Development of a Flow Cytometer For Use During Spaceflight
(Editor's note: This is the second part of a two-part series. Part 1 is available here. The figures for this series are available here.)
Prototype Cytometer Validation
A two-color flow cytometry antibody matrix compatible with the laser/optics of the prototype instrument was developed to allow a leukocyte differential and the relative percentages of primary peripheral leukocyte/lymphocyte subsets to be assessed (table). We anticipate this panel may be expanded to accommodate any research need for which the appropriate labeled antibodies exist, as well as various intracellular or physiologic assays for which microgravity-compatible sample processing may be developed.
All microgravity evaluations of the prototype instrument were performed onboard NASA parabolic flight aircraft, which generate approximately forty 30-second intervals of microgravity by flying repeated high-angle parabolas. There are corresponding periods of 2x gravity between each parabola.
The PFC was found to function extremely well in zero, lunar and Mars gravity conditions (figure 4). During these flights, a complete blood-to-data validation was completed by performing all sample processing and instrument operations during only the zero gravity phases of flight.
Three individual users indicated the touch-screen instrument computer control was a significant improvement for reduced gravity operations. The lightweight portable and stabilized aspects of the instrument were validated, as for each flight the PFC was stored in the overhead aircraft storage bins prior to in-air deployment and utilization.
Click image above to view the larger table.
The ability to analyze soluble factors (plasma, culture supernatant) would be a beneficial additional capability for an on-orbit cytometer. As a representative cytometry-based platform for measuring soluble analytes, the Becton Dickinson Cytometric Bead Array (CBA) kit determines the concentration of cytokines in plasma or culture supernatants.
The assay uses a matrix of six same-size bead populations that differ in fluorescence along a single wavelength (corresponding with a cytometer PMT). Using a fluorescent capture antibody (using another cytometer PMT), a sample may be assayed for six secreted cytokines simultaneously. The feasibility of performing the CBA assay on the PFC with microgravity-compatible sample processing was evaluated.
Initial testing revealed the PFC could not successfully resolve all six bead populations; however three bead populations could be resolved in an acceptable fashion (figure 5). This does not mean that the assay is incompatible with the PFC, but only that with the current optical resolution there is a limit to the number of simultaneous accurate analysis that are possible.
For preparation of CBA samples, the terrestrial assay protocol requires multiple washes and centrifugations. As these are unlikely to be available during (or compatible with) spaceflight, the assay preparation required modification for reduced gravity conditions. The flexible WBSD2 platform was adapted for a microgravity-compatible CBA sample preparation.
A two-chambered 'WBSD-CBA' was created that would allow a modified secreted assay preparation during microgravity. The first chamber contained 50µL of cytokine capture beads and 50ul of PE detection reagent. The second chamber contained 1.0 mL of the wash buffer provided in the kit. Analysis samples were injected into the first chamber and incubated at room temperature for 3 hours.
Following incubation, the clip was removed and the sample mixed with buffer to dilute the unbound fluorescent reagents. No washing or centrifugation was performed before cytometric analysis. The primary drawback to this approach is that, due to inability to wash our free detection antibody, the background fluorescence is much higher. However, no wash cytometry is an accepted practice for selected high-expression surface markers.
The modified WBSD-based assay was still found to be workable despite the no-wash constraint. Demonstration of successful secreted cytokine analysis on the PFC instrument is presented in figure 5. Three distinct bead populations were resolved, and cytokine expression was readily detected.
The higher background is evident and may be adjusted for when assessing blanks and standards. There is an obvious compensation issue (spanning everything performed on the PFC, as it does not currently have compensation capability). It is likely that the next generation PFC would address this limitation. However, even in current form useful and accurate data was derived using the WBSD-CBA protocol and the PFC. An actual flight cytometer would incorporate additional capabilities (side scatter, fluorescence compensation, custom gating protocols) absent in the PFC that would greatly facilitate bead-based secreted molecule analysis. These capabilities are already available in similar second-generation flow cytometers.
Prototype Flight Cytometer validation during both
ground and zero-gravity (flight) conditions.
The Prototype Flight Cytometer (PFC) that functions in microgravity conditions was developed by the JSC Immunology Laboratory. The prototype is based on a recently developed commercial flow cytometer with significant additional engineering modifications.
The commercial instrument base possesses a novel flow cell design that creates single-particle laser scanning and evaluation without the need for sheath-fluid based hydrodynamic focusing. The final prototype cytometer is miniaturized and lightweight, uses a low energy diode laser, has a small number of moving parts and does not generate significant liquid waste.
The completed prototype functions operationally similar to a standard benchtop laboratory flow cytometer, aspirating liquid particle samples and generating histogram or dot-plot data in standard 'FCS' file format. The PFC is capable of a number of hematology and immunology assays, including WBC, differential, phenotyping, absolute counts and secreted molecule analysis. An associated microgravity-compatible sample processing hardware was also created that interfaces directly with the flight cytometer and is extremely flexible to adaptation for other assays.
The Prototype Flight Cytometer has successfully demonstrated that standard flow cytometry data may be achieved during microgravity conditions. Although other technologies may subsequently demonstrate similar capability (microfluidics, fiber optics, etc.), this effort has shown that standard cytometer technology may be adapted to the weight/power/fluid requirements for spaceflight.
Brian Crucian is an immunologist, Heather Quiriarte is a laboratory technician and Clarence Sams is the director at the NASA Johnson Space Center Immunology Laboratory. Terry Guess is a electro-mechanical engineer for the Wyle Advanced Projects Group, Houston.
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