Merging and imputation of flow cytometry data: A critical assessment.

Although most modern techniques and analysis methods in multiparameter flow cytometry (MFC) allow for increased dimensionality for the characterization and quantification of cell populations, most MFC applications depend on flow cytometers measuring relatively small (<16) numbers of parameters. When more markers than the available parameters need to be acquired, these are commonly distributed over multiple independent measurements that include a backbone of common markers. Several methods have been proposed to impute values for combinations of markers that were not measured simultaneously. These imputation methods are frequently used without proper validation and knowledge of their effects on data analysis. We evaluated the performance of existing imputation software (Infinicyt, CyTOFmerge, CytoBackBone, and cyCombine) in approximating known measured expression data in terms of similarity in visual appearance, cell expression, and gating in different datasets by splitting MFC samples into separate measurements with partially overlapping markers and re-calculating missing marker expression. Out of the assessed packages, CyTOFmerge showed the most accurate approximation of the known expression in terms of similar expression values and concordance with manual gating, with a mean F-score between 0.53 and 0.87 when retrieving cell populations in different datasets. Performance remained inadequate for all methods, with only limited similarity at the cell level. In conclusion, the use of imputed MFC data should take such limitations into account and include independent validation of results to justify conclusions.

The role of minor subpopulations within the leukemic blast compartment of AML patients at initial diagnosis in the development of relapse.

The majority of pediatric and younger adult (<60 years) AML patients achieve complete remission. However, 30-40% of patients relapse and display a dismal outcome. Recently we described a frequent instability of type I/II mutations between diagnosis and relapse. Here, we explored the hypothesis that these mutational shifts originate from clonal selection during treatment/disease progression. Subfractions of blasts from initial diagnosis samples were cell sorted and their mutational profiles were compared with those of the corresponding relapse samples of 7 CD34(+) AML patients. At diagnosis, subfractions of the CD45(dim)CD34(+)CD38(dim/-) compartment were heterogeneous in the distribution of mutations, when compared to the whole CD45(dim)CD34(+) blast compartment in 6 out of 7 patients. Moreover, within CD45(dim)CD34(+)CD38(dim/-) fraction of initial samples of 5 of these 6 AML patients, we found evidence for the presence of a minor, initially undetected subpopulation with a specific mutational profile that dominated the bulk of leukemic blasts at relapse. In conclusion, our findings lend support to the AML oligoclonality concept and provide molecular evidence for selection and expansion of a chemo-resistant subpopulation towards development of relapse. These results imply that early detection of pre-existing drug-resistant leukemic subpopulations is crucial for relapse prevention by proper timing of targeted treatment.