268 research outputs found
Integration of Magnetic Bead-Based Cell Selection into Complex Isolations
Magnetic bead-based analyte capture
has emerged as a ubiquitous
method in cell isolation, enabling the highly specific capture of
target populations through simple magnetic manipulation. To date,
no “one-size fits all” magnetic bead has been widely
adopted leading to an overwhelming number of commercial beads. Ultimately,
the ideal bead is one that not only facilitates cell isolation but
also proves compatible with the widest range of downstream applications
and analytic endpoints. Despite the diverse offering of sizes, coatings,
and conjugation chemistries, few studies exist to benchmark the performance
characteristics of different commercially available beads; importantly,
these bead characteristics ultimately determine the ability of a bead
to integrate into the user’s assay. In this report, we evaluate
bead-based cell isolation considerations, approaches, and results
across a subset of commercially available magnetic beads (Dynabeads
FlowComps, Dynabeads CELLection, GE Healthcare Sera-Mag SpeedBeads
streptavidin-blocked magnetic particles, Dynabeads M-270s, Dynabeads
M-280s) to compare and contrast both capture-specific traits (i.e.,
purity, capture efficacy, and contaminant isolations) and endpoint
compatibility (i.e., protein localization, fluorescence imaging, and
nucleic acid extraction). We identify specific advantages and contexts
of use in which distinct bead products may facilitate experimental
goals and integrate into downstream applications
AirJump: Using Interfaces to Instantly Perform Simultaneous Extractions
Analyte
isolation is an important process that spans a range of biomedical
disciplines, including diagnostics, research, and forensics. While
downstream analytical techniques have advanced in terms of both capability
and throughput, analyte isolation technology has lagged behind, increasingly
becoming the bottleneck in these processes. Thus, there exists a need
for simple, fast, and easy to integrate analyte separation protocols
to alleviate this bottleneck. Recently, a new class of technologies
has emerged that leverages the movement of paramagnetic particle (PMP)-bound
analytes through phase barriers to achieve a high efficiency separation
in a single or a few steps. Specifically, the passage of a PMP/analyte
aggregate through a phase interface (aqueous/air in this case) acts
to efficiently “exclude” unbound (contaminant) material
from PMP-bound analytes with higher efficiency than traditional washing-based
solid-phase extraction (SPE) protocols (i.e., bind, wash several times,
elute). Here, we describe for the first time a new type of “exclusion-based”
sample preparation, which we term “AirJump”. Upon realizing
that much of the contaminant carryover stems from interactions with
the sample vessel surface (e.g., pipetting residue, wetting), we aim
to eliminate the influence of that factor. Thus, AirJump isolates
PMP-bound analyte by “jumping” analyte directly out
of a free liquid/air interface. Through careful characterization,
we have demonstrated the validity of AirJump isolation through comparison
to traditional washing-based isolations. Additionally, we have confirmed
the suitability of AirJump in three important independent biological
isolations, including protein immunoprecipitation, viral RNA isolation,
and cell culture gene expression analysis. Taken together, these data
sets demonstrate that AirJump performs efficiently, with high analyte
yield, high purity, no cross contamination, rapid time-to-isolation,
and excellent reproducibility
Using Exclusion-Based Sample Preparation (ESP) to Reduce Viral Load Assay Cost.
Viral load (VL) measurements are critical to the proper management of HIV in developing countries. However, access to VL assays is limited by the high cost and complexity of existing assays. While there is a need for low cost VL assays, performance must not be compromised. Thus, new assays must be validated on metrics of limit of detection (LOD), accuracy, and dynamic range. Patient plasma samples from the Joint Clinical Research Centre in Uganda were de-identified and measured using both an existing VL assay (Abbott RealTime HIV-1) and our assay, which combines low cost reagents with a simplified method of RNA isolation termed Exclusion-Based Sample Preparation (ESP).71 patient samples with VLs ranging from 3,000,000 copies/mL were used to compare the two methods. We demonstrated equivalent LOD (~50 copies/mL) and high accuracy (average difference between methods of 0.08 log, R2 = 0.97). Using expenditures from this trial, we estimate that the cost of the reagents and consumables for this assay to be approximately $5 USD. As cost is a significant barrier to implementation of VL testing, we anticipate that our assay will enhance access to this critical monitoring test in developing countries
Magnetic System for Automated Manipulation of Paramagnetic Particles
The simple, rapid magnetic manipulation
of paramagnetic particles
(PMPs) paired with the wide range of available surface chemistries
has strongly positioned PMPs in the field of analyte isolation. One
recent technology, sliding lid for immobilized droplet extractions
(SLIDE), presents a simple, rapid alternative to traditional PMP isolation
protocols. Rather than remove fluid from PMP-bound analyte, SLIDE
directly removes the PMPs from the fluid. SLIDE collects the PMPs
on a hydrophobic, removable surface, which allows PMPs to be captured
from one well and then transferred and released into a second well.
Despite several key advantages, SLIDE remains limited by its passive
magnetic manipulation that only allows for a one-time capture-and-release
of PMPs, preventing wash steps and limiting purity. Furthermore, the
strategy employed by SLIDE constrains the position of the wells, thereby
limiting throughput and integration into automated systems. Here,
we introduce a new, mechanically and operationally simplistic magnetic
manipulation system for integration with the SLIDE technology to overcome
the previously stated limitations. This magnetic system is compatible
with nearly any plate design, can be integrated into automated workflows,
enables high-throughput formats, simplifies mechanical requirements,
and is amenable to a range of analytes. Using this magnetic system,
PMPs can be collected, released, and resuspended throughout multiple
wells regardless of proximity. We demonstrate this system’s
capabilities to isolate whole cells, mRNA, and DNA, demonstrating
up to a 28-fold improvement of purity via the multiwash protocols
enabled by this magnetic technology
Data from: High specificity in circulating tumor cell identification is required for accurate evaluation of programmed death-ligand 1
Background: Expression of programmed-death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs) may be a less invasive method to assay tumor cells for these purposes. These liquid biopsies rely on accurate identification of CTCs from the diverse populations in the blood, where some tumor cells share characteristics with normal blood cells. While many blood cells can be excluded by their high expression of CD45, neutrophils and other immature myeloid subsets have low to absent expression of CD45 and also express PD-L1. Furthermore, cytokeratin is typically used to identify CTCs, but neutrophils may stain non-specifically for intracellular antibodies, including cytokeratin, thus preventing accurate evaluation of PD-L1 expression on tumor cells. This holds even greater significance when evaluating PD-L1 in epithelial cell adhesion molecule (EpCAM) positive and EpCAM negative CTCs (as in epithelial-mesenchymal transition (EMT)).
Methods: To evaluate the impact of CTC misidentification on PD-L1 evaluation, we utilized CD11b to identify myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP) technology.
Results: Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33–100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples.
Conclusions: Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria, thus improving the specificity of CTC identification and the accuracy of biomarker evaluation
Loading and operation of ESP devices.
<p>A) An unfilled device with each well labeled. B) Aqueous reagents are first added to the ESP device. C) Oil is added last, between each aqueous reagent. D) Manually operated devices are held by ridges located around the periphery of the ESP device to prevent contact between the reagents and the operator’s hand. E) Optionally, PMPs can be mixed within each wash well to enhance purity. F) After operation, the eluent is removed from the ESP device via pipette.</p
Results of the ESP comparison trial.
<p>A) Comparison of RNA extraction between ESP and gold standard protocols; B) Comparison of low cost RT-qPCR reagents with gold standard reagents on ESP-extracted RNA; C) Breakdown of total assay costs for ESP / low cost reagent protocol.</p
Raw Data
This file contains the raw data supporting all figures in the manuscript. Flow cytometry data is in FCS file format, and can be read with programs such as flowjo. JEX data is in arff format and can be read with the R application as described in the manuscript
HIV Viral RNA Extraction in Wax Immiscible Filtration Assisted by Surface Tension (IFAST) Devices
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