Impact of red blood cell lysing on rare event analysis

Rico, Laura; Calle, Fernando Raimúndez de la; Salvia, Roser; Ward, Mike D.; Bradford, Jolene A.; Juncà, Jordi; Sorigué, Marc; Pétriz, Jordi

doi:10.1002/cyto.a.24688

Cited by 2 publications

(3 citation statements)

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“…Applying bulk lysis protocols results in a significant decrease in non‐necrotic cells (Figure 2, lower row; and Supplementary Table 2) compared to MSP protocols. This decrease may be associated with the detrimental effects produced by erythrocyte lysis solutions containing ammonium chloride and washing steps [13–16]. Regarding target cell frequency detection, we observed a significant loss of EGFP‐K562 cells when preparing samples with BL methods for all established percentages (Figures 1 and 2, upper row; and Supplementary Table 1).…”

Section: Discussionmentioning

confidence: 87%

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Bulk lysis procedures alter target cell population counts

Rico,

Salvia,

Ward

et al. 2024

Cytometry Pt A

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To achieve high‐sensitivity cell measurements (<1 in 105 cells) by flow cytometry (FCM), the minimum number of acquired cells must be considered and conventional immunophenotyping protocols fall short of these numbers. The bulk lysis (BL) assay is a standardized erythrocyte lysing approach that allows the analysis of the millions of cells required for high‐sensitivity measurable residual disease (MRD) detection. However, this approach has been associated with significant cell loss, along with potential over or underestimates of rare cells when using this method. The aim of this study was to evaluate bulk lysis protocols and compare them with minimal sample perturbation (MSP) protocols, which are reported to better preserve the native cellular state and avoid significant cell loss due to washing steps. To achieve this purpose, we first generated an MRD model by spiking fresh peripheral blood with K562 cells, stably expressing EGFP, at known percentages of EGFP positive cells to leukocytes. Samples were then prepared with BL and MSP protocols and analyzed using FCM. For all percentages of K562 cells established and evaluated, a significant decrease of this population was detected in BL samples compared with MSP samples, even at low K562 cell percentages. Significant decreases for non‐necrotic cells were also observed in BL samples relative to MSP samples. In conclusion, the evaluation of the potential effects of BL protocols in obtaining the final count is of great interest, especially for over‐ or under‐estimation of target cells, as in the case of measurable residual disease. Since conventional flow cytometry or minimal sample perturbation assays fall short in obtaining the minimum numbers required to reach high sensitivity measurements, significant efforts may be needed to improve bulk lysis solution reagents.

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Section: Discussionmentioning

confidence: 87%

“…That alteration resulted in under‐ and over‐estimation of those target cells when calculating their frequency relative to total leukocytes [15]. Another study of our group reported that erythrocyte lysing solutions can cause an over‐ or under‐estimation of rare cells in MRD models and in chronic lymphocytic leukemia [16].…”

Section: Discussionmentioning

confidence: 99%