Flow Cytometry in Space, Part 1

0

To help mitigate risk to astronauts, spaceflight-compatible clinical laboratory instruments must be developed.

(Editor’s note: this is the first part of a two-part series. Click here to read part 2.)

Spaceflight can have adverse effects on the human body, including dysregulation of hematology and immunology parameters.1,2 Exploration class deep-space missions (Moon, Asteroids, Mars), with limited clinical care capability and elevated radiation levels, may result in significant clinical risk to crewmembers.

To help mitigate this risk, spaceflight-compatible clinical laboratory instruments must be developed. The International Space Station represents an excellent platform for such development work. Unfortunately, current on-orbit diagnostic laboratory capability is extremely limited. To date, only one blood analysis instrument was utilized during spaceflight, limited to basic clinical chemistry data. It is currently not possible to obtain even a simple white blood cell count during spaceflight. The capability to measure some hematology parameters have been identified as a medical requirement, prior to the initiation of deep-space missions.3

A flow cytometer is capable of providing a wide range of cellular analysis, and a spaceflight-compatible flow cytometer could potentially satisfy some unmet flight medical requirements. Unfortunately, standard commercially available flow cytometers are large, complex instruments that use high-energy lasers and require liters of liquid sheath fluid to operate.

They also generate a significant amount of liquid biohazardous waste and require significant training to operate. Standard cytometers use the fluid mechanical property of hydrodynamic focusing to place stained blood cells in single-file (laminar flow) as they pass through a laser beam for scanning and evaluation.

Many spaceflight experiments have demonstrated that fluid physics is dramatically altered in microgravity, and a previous NASA student experiment demonstrated that sheath-fluid based hydrodynamic focusing may also be altered during microgravity.4

For these reasons, it is likely that any spaceflight compatible design for a flow cytometer would abandon the sheath fluid requirement. The elimination of sheath fluid significantly reduces total system operational weight, including generation of much less liquid biohazardous waste. It would, however, require creation of laminar particle flow distinct from the standard sheath-fluid based method.

Prototype flight cytometer developed at the
NASA Johnson Space Center

A two color/three parameter (FSC, 2 PMT) commercially available cytometer was recently developed by Guava Technologies, which is extremely miniaturized and does not use sheath fluid. Although innovative, this instrument is not microgravity compatible.

The specific aims for the current development effort were to: (1) engineer a prototype flight cytometer based on the optics/fluidics of the commercial cytometer; (2) perform ground-based and microgravity validation of the prototype; (3) design and validate a set of medical assays compatible with the prototype; (4) design and validate a microgravity compatible cell staining device for sample processing that can interface with the prototype.

Prototype Cytometer Development

Initial evaluation of the cytometer core fluidics and optical resolution during reduced gravity confirmed that the design had the capability to acquire cytometry data during microgravity conditions.5

The unaltered commercial cytometer possessed a sample collection and waste containment design that was not microgravity compatible. Also, the instrument was too large and heavy to meet ISS launch and rack stowage requirements. Our laboratory developed a prototype spaceflight-compatible flow cytometer using the core of the commercial instrument.

All modifications centered on further miniaturization, enhanced robustness and altered fluidics. The unit was significantly reduced in size, electronic boards were relocated, unused internal volume was reduced, and the heavy steel casing was replaced with a lightweight rack-size aluminum casing.

For microgravity compatibility, the sample delivery method, formerly a glass straw that dipped into an open microfuge sample tube, was re-engineered to an external hard metal port via PEEK-based sample tube. The port was sized to interface directly with the NASA-developed microgravity-compatible cell staining device.

The laptop control system was replaced with a touchscreen-based computer control system. This makes the Prototype Flight Cytometer significantly easier to use during reduced gravity conditions. A vulnerability to launch vibration was identified in the commercial instrument optical path, which included two mirrors to guide the laser beam to the flow cell.

For the PFC, the mirrors were removed and the laser manually aligned and hard mounted to the optical bench directly in front of the flow cell.

Upon completion of all engineering changes, the final prototype was completed and is shown in figure 1. A diagrammatic summary of all engineering changes is presented in figure 2.

Prototype Flight Cytometer developed at the NASA Johnson Space Center. Whole blood samples may be processed during spaceflight using the Whole Blood Staining Device (foreground), and WBSD units interface directly with sample ports on the PFC front panel.

Diagrammatic representation of major engineering alterations, off the commercial cytometer core, to develop the Prototype Flight Cytometer.

Improved Whole Blood Staining Device, based on original WBSD design, but augmented to interface with the PFC instrument and enable absolute count determination. This platform may be modified to accommodate other cell sample staining protocols, including secreted analyte analysis.

Prototype Flight Cytometer validation during both ground and zero-gravity (flight) conditions. Representative two-parameter dot-plots for bead based linearity/optical precision (A, D), analysis of granulocytes, lymphocytes and monocytes (B, E), and T cell subsets (C, F) are shown.

Arrow
Arrow
1 - Prototype Flight Cytometer developed at the NASA Johnson Space Center. Whole blood samples may be processed during spaceflight using the Whole Blood Staining Device (foreground), and WBSD units interface directly with sample ports on the PFC front panel.
Slider

Staining Blood Samples in Microgravity

A working flow cytometer has little utility without a working method to stain and process blood leukocytes. The Whole Blood Staining Device (WBSD) was designed by NASA to stain peripheral white blood cells with fluorescent-labeled monoclonal antibodies, lyse the RBC population, and fix the stained WBCs.6

The WBSD was designed so that all steps could be performed in microgravity conditions. The original device consisted of a section of hard tygon tubing divided into chamber with clips, and an injection port fused to one end.

Basically, whole blood is loaded into one end, containing the monoclonal staining antibodies. Cells are then moved through the various steps by removing the clips that separate the reagent chambers. This WBSD configuration was evaluated for use with the PFC, however it was found that the hard tubing body created a negative pressure that skewed the absolute count measurements.

To mitigate this issue, a second-generation device (WBSD2) was created. The WBSD2 consists of a sterile 12.9×2.8cm teflon bag with an ultrasonically welded leur-lock port (American Fluoroseal) divided into chambers using plastic clips, and with an interlink injection site (Baxter) attached to one end (figure 3).

Operation is similar to the original WBSD.6 As the chambers of the WBSD2 inflate and deflate freely, it was found that use of the WBSD2 with the PFC gave perfectly acceptable absolute count data (data not shown). Absolute counts are possible using the PFC, since it does not dilute the cell sample in sheath fluid. Whole blood samples are delivered to the WBSD2 using a 100uL positive displacement pipette (Rainin) from EDTA monovettes (Sarstedt).

The plastic monovette is safer for microgravity operations and does not contain vacuum until primed before use. This significantly extends shelf life. Stability testing has verified that the WBSD-2 devices, loaded with all reagents and fluorescent antibodies, are stable for at least six months (data not shown).

References

  1. Sonnenfeld G, Shearer WT. Immune function during space flight. Nutrition 2002;18(10):899-903.
  2. Gueguinou N, Huin-Schohn C, Bascove M, et al. Could spaceflight-associated immune system weakening preclude the expansion of human presence beyond Earth’s orbit? J Leukoc Biol 2009;86:1027-38.
  3. Willians, R. NASA Space Flight Human System Standard Volume 1: Crew Health. Available at: https://standards.nasa.gov/documents/viewdoc/3315622/331562. Last accessed Aug 10, 2012.
  4. Crucian BE, Norman J, Brentz, J, Pietrzyk R, Sams CF: Laboratory outreach: student assessment of flow cytometer fluidics in zero-gravity. Laboratory Medicine 2000, 31:569-572.
  5. Crucian BE, Sams CF: Microgravity evaluation of a potential spaceflight-compatible flow cytometer. Cytometry, 2005:66(1):1-9.
  6. Sams CF, Crucian BE, Clift VL, Meinelt EM. Development of a whole blood staining device for use during space shuttle flights. Cytometry 1999;37(1):74-80.
Share.
// Uncomment below to display word count of article //

1156 words

About Author

Brian Crucian

Brian Crucian is an immunologist at the NASA Johnson Space Center Immunology Laboratory.

Heather Quiriarte

Heather Quiriarte is a laboratory technician at the NASA Johnson Space Center Immunology Laboratory.

Terry Guess

Terry Guess is a electro-mechanical engineer for the Wyle Advanced Projects Group, Houston.

Clarence Sams

Clarence Sams is the director at the NASA Johnson Space Center Immunology Laboratory.

Leave A Reply

Log in or register to comment on this article.