Bulk lysis procedures alter target cell population counts.

Area of Science:

• Hematology
• Immunology
• Cellular Biology

Background:

• High-sensitivity cell measurements (<1 in 10^5 cells) by flow cytometry (FCM) require analyzing millions of cells, particularly for measurable residual disease (MRD) detection.
• Conventional immunophenotyping protocols and bulk lysis (BL) assays often fail to acquire sufficient cell numbers and can lead to significant cell loss.
• Minimal sample perturbation (MSP) protocols are proposed to better preserve cellular integrity and avoid cell loss during sample preparation.

Purpose of the Study:

• To evaluate and compare the efficacy of bulk lysis (BL) protocols against minimal sample perturbation (MSP) protocols for high-sensitivity cell analysis.
• To assess the impact of BL protocols on cell counts, particularly for rare cell populations like those in MRD detection.

Main Methods:

• An MRD model was created by spiking peripheral blood with known percentages of EGFP-expressing K562 cells.
• Samples were processed using both bulk lysis (BL) and minimal sample perturbation (MSP) protocols.
• Flow cytometry (FCM) was used to analyze and compare cell populations between the two protocols.

Main Results:

• A significant decrease in K562 cell populations was observed in BL samples compared to MSP samples across all tested percentages.
• Bulk lysis (BL) protocols resulted in significant loss of non-necrotic cells relative to MSP protocols.
• These findings indicate potential over- or under-estimation of rare target cells when using BL methods.

Conclusions:

• Bulk lysis (BL) protocols significantly reduce cell counts, impacting the accuracy of high-sensitivity measurements, especially for rare cells in MRD detection.
• Minimal sample perturbation (MSP) protocols demonstrate superior cell recovery and preservation compared to BL methods.
• Improvements in bulk lysis reagents and solutions are necessary to enhance cell recovery and ensure accurate quantification for high-sensitivity FCM applications.