Stabilization of pre‐optimized multicolor antibody cocktails for flow cytometry applications

Chan, Roger W.; Kotner, Joshua S.; Chuang, Christine Ming-Hui; Gaur, Amitabh

doi:10.1002/cyto.b.21371

Cited by 19 publications

(29 citation statements)

References 34 publications

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“… 13 − 15 Nevertheless, only a few commercial methods are available for lyophilizing multiple-antibody cocktails for diagnostic assays, all of which rely on proprietary buffer formulations and techniques. 16 , 17 Here, we demonstrated a simple, low-cost strategy for preparing lyophilized, ready-to-use antibody cocktails containing up to five different antibody markers, stabilized with only common laboratory reagents.…”

Section: Discussionmentioning

confidence: 99%

Glass Chemistry to Analyze Human Cells under Adverse Conditions

2019

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Emerging point-of-care diagnostic tests capable of analyzing whole mammalian cells often rely on the attachment of harvested cells to glass surfaces for subsequent molecular characterization. We set out to develop and optimize a kit for the diagnosis of lymphoma in low- and middle-income countries where access to advanced healthcare testing is often absent or prone to error. Here, we optimized a process for the lyophilization of neutravidin-coated glass and cocktails of antibodies relevant to lymphoma diagnosis to establish long-term stability of reagents required for point-of-care cell capture technology. Lyophilized glass slides showed no decline in their performance compared to freshly prepared neutravidin glass and preserved capture efficiency for 5 weeks under easily attainable storage conditions. We demonstrate the successful performance of the low-cost, lyophilized kit in a cell capture assay to enable true point-of-care analyses under adverse conditions. We anticipate that the strategy can be expanded to other cancer cell types or cell-derived vesicle analysis.

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Section: Discussionmentioning

confidence: 99%

Glass Chemistry to Analyze Human Cells under Adverse Conditions

2019

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“…Indeed, lyophilized products, particularly Lyoplates V R and Lyotubes V R from BD Biosciences, have already been shown to be very stable overtime (15,16), to strongly correlate with liquid formulations (15,17) and to allow a very satisfactory standardization in cross-center studies (13,17). Indeed, lyophilized products, particularly Lyoplates V R and Lyotubes V R from BD Biosciences, have already been shown to be very stable overtime (15,16), to strongly correlate with liquid formulations (15,17) and to allow a very satisfactory standardization in cross-center studies (13,17).…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we used the Duraclone formulation, but other manufacturers currently propose comparable products, such as lyophilized antibodies, that would probably yield similar efficiency regarding the stability and reproducibility of the results. Indeed, lyophilized products, particularly Lyoplates V R and Lyotubes V R from BD Biosciences, have already been shown to be very stable overtime (15,16), to strongly correlate with liquid formulations (15,17) and to allow a very satisfactory standardization in cross-center studies (13,17). Together with our results, these studies tend to demonstrate that the new generation of stabilized reagents, either dried or lyophilized, will be critical in the future to reduce variability over time and in inter-institutional studies.…”

Section: Discussionmentioning

confidence: 99%

A standardized flow cytometry procedure for the monitoring of regulatory T cells in clinical trials

Pitoiset

Barbié

Monneret

et al. 2018

Cytometry Part B Clinical

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“…Flow cytometry has become a standard tool for the analysis of cellular phenotype and function within the immune system, among other applications. As more fluorochromes have been developed with advances in cytometer design and lasers, the identification of more rare cell populations has become achievable, and the sensitivity of existing assays has increased ( 1 , 2 ). For PNH, as a rare disease, a high sensitivity assay with short acquisition time is required.…”

Section: Discussionmentioning

confidence: 99%

“…It allows for the identification of cellular subsets based on specific markers both on the surface of and inside cells ( 1 ). With the development of novel classes of fluorochromes and advancements in cytometer design, the use of more than 8 colors for flow cytometry should become common practice in clinical labs and increase the sensitivity of current assays ( 2 ).…”

Section: Introductionmentioning

confidence: 99%

High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections

et al. 2018

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Flow cytometry is the gold standard in diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) by detecting the absence of glycol-phosphatidyl inositol (GPI)-linked protein expression on granulocyte and monocyte surfaces. However, the current assays are not optimized and require improvement, particularly in reducing background fluorescence and optimizing sensitivity and specificity. With more fluorochromes available and with advances in instrument engineering, rare populations may be identified with high sensitivity. The present study assessed an 8-color combination of comprehensive GPI-linked markers, namely fluorescein-labeled proaerolysin (FLAER), cluster of differentiation 157 (CD157), CD24 and CD14, and the lineage markers for granulocyte (CD15) and monocyte (CD64) cells to detect PNH clones. Additionally, to optimize the PNH flow assay, a ‘dump’ channel was used, comprised of CD5 and CD19, to exclude non-specific binding in order to reduce background. This method aimed to improve sensitivity and reduce the background to create an optimized PNH flow cocktail. The results demonstrated that the current 4-color PNH combination identifies a CD55− and FLAER+ population that is not PNH clones. By contrast, the 8-color panel delineated PNH clones from both monocyte and granulocytes by using granulocyte antigen (CD15) and monocyte antigen (CD64) as a gating strategy. The sensitivity was 0.01% for granulocytes and 0.05% for monocytes with an acquisition of 100,000 monocyte and granulocyte events. The background on a normal whole blood sample was 0.00076% on monocytes and 0.00277% on granulocytes. Thus, overall, the 8-color PNH assay exhibited high levels of specificity and sensitivity. The 8-color combination facilitated the improvement and enhancement of sensitivity in PNH clone identification, and may provide a useful tool for pathologists in PNH diagnosis and for monitoring patients at risk of developing classical/hemolytic PNH, to enable treatment to be delivered promptly.

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Stabilization of pre‐optimized multicolor antibody cocktails for flow cytometry applications

Cited by 19 publications

References 34 publications

Glass Chemistry to Analyze Human Cells under Adverse Conditions

Glass Chemistry to Analyze Human Cells under Adverse Conditions

A standardized flow cytometry procedure for the monitoring of regulatory T cells in clinical trials

High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections

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