Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials

Abrecht, Charlotte; Hallisey, Margaret; Dennis, Jenna; Nazzaro, Matthew; Brainard, Martha; Hathaway, Emma; Schork, Abigail N.; Hodi, F. Stephen; Severgnini, Mariano; Bagińska, Joanna

doi:10.1016/j.xpro.2022.101362

Cited by 2 publications

(4 citation statements)

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“…To the best of our knowledge, this is the first time these differences are reported, mainly because in previous studies, fixed numbers of cells were measured per sample, whereas all the stained sample was analyzed in our study (12,19). The lower cell recovery by MC vs. SFC is likely caused by the longer sample preparation and staining protocols with more centrifugation steps, which lead to the loss of up to 50% of cells (50). In addition, a limited instrument-related cell recovery has also been reported (30% to 50%) for MC (17,50).…”

Section: Discussionmentioning

confidence: 99%

“…The lower cell recovery by MC vs. SFC is likely caused by the longer sample preparation and staining protocols with more centrifugation steps, which lead to the loss of up to 50% of cells (50). In addition, a limited instrument-related cell recovery has also been reported (30% to 50%) for MC (17,50).…”

Section: Discussionmentioning

confidence: 99%

“…Despite this, our results indicate that, compared to MC, SFC might be a more robust technology for situations where samples need to be processed and analyzed immediately after collection. However, the use of additional strategies aimed at reducing the higher daily technical variability observed with MC, such as the use of batch correction methods or sample barcoding/multiplexing needs to be evaluated (29,30,50,56).…”

Section: Discussionmentioning

confidence: 99%

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Performance of spectral flow cytometry and mass cytometry for the study of innate myeloid cell populations

et al. 2023

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IntroductionMonitoring of innate myeloid cells (IMC) is broadly applied in basic and translational research, as well as in diagnostic patient care. Due to their immunophenotypic heterogeneity and biological plasticity, analysis of IMC populations typically requires large panels of markers. Currently, two cytometry-based techniques allow for the simultaneous detection of ≥40 markers: spectral flow cytometry (SFC) and mass cytometry (MC). However, little is known about the comparability of SFC and MC in studying IMC populations.MethodsWe evaluated the performance of two SFC and MC panels, which contained 21 common markers, for the identification and subsetting of blood IMC populations. Based on unsupervised clustering analysis, we systematically identified 24 leukocyte populations, including 21 IMC subsets, regardless of the cytometry technique.ResultsOverall, comparable results were observed between the two technologies regarding the relative distribution of these cell populations and the staining resolution of individual markers (Pearson’s ρ=0.99 and 0.55, respectively). However, minor differences were observed between the two techniques regarding intra-measurement variability (median coefficient of variation of 42.5% vs. 68.0% in SFC and MC, respectively; p<0.0001) and reproducibility, which were most likely due to the significantly longer acquisition times (median 16 min vs. 159 min) and lower recovery rates (median 53.1% vs. 26.8%) associated with SFC vs. MC.DiscussionAltogether, our results show a good correlation between SFC and MC for the identification, enumeration and characterization of IMC in blood, based on large panels (>20) of antibody reagents.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Performance of spectral flow cytometry and mass cytometry for the study of innate myeloid cell populations

et al. 2023

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“…The data were normalized using the FCS Processing tab of the Fluidigm CyTOF Software 7.0.8493. The detailed protocol is available ( 19 ), and Supplementary Table S1 lists the antibodies used in the panel. The CyTOF data were quantified and analyzed using the CATALYST Pipeline ( 17, 18 ).…”

Section: Methodsmentioning

confidence: 99%

Phase II Study of Eribulin plus Pembrolizumab in Metastatic Soft-tissue Sarcomas: Clinical Outcomes and Biological Correlates

Haddox,

Nathenson,

Mazzola

et al. 2024

Clinical Cancer Research

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Purpose: Eribulin modulates the tumor-immune microenvironment via cGAS-STING signaling in preclinical models. This non-randomized phase II trial evaluated the combination of eribulin and pembrolizumab in patients with soft tissue sarcomas (STS). Patients and Methods: Patients enrolled in one of three cohorts: leiomyosarcoma (LMS), liposarcomas (LPS), or other STS that may benefit from PD-1 inhibitors, including undifferentiated pleomorphic sarcoma (UPS). Eribulin was administered at 1.4 mg/m2 IV (days 1 and 8) with fixed-dose pembrolizumab 200 mg IV (day 1) of each 21-day cycle, until progression, unacceptable toxicity, or completion of 2 years of treatment. The primary endpoint was the 12-week progression-free survival rate (PFS-12) in each cohort. Secondary endpoints included the objective response rate (ORR), median PFS, safety profile, and overall survival (OS). Pre- and on-treatment blood specimens were evaluated in patients who achieved durable disease control (DDC) or progression within 12 weeks (early progression [EP]). Multiplexed immunofluorescence was performed on archival LPS samples from patients with DDC or EP. Results: Fifty-seven patients enrolled (LMS, n=19; LPS, n=20; UPS/other, n=18). The PFS-12 was 36.8% (90% confidence interval [CI] 22.5-60.4%) for LMS, 69.6% (54.5-89.0%) for LPS, and 52.6% (36.8-75.3%) for UPS/other cohorts. All 3 patients in the UPS/other cohort with angiosarcoma achieved RECIST responses. Toxicity was manageable. Higher interferon (IFN) alpha and IL-4 serum levels were associated with clinical benefit. Immune aggregates expressing PD-1 and PD-L1 were observed in a patient that completed 2 years of treatment. Conclusions: The combination of eribulin and pembrolizumab demonstrated promising activity in LPS and angiosarcoma.

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Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials

Cited by 2 publications

References 5 publications

Performance of spectral flow cytometry and mass cytometry for the study of innate myeloid cell populations

Performance of spectral flow cytometry and mass cytometry for the study of innate myeloid cell populations

Phase II Study of Eribulin plus Pembrolizumab in Metastatic Soft-tissue Sarcomas: Clinical Outcomes and Biological Correlates

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