Spaceflight blood diagnostics need innovation. Few demonstrations have been published illustrating in-flight, reduced-gravity health diagnostic technology. Here we present a method for construction and operation of a parabolic flight test rig for a prototype point-of-care flow-cytometry design, with components and preparation strategies adaptable to other setups.

The overall goal of this procedure is to operate a miniaturized flow cytometer on board a reduced gravity parabolic flight using components preparation and in-flight procedures potentially adaptable to other setups. This is accomplished by first carefully selecting off the shelf and custom fabricated components for ease of use and safety in reduced gravity. The second step is to assemble components within a parabolic flight test rig containing additional elements for containment, viewing automation and facilitation of multiple demonstrations.

Next, the team prepares for successful in-flight experimentation through meticulous planning, protocol development and training. The final step is multi-component demonstrations in flight. Ultimately, parabolic flight testing is used to showcase the potential space applications of technology and to identify the effects of weightlessness gravity changes and vibration on performance.

Although this method can be applied to flow cytometry and related technology, it can also be applied in parts to other types of individual diagnostic testing in reduced gravity, particularly anything with multiple demonstrations or trigger procedures. So we decided to do this video for JO on parabolic flight testing because when we were preparing for our parabolic flights with nasa, essentially there weren't that many videos or even literature that described how to actually best prepare for the experiments. We actually had to try very, very hard to talk to the right people at various locations within NASA to dig up that information Here.

In this case, we want to be able to share this information for the readership of Joe so that they can also prepare for these flights appropriately. Constructing a simple flow cytometry system for use in reduced gravity conditions requires multiple fluidics, optical and electronic prototype components. Start by preparing a pressure system with minimal weight and power needs to drive the system.

Fluidics connect a miniaturized air pump to a differential pressure sensor. Next, assemble a fluid source container that can be loaded without trapping air fit. A rigid plastic vial with an elastic rubber diaphragm, firmly securable cap and inlet air tubing at the vial base.

Seal the inlet air tubing connection using optical adhesive. Place a temporary slide clamp over the cap exit tubing to prevent fluid expulsion during and after cap insertion. To load the vial, expand the diaphragm with a syringe connected to the air inlet.

Pour in fluid to the top and insert the cap at an angle such that no air is trapped underneath. Briefly remove the slide clamp to prime the outlet tubing and release collapsing pressure exerted by the diaphragm. Ensure that the pump pressurizes the vial without air or fluid leaks.

Compressing the diaphragm to drive fluid flow out of the cap exit tubing. The third component needed is a fluid waste container to collect waste without building a back pressure that will compromise flow. Use a vial glued within a vial designed for double containment.

Cap the vials with a secured foam sponge window that traps floating liquids, but allows air pressure equalization with the cabin environment to make a sample loader for use and reduced gravity machine and assemble a spring loaded clamp design with guide rails such that it reliably clamps as sheath fitted capillary between two O-rings in the fluid line. Ensure that in the absence of a capillary, the springs press the O-rings together to complete the fluid line and enable priming without leaking. Design a micro mixer that does not rely on powered mechanical sub-components to function.

Using the rapid prototype Polymethyl soane method, a two inlet spiral vortex micro mixer is chosen and fabricated to detect individual flowing particles. Mount a custom fabricated palm sized miniature optical block to a microscope breadboard plate using commercially available optomechanical components. The final step in prototype assembly is to design electronics and software for device control and data acquisition.

For convenience and early prototyping, utilize hand soldered pieces connected to commercially available data acquisition cards, code and program, a custom software to operate rig devices and synchronize all data. Remove the laptop battery and set the laptop to operate through the power cable alone. For safety reasons on reduced gravity flights, the electrical power scheme for powering all devices must include a mechanism for quick and complete electronic shutdown.

In flight. A single power strip with a single on off button is connected to the aircraft power distribution panel for successful in-flight performance, the total space available and how it will be divided between experimental rig space and user space surrounding the rig must be considered. The total space available is limited to a smaller area than provided for a similar demonstration on the ground.

Determine which components are more appropriately accessed at a floor kneeling or standing height. It is also important to consider which components will benefit most from the protection attained within a support structure. The rig support structure here is a vertical equipment rack that can withstand flight accelerations and be securely attached to the intended aircraft.

Cabin floor assigned components to levels within the rack, A top level to place the laptop, a mid rack level to contain prototype sub components and a floor level to contain extra wipes, gloves, and a miscellaneous waste container. To secure and contain the prototype and to view samples, various non prototype components must be fabricated or adapted. These include a custom acrylic box to contain the electronics and a custom acrylic glove box with arm access holes to provide a cubic space in which to perform a loader demonstration without risking contamination of the flight cabin.

To record a micro mixer demonstration bolt, a stereo microscope to the breadboard plate fitted with a custom acrylic chip holder and CCD camera. To allow safe demonstration of the optical block, use a custom opaque acrylic box to block ambient light and control laser hazards. Some simple design strategies can eliminate the need for manual tubing, adjustments in flight or other actions that require significant dexterity.

For example, to pressurize multiple source files simultaneously use a custom machine to pressure manifold consisting of a hollowed out cylinder, adapted to an inlet needle and multiple outlet tubings for controlling direction of fluid flow. Using the computer, assemble a panel of three-way solenoid valves. Controlled by tandem MOSFET switches wired to a DAQ card.

The three-way solenoid valves have a common port that is always connected to either the default off port or on port. The switch to the on state is triggered with a five volt signal. Programmed the software to proceed through the demonstrations using single button interventions such as a single click on the laptop to switch valve states or change pump driving pressure.

This avoids the need for manual tubing adjustments that may incur leakages into the environment and the loss of experiment time in a chaotic environment. The sample loader demonstration includes loading a sample and driving the sample to the optical block or OB for detection. The setup utilizes two valves, one before, and one after the loader.

During loading, both valves are set to off preventing fluid movement as the loader is utilized, turning the valves on opens the fluid X pathway extending from the saline vial to the waste vial, allowing the pump to drive the sample for analysis. The optical block demonstration includes sequential detection of three different sample types Without needing to manually change tubing connections, saline is able to flush the system between samples. The micro mixer demonstration includes blood saline mixing and blue yellow dye mixing segments.

The setup uses two valves to guide pressure to either the blood and saline vials or the dye vials so that only one mixing demonstration is active at a time. An additional valve enables air bubble injection into the blood saline mixing chip. The system must be prepared for sudden jolting forces vibration or passenger collision in flight.

For alignment stabilization, apply quick drying epoxy to aligned components that are easily misadjusted, particularly the optical components. Apply industrial grade epoxy over the quick dry epoxy as well to secure other components as necessary, including the CCD camera attachment to the microscope I piece for physical disturbance testing. Shake the rig support structure with all components in place.

Check individual component functionality after subjecting the rig to the disturbance, particularly the aligned optical components trained for unexpected in-flight occurrences, including the plane suddenly leveling out in the middle of an experiment or sudden forces hitting the rig. Protect floating passengers by adding padding to the rack. Corners train multiple individuals as primary operators to expertly operate the device in flight.

It is unpredictable who will get sick during the parais and a given user may be unaffected on one flight and become sick on another. Check the rig after transport to the flight location, making any necessary fixes and setting tubing connections before loading onto the aircraft. On each flight day, prepare and hook in sample vials corresponding to the day's demonstrations.

Prepare for possibly long intervals between setup and experimentation as well as high ambient temperatures depending on flight location. Avoid sickness in flight by taking provided medications such as scopolamine with text amphetamine, and use several early parabola to adjust to the gravity transitions by rising slowly parallel with the floor and lying flat during high gravity. Once in flight position, rig operators when nearing dedicated parabola airspace provide enough space to allow rig operators to lie down during high gravitation intervals and enable access to leg straps once parabola begin.

Do not apply strong forces on the body during reduced gravity. As this may send the body up too quickly and somewhat dangerously to perform the sample loader demonstration. When the plane enters reduced gravity, use the sample syringe to place a drop of the counting bead dye mixture on a fingertip To simulate a finger prick sample, use a capillary consumable to pick up the sample off the finger and load the sample into the capillary loader.

Drive the sample into the optical system for detection. Perform the microfluidic mixer demonstration set up beneath the microscope. Mix blood and saline in a one-to-one ratio at 1.52345, and six PSI for at least two parabola each.

Recording video data synchronized to other readings. Actual in-flight footage of a mixer demonstration is shown here. Inject air into the saline inlet to test whether channel architecture will trap a bubble that could prevent optimal mixing.Mix.

Blue and yellow food dies at 1.52345 and six PSI for at least two parabola each. Again, recording synchronized data shown here are representative results for two micro mixer demonstrations as viewed by a CCD camera fitted to the stereo microscope panel. A shows blue and yellow dye mixing under microgravity conditions, and panel B shows blood and saline mixing under lunar gravity conditions.

Mixing can be visually assessed at any point along the spiral as well as in the exit channel in a demonstration of optical block detection of fluorescently labeled white blood cells during microgravity flight. The critical performance metrics for the flow cytometry data include the coefficient of variation of the peak intensities signal to noise ratios, peak counting rates, and detection efficiency as shown here. Optical block detection appears relatively unperturbed by a transition from approximately 1.5 G to nearly zero G and continues during the transition back to 1.5 g.

Detection of fluorescent counting beads spiked into a loaded sample following demonstration of the loader in lunar gravity indicates that the sample was successfully loaded and reached the optical block for detection. After watching this video, you should have a better understanding of how to perform device testing and reduce gravity on board a parabolic flight, and particularly what sorts of procedures are feasible, careful planning, part selection and testing implementation, all help to ensure a high yield from your experience.