Isolation of acute myeloid leukemia blasts from blood using a microfluidic device.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and associated with poor prognosis. Unfortunately, most of the patients that achieve clinical complete remission after the treatment will ultimately relapse due to the persistence of minimal residual disease (MRD), that is not measurable using conventional technologies in the clinic. Microfluidics is a potential tool to improve the diagnosis by providing early detection of MRD. Herein, different designs of microfluidic devices were developed to promote lateral and vertical mixing of cells in microchannels to increase the contact area of the cells of interest with the inner surface of the device. Possible interactions between the cells and the surface were studied using fluid simulations. For the isolation of leukemic blasts, a positive selection strategy was used, targeting the cells of interest using a panel of specific biomarkers expressed in immature and aberrant blasts. Finally, once the optimisation was complete, the best conditions were used to process patient samples for downstream analysis and benchmarking, including phenotypic and genetic characterisation. The potential of these microfluidic devices to isolate and detect AML blasts may be exploited for the monitoring of AML patients at different stages of the disease.

Evolution in Automatized Detection of Cells: Advances in Magnetic Microcytometers for Cancer Cells.

Flow cytometers are well-established tools with fundamental importance in biology and medicine to examine and identify cell populations, density, size distributions, compositions, and disease diagnosis and monitoring. Still, these devices are expensive with a low level of integration for sample preparation. Miniaturized microfluidic cytometers, i.e., microcytometers, for monitoring cells in a wide range of biological samples are currently being developed, providing more affordable and integrated solutions. Several detection methods have been developed and applied in microcytometers such as electrical, optical, and magnetic sensing techniques, which are integrated with microfluidic technology. Magnetic microcytometers present several advantages when compared to optical systems such as the fact that these devices provide more stable labeling by using magnetic nanoparticles (MNPs) or beads (MBs) instead of fluorophores. In this chapter, we explore the evolution of the automation of whole cell detection and enumeration that led to the development of microcytometers and particularly examine the anatomy of magnetic microcytometers applied to cancer research. We then give an overview of the challenges of Circulating Tumor Cells enrichment and enumeration, and the progress of magnetic microcytometers in this field.

Discriminating Epithelial to Mesenchymal Transition Phenotypes in Circulating Tumor Cells Isolated from Advanced Gastrointestinal Cancer Patients.

Gastrointestinal (GI) cancers constitute a group of highest morbidity worldwide, with colorectal cancer (CRC) and gastric cancer being among the most frequently diagnosed. The majority of gastrointestinal cancer patients already present metastasis by the time of diagnosis, which is widely associated with cancer-related death. Accumulating evidence suggests that epithelial-to-mesenchymal transition (EMT) in cancer promotes circulating tumor cell (CTCs) formation, which ultimately drives metastasis development. These cells have emerged as a fundamental tool for cancer diagnosis and monitoring, as they reflect tumor heterogeneity and the clonal evolution of cancer in real-time. In particular, EMT phenotypes are commonly associated with therapy resistance. Thus, capturing these CTCs is expected to reveal important clinical information. However, currently available CTC isolation approaches are suboptimal and are often targeted to capture epithelial CTCs, leading to the loss of EMT or mesenchymal CTCs. Here, we describe size-based CTCs isolation using the RUBYchip™, a label-free microfluidic device, aiming to detect EMT biomarkers in CTCs from whole blood samples of GI cancer patients. We found that, for most cases, the mesenchymal phenotype was predominant, and in fact a considerable fraction of isolated CTCs did not express epithelial markers. The RUBYchip™ can overcome the limitations of label-dependent technologies and improve the identification of CTC subpopulations that may be related to different clinical outcomes.

HER2 Expression in Circulating Tumour Cells Isolated from Metastatic Breast Cancer Patients Using a Size-Based Microfluidic Device.

HER2 is a prognostic and predictive biomarker in breast cancer, normally assessed in tumour biopsy and used to guide treatment choices. Circulating tumour cells (CTCs) escape the primary tumour and enter the bloodstream, exhibiting great metastatic potential and representing a real-time snapshot of the tumour burden. Liquid biopsy offers the unique opportunity for low invasive sampling in cancer patients and holds the potential to provide valuable information for the clinical management of cancer patients. This study assesses the performance of the RUBYchip™, a microfluidic system for CTC capture based on cell size and deformability, and compares it with the only FDA-approved technology for CTC enumeration, CellSearch®. After optimising device performance, 30 whole blood samples from metastatic breast cancer patients were processed with both technologies. The expression of HER2 was assessed in isolated CTCs and compared to tissue biopsy. Results show that the RUBYchipTM was able to isolate CTCs with higher efficiency than CellSearch®, up to 10 times more, averaging all samples. An accurate evaluation of different CTC subpopulations, including HER2+ CTCs, was provided. Liquid biopsy through the use of the RUBYchipTM in the clinic can overcome the limitations of histological testing and evaluate HER2 status in patients in real-time, helping to tailor treatment during disease evolution.

Enhanced magnetic microcytometer with 3D flow focusing for cell enumeration.

We report the design and characterization of a lateral and vertical hydrodynamic focusing feature for whole cell detection on a miniaturized flow cytometer. The developed system, based on magnetic sensing, incorporates spin valve sensors on the bottom of the microfluidic channels that detect cells labeled with magnetic beads. An adaptable 3D hydrodynamic focusing system was developed that pushes labeled cells towards the bottom of the microchannel, closer to the sensors, allowing increased signal amplitude for cells labeled with magnetic beads and enhanced discrimination of labeled cells. Fluorescence microscopy indicates that the lateral and vertical hydrodynamic focusing effect was adequately implemented, consistent with simulation predictions. The sensitivity of the system to detect labeled cells was improved by at least two-fold. By estimating the coverage of magnetic beads on cells, the signal from labeled cells could be predicted using a mathematical model, which also demonstrated the sensitivity of the signal to the height of the cells relative to the sensor. The system is versatile allowing interchangeable flow rates for cells with different diameters.