Single drop cytometry onboard the International Space Station

NASA plans for human missions to Mars necessitate long-duration spaceflight with significant health risks and limited opportunities for sample return and Earth-based diagnostics. Current reliance on downmassing biological samples introduces months-long delays and risks of degradation due to variable storage, vibration, and temperature during transport, limiting timely clinical decision-making and research insights. Flow cytometry offers broad analytical capability (e.g., blood counts, hormones, chemistry, nucleic acids, proteins, and multiplexed biomarkers) and high data density from minimal sample volumes, making it desirable for in-flight use. However, conventional cytometers are too large, power-hungry, require precise laser alignment, and are sensitive to air bubbles and maintenance—rendering them unsuitable for microgravity and rocket launch environments. The study aims to demonstrate that a highly miniaturized, alignment-free, low-resource flow cytometer (rHEALTH ONE), adapted for microgravity fluidics and EMI constraints, can perform single-drop (10 µL) cytometry on the ISS with performance comparable to ground-based systems.

Interest in space-capable cytometry dates back decades (e.g., Jett et al., 1985), recognizing potential applications spanning hematology, chemistry, enzymes, nucleic acids, proteins, and multiplexed biomarkers. Prior efforts include Microflow1 (CSA) on ISS, which demonstrated feasibility but lacked microgravity-compatible small-volume sample loading and omitted forward scatter channels due to fixed fiber-optic geometry. Additional work showed cytometry performance in reduced gravity using miniaturized and modified platforms (e.g., Guava-based systems, LED-based cytometers, and plastic chip/fiber optic cytometers), often employing sheathless approaches. The authors previously demonstrated early rHEALTH prototypes on parabolic flights, including in-line microvolume sample loading and microfluidic vortex mixing, as well as multiplexing using dyed microspheres and barcoded hydrogel microparticles/nanostrips enabling hundreds-to-thousands of simultaneous analytes. Standard hydrodynamic focusing with sheath flow remains advantageous for centering samples away from channel walls to improve uniformity and reduce fouling, but is challenging in microgravity due to bubble formation without buoyancy and demands fixed, ruggedized alignment to survive launch vibrations and g-loads.

Device and payload: The rHEALTH ONE is a 1.5 kg, 13.4 × 17.8 × 13.0 cm, 2.9 W, dual-laser (405 nm, 5 mW; 532 nm, 20 mW), five-detector silicon photomultiplier (SiPM) cytometer using sheath-flow hydrodynamic focusing. It employs an alignment-free optical module with all components epoxied in place, a fused silica rectangular flow cell with integrated lens and brass top for sheath/sample/burp connections, and photon counting at 10 µs bins across five channels (FSC, SSC, blue, green, orange). The optical module is cooled by a fan (required in microgravity due to lack of natural convection). Electronics include a SmartFusion SoC (FPGA) main board and a dsPIC33-based detector board enabling 10 µs binning and real-time USB streaming to a laptop running rHEALTH Capture and Viewer software. Spaceflight-specific modifications: A pressurized microgravity fluidics system used flexible medical-grade internal bags inside external bottles for sheath and cleaning fluids, pressurized at ~70 mbar (1 psig). Astronauts filled bags with filtered, cell culture-grade water and used a figure-eight maneuver to remove bubbles. Disposable waste was captured in a check-valved waste bag. The sample loader creates a defined fluid profile: a fluid-fluid leading interface with Poiseuille flow and a trailing intentional air bubble to ensure complete delivery of the 10 µL sample for absolute volumetric counts. EMI mitigation included internal copper tape shielding and an additional grounding cable; all joints and connections were ruggedized (zip ties, heat-shrink, RTV silicones, epoxy) for launch/return vibrations. Sample preparation and operations: Four blinded, safe (TOX 0) contrived bead-based samples were prepared and flown: (A) compensation beads labeled with anti-CD3 V500, anti-CD14 V450, anti-CD19 PE; (B) size standards mix (4, 6, 10, 15 µm); (C) three-intensity rainbow fluorescent standards (blue/green/orange); (D) Flow-Set Pro fluorospheres. All were bottled separately (1 mL) with blue dye for visual fill verification, stored at 2–8°C prelaunch, then transported/operated at ambient temperatures. On-orbit, astronauts dispensed droplets onto polyimide tape, wicked 10 µL into a capillary sample consumable by touch, and loaded it into the in-line zero-dead-volume sample loader. Pressure-driven flow at 70 mbar delivered samples through the optical block; cleaning water rinsed between runs. Data were streamed in real time and saved (TDMS and exported to FCS v3.1) for analysis (FlowJo, GraphPad). Experimental timeline and data acquisition: rHEALTH ONE flew on NG-17 (launched 2022-02-19) and was operated by ESA astronaut Samantha Cristoforetti on 2022-05-13 and 2022-05-16. Each run lasted ~2–3 min, producing ~71.55 million raw data points per run (10 µs bins). Preflight ground comparisons used a benchmark Gallios cytometer (multiple lasers; large mass/volume/power). Postflight, returned samples were re-analyzed on Gallios when an on-orbit anomaly was observed. Data analysis and statistics: rHEALTH Viewer peak-calling parameters were preset and kept constant for ground vs flight datasets. Histograms (log10 burst intensity), XY scatter plots, gating for singlets/doublets/triplets, percent robust coefficient of variance (%RCV), and linearity vs MEF were evaluated. At least triplicates (N ≥ 3) per sample type were run; two runs were excluded (one practice produced no data; one incomplete due to low sheath). Sample order was not randomized; no prior power calculation was performed.

• Microgravity single-drop cytometry: Successful cytometry from individual 10 µL sample drops on ISS with five simultaneous channels at 10 µs binning, yielding on average ~71.55–72 million raw data points per ~2 min run; real-time data visualization onboard. • Resource efficiency vs conventional cytometer (Gallios): 183× less volume (2,808 vs 513,650 cm³), 517× less power (2.9 vs 1,500 W), 92× less mass (1.5 vs 138 kg), and 166× smaller sheath reservoir (0.060 vs 10 L). • Throughput and reliability: 32 total runs over two days (4 blanks, 28 samples); 26/28 sample runs produced good-quality data across all channels; average run duration 143.10 s. • Optical and intensity performance (Flow-Set beads): Ground vs flight showed similar single-peak histograms across channels; fluorescent channel %RCVs differed by ≤2%; flight SSC and blue %RCVs were lower; FSC %RCV was 1.44% higher in flight with slightly elevated background; orange and SSC channels were brighter in flight by >+100 detected photons; raw counts per channel matched within <1% across ground and flight, indicating uniform detection of pan-fluorescent beads in all channels. • Fluorescence resolution and linearity (3-intensity rainbow standards): Distinct low/medium/high populations clearly resolved for blue, green, and orange channels; log10 burst intensity vs log10 MEF showed linear relationships both on ground and in flight; dim populations (25–164 MEFL) were fully resolved on orbit, demonstrating high sensitivity to dim events. • Size discrimination (4, 6, 10, 15 µm beads): FSC increased with particle size; clear gating of four bead populations in FSC vs SSC scatter; on-orbit data showed slightly less separation between 10 and 15 µm but better separation between 4 and 6 µm; Gallios corroborated increasing FSC trend with size. • Multicolor capability (compensation beads with anti-CD3 V500, anti-CD14 V450, anti-CD19 PE): Correct quadrant separations observed on ground. In flight, a new lower-intensity orange peak appeared, with increased orange-green and orange-blue coincidences; total bead counts and labeled/unlabeled fractions were similar to ground. Postflight Gallios analyses (after ~3 months ambient return) confirmed altered orange distribution, consistent with on-orbit findings, suggesting in-transit sample change/degradation. • Operational feasibility in microgravity: Pressurized bag-in-bottle sheath/cleaning system, microgravity-compatible wicking/sample loading, and EMI shielding enabled robust, alignment-free operation. Hydrodynamic focusing with water sheath was achieved at low pressure (70 mbar), minimizing fluid use (e.g., ~2.44 mL prime, ~1.13 mL per subsequent run; best-case).

The study demonstrates that a compact, ruggedized, alignment-free, sheath-flow cytometer can operate in microgravity using just 10 µL of sample per run, delivering multi-channel, high-temporal-resolution data comparable to ground-based systems. By addressing microgravity fluidics (bubble mitigation, pressurized bag system), EMI requirements, and fixed optical alignment, rHEALTH ONE overcame key barriers that preclude conventional cytometers from spaceflight use. The device resolved dim fluorescence populations, sized beads across multiple diameters, and multiplexed fluorophores, validating core analytical capabilities necessary for diverse biomarker, cell, and particle analyses. The on-orbit detection of altered orange fluorescence in compensation beads, later confirmed postflight, underscores the value of immediate in-flight analysis to identify sample degradation that typically remains unnoticed with delayed downmass workflows. Compared to prior ISS cytometry efforts (e.g., Microflow1), rHEALTH ONE adds sheath-flow hydrodynamic focusing and forward scatter, enabling standard cytometry performance characteristics in microgravity. These results support the feasibility of routine, in-flight cytometry for astronaut health monitoring and space biology research, eliminating delays and artifacts associated with sample return and enabling timely, actionable decision-making during exploration missions.

This work establishes the first demonstration of microvolume, single-drop, sheath-flow cytometry on the ISS using a highly miniaturized, low-power rHEALTH ONE device with fixed alignment optics, bubble-resilient microgravity fluidics, and EMI shielding. Performance on orbit largely matched ground results across fluorescence intensity, linearity, and size discrimination, while also detecting sample changes in-flight that were confirmed postflight. The instrument’s drastic reductions in mass, volume, power, and sheath consumption make routine deployment feasible for space missions. Future directions include: automating priming and fluid handling with pre-filled, gas-impermeable bags; further hardening optics to reduce FSC noise; expanding assays to biological samples (e.g., blood, urine) within NASA biosafety constraints and microgravity-compatible preparation; and enhancing onboard software for automated gating, thresholding, compensation, and clinical reporting. The approach is poised to support astronaut health diagnostics and spaceflight biology, and has analogous benefits for point-of-care testing in resource-limited terrestrial settings.

• Biological samples were not used on orbit due to biosafety constraints; performance was evaluated with contrived bead standards, limiting conclusions about cell-based assays in microgravity. • Fluid bag filling and bubble removal were operator-dependent and time-consuming; improper filling risks introducing bubbles that degrade data quality. • Manual priming via a burp port was required at startup; automation could improve usability and repeatability. • Slightly higher FSC noise on-orbit suggests minor mechanical shifts; more rigid optical/mechanical elements may further stabilize performance. • Limited crew time constrained total runs (two sessions over two days); practice and one incomplete run were excluded. • No randomization of run order and no a priori power calculation; sample sizes were designed for triplicates due to operational constraints. • Observed sample degradation in compensation beads highlights sensitivity to storage/transit conditions; on-orbit cold chain and radiation effects require further study.