Cell Enrichment from Human Blood through Red Cell Lysis

Miller, Brandon; Chalmers, Jeffrey J.; Lowrie, William G.

doi:10.1016/b978-0-12-405914-6.00009-3

Cited by 3 publications

(4 citation statements)

References 2 publications

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“…The remaining blood volume (7-9 mL) was then used for CTC staining and collection. After validating our FACS protocol using spike-in experiments (see Supplementary note 1 ), we followed a similar approach to Miller et al (2012) and first lysed the red blood cells using BD Pharm Lyse lysing solution (BD Biosciences, NJ, USA) following the fabricant recommendations. When needed, we repeated the lysing step up to three times.…”

Section: Methodsmentioning

confidence: 99%

Comparative analysis of capture methods for genomic profiling of circulating tumor cells in colorectal cancer

Alves

Estévez-Gómez

Valecha

et al. 2022

Preprint

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The genomic profiling of circulating tumor cells (CTCs) in the bloodstream should provide clinically relevant information on therapeutic efficacy and help predict cancer survival. However, the molecular characterization of CTCs has so far proven extremely difficult. A variety of technologies have been developed for CTC isolation, but so far the impact on the genomic assessment of CTCs has not been fully evaluated. To fill this gap, here we contrasted the genomic profiles of CTC pools recovered from blood samples obtained from four metastatic colorectal cancer (mCRC) patients using three different enrichment strategies (CellSearch, Parsortix, and FACS). Our results suggest clear differences in the mutational burden of CTC pools depending on the enrichment method used, with all evaluated methods returning a somewhat limited representation of the mutational spectrum of individual tumors, potentially due to allelic dropout during whole-genome amplification. Nevertheless, the CTC pools from Parsortix, and in part, CellSearch, showed diversity estimates, mutational signatures and drug-suitability scores remarkably close to the ones found in matching primary tumor samples. In contrast, FACS CTC pools were substantially enriched in apparent sequencing artifacts, which led to much higher estimates of genomic diversity. Although CTC genomics still faces technical challenges, our results suggest that CTC-derived metrics can reflect the diversity scores seen in primary tumor lesions thus highlighting the utility of CTCs to assess the heterogeneity status of individual tumors, and to help clinicians prioritize drugs in mCRC.

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Section: Methodsmentioning

confidence: 99%

Comparative analysis of capture methods for genomic profiling of circulating tumor cells in colorectal cancer

Alves

Estévez-Gómez

Valecha

et al. 2022

Preprint

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“…Human blood was collected in K2-EDTA tubes according to protocol #2018H0268 approved by the Biomedical Sciences Committee Institutional Review Board (IRB) at The Ohio State University. For experiments using the PBLs, the red blood cells (RBCs) were depleted through RBC lysis ( 29 ). A cell lysis buffer of 150 mM ammonium chloride, 10 mM sodium bicarbonate, and 0.1 mM EDTA at 7.4 pH was prepared.…”

Section: Methodsmentioning

confidence: 99%

Analyzing Inter-Leukocyte Communication and Migration In Vitro: Neutrophils Play an Essential Role in Monocyte Activation During Swarming

et al. 2021

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Neutrophils are known to be the first responders to infection or injury. However, as inflammation progresses, other leukocytes become increasingly important in inflammation propagation, tissue reconstruction, and inflammation resolution. In recent years, there has been an increase in publications that analyze neutrophil behavior in vitro, but there remains a gap in the literature for in vitro technologies that enable quantitatively measuring interactions between different types of human leukocytes. Here, we used an in vitro platform that mimics inflammation by inducing neutrophil swarming to analyze the behavior of various leukocytes in a swarming setting. Using human peripheral blood leukocytes isolated directly from whole blood, we found that myeloid cells and lymphoid cells had different migratory behaviors. Myeloid cells, which are predominately neutrophils, exhibited swarming behavior. This behavior was not seen with lymphoid cells. We perturbed the peripheral blood leukocyte system by adding exogenous leukotriene B4 (LTB4) to the medium. Notably, only the myeloid cell compartment was significantly changed by the addition of LTB4. Additionally, LTB4 had no significant impact on myeloid cell migration during the recruitment phase of swarming. To further investigate the myeloid cell compartment, we isolated neutrophils and monocytes to analyze their interaction on the platform. We found that neutrophils increase monocyte migration toward the bioparticle clusters, as measured through speed, chemotactic index, track straightness, and swarm size. These results were confirmed with in vivo mouse experiments, where monocyte accumulation only occurred when neutrophils were present. Additionally, we found that both neutrophils and monocytes release the monocyte chemoattractant proteins CCL2 and CCL3 in the presence of Staphylococcus aureus bioparticles. Furthermore, extracellular vesicles from swarming neutrophils caused monocyte activation. These findings suggest that neutrophils play an essential role in the onset of inflammation not only by sealing off the site of infection or injury, but also by recruiting additional leukocytes to the site.

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“…Ammonium chloride lysis buffer is used, which exploits the RBC’s relative weakness to osmotic pressure compared to other cell types. Previous spiking studies have demonstrated the superior recovery of spiked MCF7 cancer cells by chemical lysis in comparison to the Ficoll–Hypaque density gradient separation technique [8]. …”

Section: Methodsmentioning

confidence: 99%

“…The lithium heparin coating is beneficial in preventing clumping of the white blood cells prior to analysis. The RBC lysis procedure was carried out as previously published [8]. Briefly, the blood is mixed with 1× lysis buffer at a ratio of 1:20 to the total volume.…”

Section: Methodsmentioning

confidence: 99%

Isolation and analysis of rare cells in the blood of cancer patients using a negative depletion methodology

Deighan

Miller

et al. 2013

Methods

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A variety of enrichment/isolation technologies exist for the characterization of rare cells in the blood of cancer patients. In this article, a negative depletion process is presented and discussed which consists of red blood cell (RBC) lysis and the subsequent removal of CD45 expressing cells through immunomagnetic depletion. Using this optimized assembly on 120 whole blood specimens, from 71 metastatic breast cancer patients, after RBC lysis, the average nucleated cell log depletion was 2.56 with a 77% recovery of the nucleated cells. The necessity of exploring different anti-CD45 antibody clones to label CD45 expressing cells in this enrichment scheme is also presented and discussed. An optimized, four-color immunofluorescence staining is conducted on the cells retained after the CD45-based immunomagnetic depletion process. Different types of rare non-hematopoietic cells are found in these enriched peripheral blood samples and a wide range of external and internal markers have been characterized, which demonstrates the range and heterogeneity of the rare cells.

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Cell Enrichment from Human Blood through Red Cell Lysis

Cited by 3 publications

References 2 publications

Comparative analysis of capture methods for genomic profiling of circulating tumor cells in colorectal cancer

Comparative analysis of capture methods for genomic profiling of circulating tumor cells in colorectal cancer

Analyzing Inter-Leukocyte Communication and Migration In Vitro: Neutrophils Play an Essential Role in Monocyte Activation During Swarming

Isolation and analysis of rare cells in the blood of cancer patients using a negative depletion methodology

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