Attune NxT Demo Video

“Traditional antibacterial sensitivity testing can take 1–3 days depending on how difficult the bacteria are to grow, isolate, and then test for sensitivity. The Attune NxT allows us, within 1 hour, to tell whether or not the drug will effectively treat the infection. Within 3 hours, accurately assay the minimum drug concentration needed to inhibit bacterial growth.”

Kieran Mulroney

Biomedical Sciences Researcher

Harry Perkins Medical Research Institute

University of Western Australia


Acoustic focusing for sensitivity

The Attune NxT Flow Cytometer combines ultrasonic waves like those used in medical imaging with hydrodynamic forces to precisely position cells into a single, focused line in the central axis. Enabling cells to be tightly focused at the point of laser interrogation allows the system to collect more photons, helping to ensure data quality regardless of the sample-to-sheath ratio.

Acoustic focusing

Acoustic focusing (left) precisely positions cells in tight alignment even at higher sample rates resulting in less signal variation and better data quality. Traditional hydrodynamic focusing (right) widens the sample stream core at high sample rates that results in signal variation and compromised data quality.

“When all is said and done, it outperforms [the rest] in that it can analyze 35,000 cells per second with acoustic focusing technology. Its accuracy is not diminished even if you conduct analysis at a high flow rate of 100–1,000 µL/min, which drastically shortens the time required for an experiment.”

Dr. Takashi Satoh

IFReC Assistant Professor

Osaka University, Japan


Fast accurate acquisition

The Attune NxT system’s acoustic focusing also lets your lab rapidly acquire high-quality data. You can achieve sample throughput rates of 12.5 µL/min or 1,000 µL/min up 10 times faster than traditional hydrodynamic focusing systems and acquisition speeds of 35,000 events/sec. This means you can process all of your samples—including low-concentration and precious samples—more quickly and accurately with minimal loss in quality.

Rapid data acquisition

 

Compares the time required to acquire 1,000,000 events over three competitor instruments running at maximum sample rates.

Acoustic focusing minimizes variation regardless of the sample rate, so you don't have to make the tradeoff between throughput and sensitivity. This is demonstrated in the cell cycle analysis example below, where it's critical to precisely detect differences in fluorescence intensity between multiple cell populations.

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Minimal data variation

 

Consistent results were achieved at high sample rates using Jurkat cells fixed and stained with propidium iodide, treated with RNase and analyzed at a concentration of 1 x 10 cells/mL. The coefficient of variation (CV) of cells in the G0/G1 and G2/M phases remain consistent, even at the highest sample rate of 1,000 μL/min.


Handling rare events

Detection of rare events requires acquisition of high numbers of cells to attain a reliable measure of accuracy. The Attune NxT cytometer allows dilute samples to be processed quickly at sample input speeds of up to 1 mL/min, significantly faster than conventional cytometers that support maximum sample input rates of 60–100 µL/min.  Acoustic focusing thus offers a unique combination of speed and quality, cutting the time to collect rare events significantly over long acquisition times.

 

 

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Collecting more than 1 million live cells and detecting a rare population of dendritic cells

 

Plasmacytoid dendritic cells (pDCs) are identified using immunophenotype CD19–/B220high/CD317+. Four-color staining of mouse splenocytes included CD19-Pacific Blue, CD317-Alexa Fluor 488, CD45R/B220-PE direct conjugates, and SYTOX AADvanced Dead Cell Stain. A gate was made on live cells using SYTOX AADvanced Dead Cell Stain, followed by gating on CD19– cells. A two-parameter plot of CD45R/B220 vs. CD317 was used to identify pDCs. A collection rate of 500 μL/min was used to acquire 1.3 million total cells with a cell concentration of 7.5 x 10  cells/mL. Plasmacytoid dendritic cells were identified as dual B220+/CD317+ (upper right quadrant) and constitute 0.851% of live CD19– cells, which is 0.194% of total splenocytes.

The Attune NxT Flow Cytometer supports both high speed of acquisition and high sensitivity to allow easy detection and phenotypic and functional characterization of rare cells. This step-by-step strategy for detecting rare events in our Flow Cytometry Learning Center will help you think through the best approach for pre-analytical, analytical, and data analysis phases of your research.

A design for flexibility

The Attune NxT Flow Cytometer is configurable with up to 4 spatially separated lasers and 16 parameters. Spatial separation provides flexibility for multicolor panel design and streamlines compensation.  The system offers superior speed with acquisition rates of up to 35,000 events per/second with high sensitivity to meet a range of research requirements.

 

High sensitivity distinguishes between dim signals and background, resulting in less variation and better signal separation. Fluorescent resolution coefficient variation is less than 3% for a single peak, and predicted MESF is ≤80 (FITC), ≤30 (PE), ≤70 (APC). Sensitivity in comparison to competitive systems is described below.

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Sensitivity measurements across flow rates.

 

Fluorescent microspheres (Spherotech Rainbow 3.2 μm) were run on a high-end conventional flow cytometer (A) and on the Attune NxT Flow Cytometer (B and C) using a 561 nm laser and 610/20 (A) or 610/15 (B and C) emission filters. The conventional cytometer was run using the highest sensitivity setting (~12.5 μL/min). The Attune NxT Flow Cytometer was run at 12.5 μL/min (B), which is equivalent to the traditional flow cytometer and 500 μL/min (C; 40x more sample). The Attune NxT Flow Cytometer results were equal to or better than those from the conventional flow cytometer, even at the highest flow rate.


Novel optical design

The Attune NxT features a novel optical design that delivers first-class reliability and superior performance over time. The flat-top beam profile in the solid-state lasers minimizes the effects of changes in fluidics or optics, which in turn can lead to instability or alignment issues and instrument downtime.

 

Laser misalignment is a major concern with users of conventional flow cytometers. The flat-top lasers used in the Attune NxT Flow Cytometer have an intensity profile that allows a wider window of alignment over Gaussian lasers used in traditional systems.  The flat-top lasers also have a higher tolerance for misalignment that allows them to maintain high sensitivity and low CVs.

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Emission profile of lasers 

 

Gaussian laser used in traditional cytometers (left) with misalignment and flat-top laser used in the Attune Nxt (right) showing proper alignment if shifted.


Clog-resistant technology

 

To prevent clogging and allow for volumetric analysis, the Attune NxT Flow Cytometer uses a positive displacement syringe pump to control sample volume. The system can perform volumetric cell counts in a known volume (gated or total events) and can easily gate out dead cells to count live cells only in a live/dead analysis. Unlike traditional cytometers that operate at a maximum pressure of 15 PSI, the Attune NxT Flow Cytometer controls pressure at 75 PSI, reducing the likelihood of cell-cell interactions and resulting in clog resistance.

 

“One of the problems that everyone is familiar with who works in flow cytometry is clogging. Clogging is a thing of the past with this instrument. You have so many samples that you have to run, you can’t wait between samples to clear everything out.”

Bruno Sainz

CLIP Investigator

Universidad Autónoma de Madrid

Madrid, Spain

Volumetric sample entry 

 

Using positive displacement, the syringe pump works like a pipette. Tension is created with the plunger pushing down (A). The tension is released as the plunger rises with the sample (B). Clogging is less likely to occur under this high and controlled pressure environment.

“We needed to have a flow cytometer that would allow us to take samples from tumors that had tended to clog other machines. Therefore the Attune NxT is the flow cytometer of choice for our application. The Attune NxT has a number of characteristics that are critical for our research… program. Not only does it have acoustic focusing, which will allow us to go at higher sample throughput, but it also has a larger flow cell, so when we’re looking at tumor cells and isolating cells from those tumors we don’t have to worry about clogging and having to spend a lot of downtime getting the instrument up and running again.”

Charles Prussak

PharmD, PhD

Director of the Cell Therapy Translational Laboratory (CTTL)

University of California San Diego


Software

The Attune NxT Software is designed to provide powerful data acquisition and analysis using an intuitive user-friendly interface. Experiments can easily be set up, customized, and saved for future studies. Compensation is automated and can be set up using a guide. The software is designed to maximize efficiency in performing data analysis, with fast refresh rates for large data sets (up to 20 million events per sample) with the ability to immediately visualize changes on data plots as you make adjustments

 

The software has unique tools to simplify experimental setup, including reagent selection using the filter configuration manager. This provides guidance for matching the right reagent to the optimized channel on the instrument by selecting reagents from a drop-down menu of prepopulated or customized reagents, which is then applied to plot labels.

Intuitive software interface. 

 

The user interface is divided into four panels. Ribbons and tabs in the top panel enable easy selection of key functions. Collection Panel is where you can easily set parameters to acquire data and see acquisition status. The Experiment Workspace is where you can preview your panel and choose from a variety of plot types and a range of statistics to illustrate your analysis.  This workspace is also where you can set Smart gate labeling. The Experiment Explorer makes it easy to manage samples and data. This panel is where you can set up batch processing, and instrument and compensation settings to new experiments.

“The Attune NxT software is very user friendly and intuitive, significantly reducing training time required to get new users up-and-running.”

James Hemphill

Ph.D

Senior Scientist

Kytopen

Software features

 

Core capabilities

  • Compensation—fully automated and manual compensation
  • Instrument tracking—automated baseline and performance test for all channels and linearity
  • Traceability—21 CFR part 11 for security, auditing, and electronic signature capabilities
  • File formats—FCS 3.0 and FCS 3.1

 

Maintenance

  • Automated maintenance—startup (<15 min), shutdown, rinse, wash, debubble, deep clean, monthly decontamination
  • User account maintenance—administrative and individual accounts with user log

 

Visualization

  • Gates—standard and customizable gates
  • Plot previews—easy workspace setup
  • Zoom filmstrip viewer—easy visualization and navigation of plots to set gates and plot attributes
  • Graphics resolution—publication-quality images (customizable)

 

Compensation Tools

Compensation is both rapid and accurate using a guided software system.  The system adjusts the compensation and applies it to the samples so that you only need to prepare the proper controls and adjust the voltage gates.

 

Automated compensation supports both negative and unstained parameters and allow you to setup and collect compensation controls directly from a plate if needed. This automation eliminates trial and error and delivers compensation rapidly and accurately. In addition, on-plot compensation controls allow for fine-tune adjustments, and compensation can be subsequently modified should the need arise to add or remove parameters after compensation is set up.

 

 

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(1) Negative gating is useful for heterogeneous samples that are often found in tumor and blood samples. (2) Choose fluorophore-labeled antibodies based on the emitted emission spectrum. Dyes should have minimally overlapping spectra. (3) Prepare single-stained controls. An unstained control is not required. (4) Fluorescent compensation beads can also be used to set compensation parameters when there is not enough sample. (5) Use the guided system and select for negative gate. (6) Run sample and adjust the voltages to see the fluorescent label on the plot. Press Apply Compensation

Attune Nxt Software version 4.2

Version 4.2 of Attune Nxt Software is designed for Windows 10 operating system and offers improved DPI support for high resolution monitors. Other improvements include expanded run protocol settings that allow you to manage overall experiment vs sample-level settings.  Additional capabilities include maintenance reminders for system decontamination and post-acquisition rinse that streamline workflow. This software version also enables Windows 10 support for CytKick and CytKick Max autosamplers.

 

Importantly, Attune Nxt Software version 4.2 supports access to optional 21 CFR part 11 software for security, auditing, and electronic signatures in conjunction with the Applied Biosystems SAE server.

 

The Attune Nxt Software 4.2 upgrade kit (Cat. No. A41324) and service installation (Cat. No. A46758) are required prior to installation of the version 4.2 software.

Optional 21CFR Part 11 Software 

The FDA released the Electronic Records and Signatures Rule, known as 21 CFR Part 11 in August 1997.* This rule defines the requirements for use of electronic documents in place of paper documents. The law specifies the system elements, controls, and procedures that are necessary to ensure the reliability of electronically stored records.

 

This software license for the Attune NxT acoustic focusing cytometer supports compliance with 21 CFR Part 11 FDA guidelines for Security, Auditing, and Electronic Signatures using a SAE console. The Attune 21 CFR software is available for use only on Windows 10 computers.

 

* https://www.fda.gov/regulatory-information/search-fda-guidance-documents/part-11-electronic-records-electronic-signatures-scope-and-application#i (accessed February 27, 2020)

 

Software enables administrator to restrict access to authorized personnel via user ID and password. The software allows the administrator to set expiration duration for both passwords and user IDs. In addition, the system will logout users after established duration of inactivity. 
The software requires ID and password must be re-entered to sign or modify a record. Event notification records and reports any ‘forgery’ attempts in administrator dashboard automatically.
 
The software lets the administrator set access and manipulation controls at a granular level over  various software functions and options using user role definitions.  

Auditing features track actions performed by users and changes to the SAE module settings. The SAE module automatically audits some actions silently. This allows the administrator to audit specific user actions and specify the audit mode, as well as generate reports of audited user actions, SEE module changes, software, and instrument activity.

 

An electronic signature determines if users are required to fulfill signature requirements before performing specific functions. The software allows adminstrators to configure an e-signature so that a given user can start a run only if the associated data are signed. In addition, settings also allow for an e-signature to be set so that multiple signatures are required and that only specified roles can sign.

 

 
A Sign Record feature allows record may be signed on demand. An e-signature sign record is prepared by creating a PDF preview.
Subsequently the Sign Report is prepared for signature. The experiment is signed and user is notified that subsequent edits will obsolesce the e-signature.
An e-signature sign record is prepared by creating a PDF preview. Request Signature allows end user to request an e-signature
An e-signature sign record is prepared by creating a PDF preview. Approver is notified of e-signature request at login and may view pending signatures. Approver may sign report and designate the reasons for signature.

Additional software licenses 

For research use only, not intended for use in diagnostic procedures.