Dataset Information

Comparison of Volumetric and Bead-Based Counting of CD34 Cells by Single-Platform Flow Cytometry.

ABSTRACT:

Background

Over 2,000 people a year in the United Kingdom need a bone marrow or blood stem cell transplant. It is important to accurately quantify the hematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification. The current gold standard method is single-platform flow cytometry using internal reference counting beads to determine the concentration of CD34 cells. However, volumetric flow cytometers have the ability to measure the acquisition volume, which removes the need for reference beads for calculation of cell concentrations.

Method

In this study, we compared both methods for calculating CD34 cell concentrations in volumetric cytometers, using either the volume reading or the number of reference beads for calculation. In addition, the uncertainty of measurement for each method was estimated.

Results

The results show that both methods have similar uncertainties of measurement. Regression analysis showed low to no statistical difference in CD34 cell concentrations obtained with each method.

Conclusions

Overall, this study suggests that the volumetric method is a valid approach but that the adoption of this technology may be hindered without some form of external calibration of volume readings to increase confidence in the measurement. © 2019 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals, Inc. on behalf of International Clinical Cytometry Society.

SUBMITTER: Saraiva L

PROVIDER: S-EPMC6899615 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Publications

Comparison of Volumetric and Bead-Based Counting of CD34 Cells by Single-Platform Flow Cytometry.

Saraiva Luisa L Wang Lili L Kammel Martin M Kummrow Andreas A Atkinson Eleanor E Lee Ji Youn JY Yalcinkaya Burhanettin B Akgöz Muslum M Höckner Jana J Ruf Andreas A Engel Andrea A Zhang Yu-Zhong YZ O'Shea Orla O Sassi Maria Paola MP Divieto Carla C Lekishvili Tamara T Campbell Jonathan J Liu Yingying Y Wang Jing J Stebbings Richard R Gaigalas Adolfas K AK Rigsby Peter P Neukammer Jörg J Vessillier Sandrine S

Cytometry. Part B, Clinical cytometry 20190220 6

<h4>Background</h4>Over 2,000 people a year in the United Kingdom need a bone marrow or blood stem cell transplant. It is important to accurately quantify the hematopoietic stem cells to predict whether the transplant will be successful in replenishing the immune system. However, they are present at low frequency, which complicates accurate quantification. The current gold standard method is single-platform flow cytometry using internal reference counting beads to determine the concentration of ...[more]

PMID: 30790450

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Project description:BackgroundExtracellular vesicles (EVs) originating from the central nervous system (CNS) can enter the blood stream and carry molecules characteristic of disease states. Therefore, circulating CNS-derived EVs have the potential to serve as liquid-biopsy markers for early diagnosis and follow-up of neurodegenerative diseases and brain tumors. Monitoring and profiling of CNS-derived EVs using multiparametric analysis would be a major advance for biomarker as well as basic research. Here, we explored the performance of a multiplex bead-based flow-cytometry assay (EV Neuro) for semi-quantitative detection of CNS-derived EVs in body fluids.MethodsEVs were separated from culture of glioblastoma cell lines (LN18, LN229, NCH82) and primary human astrocytes and measured at different input amounts in the MACSPlex EV Kit Neuro, human. In addition, EVs were separated from blood samples of small cohorts of glioblastoma (GB), multiple sclerosis (MS) and Alzheimer's disease patients as well as healthy controls (HC) and subjected to the EV Neuro assay. To determine statistically significant differences between relative marker signal intensities, an unpaired samples t-test or Wilcoxon rank sum test were computed. Data were subjected to tSNE, heatmap clustering, and correlation analysis to further explore the relationships between disease state and EV Neuro data.ResultsGlioblastoma cell lines and primary human astrocytes showed distinct EV profiles. Signal intensities were increasing with higher EV input. Data normalization improved identification of markers that deviate from a common profile. Overall, patient blood-derived EV marker profiles were constant, but individual EV populations were significantly increased in disease compared to healthy controls, e.g. CD36+EVs in glioblastoma and GALC+EVs in multiple sclerosis. tSNE and heatmap clustering analysis separated GB patients from HC, but not MS patients from HC. Correlation analysis revealed a potential association of CD107a+EVs with neurofilament levels in blood of MS patients and HC.ConclusionsThe semi-quantitative EV Neuro assay demonstrated its utility for EV profiling in complex samples. However, reliable statistical results in biomarker studies require large sample cohorts and high effect sizes. Nonetheless, this exploratory trial confirmed the feasibility of discovering EV-associated biomarkers and monitoring circulating EV profiles in CNS diseases using the EV Neuro assay. Video Abstract.

Dataset Information

Comparison of Volumetric and Bead-Based Counting of CD34 Cells by Single-Platform Flow Cytometry.

Background

Method

Results

Conclusions

Publications

Comparison of Volumetric and Bead-Based Counting of CD34 Cells by Single-Platform Flow Cytometry.

Similar Datasets

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