Relevance of sample preparation for flow cytometry

Abstract

We suggest to use LWSW method also at diagnosis to obtain more comparable antibody intensity expressions when samples from the same patient are processed for MRD evaluation after bulk lysis. Moreover, LWSW can prevent nonspecific bindings, shows no differences in the identification and quantitation of the populations of interest, and reduces acquisition of cell debris.

Validation of a modified pre‐lysis sample preparation technique for flow cytometric minimal residual disease assessment in multiple myeloma, chronic lymphocytic leukemia, and B‐non Hodgkin lymphoma

“…Our study findings are comparable with other recent reports, demonstrating improved sensitivity of flow cytometric MRD assays with implementation of a pre-lysis technique (Flores-Montero et al, 2017;Kalina et al, 2012;Muccio et al, 2018;Theunissen et al, 2017). Despite concerns for the potential of additional wash steps to increase cell loss, and the limited data available regarding the impact of pre-lysis on antigen-antibody stability, our study demonstrated no compromise in antigen stability across three different disease panels, and more reliably achieved a higher sensitivity.…”

“…Various methods currently available for MFC sample preparation are selected based on providing the best signal discrimination between populations, improving signal to noise ratio, minimization of nonspecific binding, auto-fluorescence, and antibody specificity (Tanqri et al, 2013;Wood, 2016). SLW protocols are widely adopted in the diagnostic setting, providing better signal discrimination (Johansson, Bloxham, Couzens, et al, 2014;Muccio et al, 2018). With novel sample preparation procedures, attaining a high number of events is a priority; however, clear distinctions between positive and negative populations remains essential.…”

“…Highly sensitive assays are required to improve the detection of MRD in bone marrow and peripheral blood samples of patients with haematological neoplasms undergoing treatment at multiple time points. Sample preparation and assay methodology play a key role in overall performance of flow cytometric MRD testing (Muccio et al, 2018;Theunissen et al, 2017), including sample stability and ability to discriminate between major cell populations (Flores-Montero et al, 2017). Our study shows that adopting a pre-lysis method results F I G U R E 6 Stability of CD138 expression.…”

“…To date, few studies have directly compared assay performance of pre-lysis against post-lysis (Kalina et al, 2012;Muccio, Saraci, Gilestro et al, 2018;Royston et al, 2016;Soh et al, 2020). In this article, we report on the evaluation of our department's standard postlysis preparation (SLW) against a modified EuroFlow pre-lysis (LSW) method to evaluate overall assay sensitivity in assessment of MRD using routine samples from patients with hematological malignancies for which MFC MRD plays an important clinical role.…”

“…Finally, multiple steps of sample preparation affect antigen expression, upon which the ability to detect abnormal cells relies. This is crucial for identification and enumeration of a target cell population at a particular level of sensitivity (Kalina et al, 2012;Muccio et al, 2018). Various preparation methods also result in differential loss of normal leukocyte subtypes as well as affecting discrimination between major cell populations to variable degrees (Kalina et al, 2012).…”