Quantitative Flow Cytometry: Concerns and Recommendations in Clinic and Research

Mizrahi, Olga; Shalom, Eliran Ish; Baniyash, Michal; Klieger, Yair

doi:10.1002/cyto.b.21515

Cited by 67 publications

(62 citation statements)

References 36 publications

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“…Staining and acquisition procedures were performed according to EuroFlow guidelines and flow cytometry was performed on FACSCanto II flow cytometers (BD, Erembodegem, Belgium) with EuroFlow instrument settings . Instrument performance was monitored on a daily basis using CS&T beads and rainbow beads . BM, PB, lymph node (LN), or pleural effusion (PE) samples were used for immunophenotyping.…”

Section: Methodsmentioning

confidence: 99%

CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping

Bras

Haas

Stigt

et al. 2018

Cytometry Part B Clinical

101

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Background While it is known that CD123 is normally strongly expressed on plasmacytoid dendritic cells and completely absent on nucleated red blood cells, detailed information regarding CD123 expression in acute leukemia is scarce and, if available, hard to compare due to different methodologies. Methods CD123 expression was evaluated using standardized EuroFlow immunophenotyping in 139 pediatric AML, 316 adult AML, 193 pediatric BCP‐ALL, 69 adult BCP‐ALL, 101 pediatric T‐ALL, and 28 adult T‐ALL patients. Paired diagnosis‐relapse samples were available for 57 AML and 19 BCP‐ALL patients. Leukemic stem cell (LSC) data was available for 32 pediatric AML patients. CD123 expression was evaluated based on mean fluorescence intensity, median fluorescence intensity, and percentage CD123 positive cells. Results EuroFlow panels were stable over time and between laboratories. CD123 was expressed in the majority of AML and BCP‐ALL patients, but absent in most T‐ALL patients. Within AML, CD123 expression was lower in erythroid/megakaryocytic leukemia, higher in NPM1 mutated and FLT3‐ITD mutated leukemia, and comparable between LSC and leukemic blasts. Within BCP‐ALL, CD123 expression was higher in patients with (high) hyperdiploid karyotypes and the BCR‐ABL fusion gene. Interestingly, CD123 expression was increased in BCP‐ALL relapses while highly variable in AML relapses (compared to CD123 expression at diagnosis). Conclusions Authors evaluated CD123 expression in a large cohort of acute leukemia patients, based on standardized and reproducible methodology. Our results may facilitate stratification of patients most likely to respond to CD123 targeted therapies and serve as reference for CD123 expression (in health and disease). © 2018 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

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Section: Methodsmentioning

confidence: 99%

CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping

Bras

Haas

Stigt

et al. 2018

Cytometry Part B Clinical

101

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“…There is now a growing body of evidence about the potential of quantitative assessment for clinical diagnostics (2). In addition to morphological examination and genetic profiling, an important role is played by immunophenotyping.…”

Section: Introductionmentioning

confidence: 99%

Quantitative assessment of informative immunophenotypic markers increases the diagnostic value of immunophenotyping in mature CD5‐positive B‐cell neoplasms

Starostka

Kriegová

Kudělka

et al. 2018

Cytometry Part B Clinical

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“…The beads are intended for standardization of fluorescence intensity and can be used for measuring absolute level of proteins on a cell by fluorescence flow cytometry, so-called quantitative flow cytometry (9). The beads are intended for standardization of fluorescence intensity and can be used for measuring absolute level of proteins on a cell by fluorescence flow cytometry, so-called quantitative flow cytometry (9).…”

mentioning

confidence: 99%

“…The standardization strategy involved quantum simply cellular (QSC) beads which are beads with known antibodybinding capacity. The beads are intended for standardization of fluorescence intensity and can be used for measuring absolute level of proteins on a cell by fluorescence flow cytometry, so-called quantitative flow cytometry (9). QSC beads were adapted to mass cytometry by adding a labeling step with osmium tetroxide (OsO 4 ).…”

mentioning

confidence: 99%

Expanding the Clinical Cytometry Toolbox—Receptor Occupancy by Mass Cytometry

Huse

2019

Cytometry Pt A

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DETERMINATION of the optimal therapeutic dose is a key challenge in drug development. Receptor occupancy assays (RO assays) measure the binding of biotherapeutics to their cellular targets and are one of the tools used to determine starting doses in clinical and preclinical studies. Flow cytometry-based RO assays have been developed for heterogeneous cell populations in suspension and are well suited for evaluating therapeutics targeting peripheral blood cells. Mass cytometry would further expand the possibilities to measure receptor occupancy of complex cell types as metallabeled antibodies and minimal overlap between channels allow for more than 40 markers to be detected simultaneously without loss of sensitivity. Bringeland et al. (1) have developed a mass cytometry-based RO assay, recently described in a technical note. With a backbone of 15 lineage markers, they identified eight cell types of interest and obtained receptor occupancy data for each cell population.Receptor occupancy assays can prevent life-threatening side effects in clinical trials, exemplified by the disastrous TGN1412 first-in-human clinical trial in 2006 (2). TGN1412 is an anti-CD28 superagonistic monoclonal antibody that stimulates T cells, intended for treatment of B-cell chronic lymphocytic leukemia and rheumatoid arthritis. In preclinical trials, cynomolgus monkeys were used to determine toxicity, and starting doses in the first human clinical trial was calculated based on the no-adverse-effect level (NOAEL) in these animals (3). However, despite using doses 500 times lower than the dose found safe in animal models, the human volunteers involved in the trial suffered severe cytokine release syndrome and multiple organ failure after receiving the drug (2). Follow-up studies revealed that biological differences between humans and monkeys, in this case different expression levels of CD28 on their respective T cells, resulted in 90% receptor occupancy and severe cytokine release syndrome with a dose that was expected to lead to only 10% receptor occupancy (4). A simple RO assay could have revealed these differences, and as a consequence of this trial, the procedures for first-in-human clinical trials were changed (5).Today, RO assays play an increasing role in drug development (3,4) and have been used in testing of therapeutic antibodies like anti-PD-1 (6) and anti-PD-L1 (7). Receptor occupancy data can contribute to determining the minimal anticipated biological effect level (MABEL) which is often better suited than NOAEL to predict safe starting doses in first-in-human clinical trials. In addition to revealing safety hazards, RO assays can limit the extent of preclinical testing, which could reduce costs for the pharmaceutical industry and benefit patients by introducing potentially life-saving drugs earlier. During later-phase trials, receptor occupancy data can monitor therapeutic response and guide optimal dosing for individual patients. Bringeland et al. (1) and others (8) have investigated the use of RO assays for dosing of natalizu...

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Quantitative Flow Cytometry: Concerns and Recommendations in Clinic and Research

Cited by 67 publications

References 36 publications

CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping

CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping

Quantitative assessment of informative immunophenotypic markers increases the diagnostic value of immunophenotyping in mature CD5‐positive B‐cell neoplasms

Expanding the Clinical Cytometry Toolbox—Receptor Occupancy by Mass Cytometry

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