Smith E, Cao X, Pounds SB, Shurtleff SA, Wang KY, et al. Comparative analysis of different approaches to measure treatment response in acute myeloid leukemia. J Clin Oncol 2012;30:3625-32.  Back to cited text no. 11
Ravandi F, Jorgensen JL. Monitoring minimal residual disease in acute myeloid leukemia: Ready for prime time? J Natl Compr Canc Netw 2012;10:1029-36.  Back to cited text no. 12
Terwijn M, Kelder A, Snel AN, Rutten AP, Scholten WJ, Oussoren YJ, et al. Minimal residual disease detection defined as the malignant fraction of the total primitive stem cell compartment offers additional prognostic information in acute myeloid leukaemia. Int J Lab Hematol 2012;34:432-41.  Back to cited text no. 13

Correspondence Address:
Aroonima Misra
Indian Council of Medical Research, National Institute of Nutrition, Tarnaka, Hyderabad - 500 005, Telangana
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_353_16

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