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Clog Resistance of non-Pressure Based Flow Cytometers By Greg Kaduchak, PhD 10.25.2017 Flow cell clogs have been a long standing issue in flow cytometry. The small dimensions of the flow cell and fluidic path are susceptible to clogs especially when using larger or ‘sticky’ cells. In addition, historically, flow cytometer systems have been pressure-based which compounds this issue even more. In pressure-based systems, the particles are transported through the system by applying pressure to the fluid. It is a straightforward method to move the fluids through the small channels. To ensure a smooth delivery of fluids and particles through the flow cell without fluctuations, the systems employ pressure regulators. For those that have used these systems, it is a proven design to deliver particles in a flow cytometer and has been successful over the years. But, in the event of a clog, there is not much these systems can do. Figure 1(a) and (b) show what happens when a clog is encountered in a pressure-based fluidic system. When the system is in normal operation (a), the fluid is pushed through the system a specified pressure. For this example, we have used 7 psi. But, as seen in (b), when a clog is encountered the regulator keeps the system at 7 psi. No additional pressure is exerted to move the clog through the flow cell and the flow stops. Figure 1 In contrast, in systems that employ positive displacement to drive the fluidic system (e.g. syringe pumps), the pressure is not held constant. These systems operate by a principle of constant volumetric flow. They are designed for fluid to flow with a specified volume delivery rate regardless of the pressure. An example of such a system facing a potential clog is shown in Figs. 1(c) and (d). As seen, the system operates at the same pressure as the pressure-based system when all is fine. But, once a clog is encountered, the system will build pressure to maintain the volumetric delivery rate. Pressure will build until the clog is displaced. The fluidic system in the Attune NxT Acoustic Focusing Flow Cytometers is based on positive displacement fluid delivery. For the purpose of robust clog removal, the system is outfit with a sensor that monitors the system pressure. When a potential clog is encountered, the pressure is allowed to build all the way up to 60 psi before safely shutting down the system. An additional benefit is used by the Attune NxT Flow Cytometer to keep the flow cell clean: a rinse cycle automatically runs between samples, this clears the sample in the flow cell with excess sheath fluid to prevent any cellular buildup. This feature has made the Attune flow cytometer platforms extremely clog resistant. Its install base has grown considerably since its initial launch more than two years ago, but still only a few clogs have been encountered by users of properly maintained instruments. Due to this resistance to clog, positive displacement systems are great from applications where cells are large and sticky, especially for tissue-based samples.
  • 2018-02-16T01:37:29

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Gene editing can help treat congenital disease before birth Updated Oct 09, 2018 | 20:02 IST | IANSPrenatal treatment could open a door to disease prevention, for HT1 and potentially for other congenital disorders. Representational image Photo Credit: ThinkstockRepresentational Image New York: In a first, a team of scientists have performed prenatal gene editing to prevent a lethal metabolic disorder in laboratory mice, offering the potential to treat human congenital diseases before birth. The study led by research from Children's Hospital of Philadelphia (CHOP) and the University of Pennsylvania used both CRISPR-Cas9 and base editor 3 (BE3) gene-editing tools and reduced cholesterol levels in healthy mice treated in utero by targeting a gene that regulates those levels. They also used prenatal gene editing to improve liver function and prevent neonatal death in a subgroup of mice that had been engineered with a mutation causing the lethal liver disease hereditary tyrosinemia type 1 (HT1). Advertising Advertising HT1 in humans usually appears during infancy, and it is often treatable with a medicine called nitisinone and a strict diet. However, when treatments fail, patients are at risk of liver failure or liver cancer. Prenatal treatment could open a door to disease prevention, for HT1 and potentially for other congenital disorders. "Our ultimate goal is to translate the approach used in these proof-of-concept studies to treat severe diseases diagnosed early in pregnancy, " said William H. Peranteau, a paediatric and foetal surgeon at CHOP. "We hope to broaden this strategy to intervene prenatally in congenital diseases that currently have no effective treatment for most patients, and result in death or severe complications in infants, " he added. In the study, published in the journal Nature Medicine, the team used BE3, joined it with a modified CRISPR-associated protein 9. After birth, the mice carried stable amounts of edited liver cells for up to three months after the prenatal treatment, with no evidence of unwanted, off-target editing at other DNA sites. In the subgroup of the mice bio-engineered to model HT1, BE3 improved liver function and preserved survival. However, "a significant amount of work needs to be done before prenatal gene editing can be translated to the clinic, including investigations into more clinically relevant delivery mechanisms and ensuring the safety of this approach", said Peranteau. He added: "Nonetheless, we are excited about the potential of this approach to treat genetic diseases of the liver and other organs for which few therapeutic options exist."
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