We previously developed a Deterministic Lateral Displacement (DLD) microfluidic method in silicon to separate cells of various sizes from blood (Davis et al., Proc Natl Acad Sci 2006;103:14779-14784; Huang et al., Science 2004;304:987-990). Here, we present the reduction-to-practice of this technology with a commercially produced, high precision plastic microfluidic chip-based device designed for automated preparation of human leukocytes (white blood cells; WBCs) for flow cytometry, without centrifugation or manual handling of samples. After a human blood sample was incubated with fluorochrome-conjugated monoclonal antibodies (mAbs), the mixture was input to a DLD microfluidic chip (microchip) where it was driven through a micropost array designed to deflect WBCs via DLD on the basis of cell size from the Input flow stream into a buffer stream, thus separating WBCs and any larger cells from smaller cells and particles and washing them simultaneously. We developed a microfluidic cell processing protocol that recovered 88% (average) of input WBCs and removed 99.985% (average) of Input erythrocytes (red blood cells) and >99% of unbound mAb in 18 min (average). Flow cytometric evaluation of the microchip Product, with no further processing, lysis or centrifugation, revealed excellent forward and side light scattering and fluorescence characteristics of immunolabeled WBCs. These results indicate that cost-effective plastic DLD microchips can speed and automate leukocyte processing for high quality flow cytometry analysis, and suggest their utility for multiple other research and clinical applications involving enrichment or depletion of common or rare cell types from blood or tissue samples.
Reliable cell recovery and expansion are fundamental to the successful scale-up of chimeric antigen receptor (CAR) T cells or any therapeutic cell-manufacturing process. Here, we extend our previous work in whole blood by manufacturing a highly parallel deterministic lateral displacement (DLD) device incorporating diamond microposts and moving into processing, for the first time, apheresis blood products. This study demonstrates key metrics of cell recovery (80%) and platelet depletion (87%), and it shows that DLD T-cell preparations have high conversion to the T-central memory phenotype and expand well in culture, resulting in twofold greater central memory cells compared to Ficoll-Hypaque (Ficoll) and direct magnetic approaches. In addition, all samples processed by DLD converted to a majority T-central memory phenotype and did so with less variation, in stark contrast to Ficoll and direct magnetic prepared samples, which had partial conversion among all donors (<50%). This initial comparison of T-cell function infers that cells prepared via DLD may have a desirable bias, generating significant potential benefits for downstream cell processing. DLD processing provides a path to develop a simple closed system that can be automated while simultaneously addressing multiple steps when there is potential for human error, microbial contamination, and other current technical challenges associated with the manufacture of therapeutic cells.
We have developed a microfluidic chip-to-chip approach to purify circulating tumor cells (CTC’s). The first, a DLD (deterministic lateral displacement) microchip contains an array of microposts arranged in sub-arrays of different gap sizes. The “product” outlet of the DLD chip is connected with a second magnetic-separation chip. In the DLD chip, cells and particles are deflected or “bumped” based on their size, whereas the second chip is designed to remove any cell or component tagged with a magnetic particle (MNP). Air pressure is applied to maintain a constant flow of sample and buffer throughout the system in a vertical fashion. First, whole blood is mixed with biotinylated antibodies to CD45 and strepavidin-MNP's for 20min, diluted in buffer and passed through the DLD chip where the blood interacts with the posts and cells are bumped based on a deterministic lateral displacement principle, thus separating blood components. The “product” fraction from the DLD chip containing white blood cells and other larger cells is directed to the magnetic chip and the “waste” fraction containing red blood cells, platelets, debris and soluble blood components is discarded. The second chip has an array of magnets, capturing the sav-MNP/CD45+-tagged cells thereby allowing the gentle free flow of rare cells towards the final product fraction. With the chip-to-chip configuration we are able to process up to 8ml of blood, removing >99.8% of red blood cells and >98% of white blood cells, with the remaining purified cell populations suitable for further analysis and characterization. Validation of the chip-to-chip approach using tumor-derived cancer cells, including MDA-MB-231, PC-3 and SKBR-3 cells, confirms a linear recovery of >80% of the spiked-cells with a viability of ∼90%. The minimum cell size captured by our approach is approximately 6μm ensuring the isolation of small-sized CTC’s. Multi-color flow cytometry and imaging analysis of the chip-to-chip isolated cells from blood of breast cancer patients confirms the purification and identification of unique populations of HER2+/CD45-/EpCAM+ and CD146+/CD44+ cells characteristic of breast carcinomas-among other rare cell populations. Our chip-to-chip approach allows a gentle purification of intact and viable rare cells from cancer patients’ blood, suitable for functional analysis, drug sensitivity tests and genetic characterization. Thus, we have developed a hands-free liquid biopsy capable of isolating and purifying rare circulating tumor cells. Citation Format: Myra Koesdjojo, Zendra Lee, Christopher Dosier, Tanisha Saini, Khushroo Gandhi, Alison Skelley, Lee Aurich, Gregory Yang, Tony Ward, Roberto Campos-González. DLD microfluidic purification and characterization of intact and viable circulating tumor cells in peripheral blood. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3956.
when considering the admissibility and probity of voice recording evidence. This is particularly the case when the court is being asked to consider the factors set out in paragraph 16 of Flynn in relation to 'lay listener' evidence. Although Kapikanya did not involve 'lay listener' evidence, it is a good example of compelling voice recording evidence. It was admitted despite the absence of formal witness evidence purporting to identify the voice. This case involved good-quality recording of speech, of a nature and duration to be sufficient for comparison, with no gap in time between the listener hearing the recording and the individual that the voice on the recording was being attributed to. R v Kapikanya could, therefore, be a useful authority for arguing that a voice recording ought to be admitted, as it demonstrates the kind of characteristics that may justify admitting it. This may be the case if either expert or 'lay listener' evidence identifying the voice were to be adduced as well as in circumstances when the jury are simply being invited to compare the recording to the person they have heard give evidence in the witness box.
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