Abstract
Simple SummaryObjective interpretation of flow cytometry may be hampered by a lack of standardized sample preparation procedures. The EuroFlow consortium conducted a series of experiments to determine the potential impact of different pre-analytical and analytical factors on the variability of results in terms of relative cell populations distribution and marker expression levels. The experiments were performed on healthy donors and patients with different hematological malignancies (e.g., acute leukemia, lymphoma, multiple myeloma, and myelodysplastic syndrome) to mimic real-world clinical settings. Overall, the results showed that sample storage conditions, anticoagulant use, and sample processing protocol might need to be tailored for sample and cell type(s), as well as to the specific markers evaluated. However, defining of well-balanced boundaries for storage time to 24 h, staining-acquisition delay to 3 h, and choosing a washing buffer of pH within the range of 7.2 to 7.8 would be a valid recommendation for most applications and circumstances described herein.Objective interpretation of FC results may still be hampered by limited technical standardization. The EuroFlow consortium conducted a series of experiments to determine the impact of different variables on the relative distribution and the median fluorescence intensity (MFI) of markers stained on different cell populations, from both healthy donors and patients’ samples with distinct hematological malignancies. The use of different anticoagulants; the time interval between sample collection, preparation, and acquisition; pH of washing buffers; and the use of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) staining protocols, were evaluated. Our results showed that only monocytes were represented at higher percentages in EDTA- vs. heparin-anticoagulated samples. Application of SM or SM+CY protocols resulted in slight differences in the percentage of neutrophils and debris determined only with particular antibody combinations. In turn, storage of samples for 24 h at RT was associated with greater percentage of debris and cell doublets when the plasma cell disorder panel was used. Furthermore, 24 h storage of stained cells at RT was selectively detrimental for MFI levels of CD19 and CD45 on mature B- and T-cells (but not on leukemic blasts, clonal B- and plasma cells, neutrophils, and NK cells). The obtained results showed that the variables evaluated might need to be tailored for sample and cell type(s) as well as to the specific markers compared; however, defining of well-balanced boundaries for storage time, staining-to-acquisition delay, and pH of washing buffer would be a valid recommendation for most applications and circumstances described herein.
Highlights
Flow cytometry (FC) is a key diagnostic tool in hemato-oncology
Careful review of guideline papers on technical aspects related to FC immunophenotyping, such as those including recommendations on sample preparation protocols, anticoagulants, optimal sample staining, and storage time and temperature [12,13,16,24,27,28] revealed that universal recommendations that are valid for virtually every sample and cell type and/or even every marker to be evaluated on a variety of cell types, can hardly be found
We investigated the impact of aging of bone marrow (BM) and peripheral blood (PB) samples stored at room temperature (RT) on the relative distribution and marker expression levels of multiple normal and abnormal cell populations, as identified with the EuroFlow Acute Leukemia Orientation Tube (ALOT), B-cell precursor acute lymphoblastic leukemia (BCP-ALL), lymphoid screening tube (LST), B-CLPDtube 1, and plasma cell disorder (PCD) antibody combinations
Summary
Flow cytometry (FC) is a key diagnostic tool in hemato-oncology. objective interpretation of FC results may still be hampered by limited technical standardization. Based on the long-term experience of the laboratories gathered within the EuroFlow Consortium, several factors related to sample preparation were identified as a potential source of unwanted levels of variability, which might negatively impact intra- and inter-laboratory reproducibility and comparability of results. In detail, these factors included, among other variables, the use of different anticoagulants for sample collection, distinct time intervals between sample collection, sample preparation and data acquisition, storage temperatures (room temperature (RT) vs 4 ◦C), the specific approaches used for staining of cell surface membrane-only (SM) vs cell surface plus intracytoplasmic (SM+CY) markers, and the pH of buffers used during sample preparation and/or acquisition in the flow cytometer These factors included, among other variables, the use of different anticoagulants for sample collection, distinct time intervals between sample collection, sample preparation and data acquisition, storage temperatures (room temperature (RT) vs. 4 ◦C), the specific approaches used for staining of cell surface membrane-only (SM) vs. cell surface plus intracytoplasmic (SM+CY) markers, and the pH of buffers used during sample preparation and/or acquisition in the flow cytometer
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