|| Medical events happened frequently to astronauts in space. For example, even the Space Shuttle Program alone reported 1867 incidences 1981-1998. Moreover, some events were serious viral/bacterial diseases such as urinary tract, conjunctivitis, acute respiratory, dental, and Varicella Zoster virus infections. Ideally, treatment on astronauts should be based on precise medical information. Meanwhile, blood is one of the most important body fluids related to health and there's tremendous information in blood. Blood analysis, if possible, should be the first important step of health monitoring for sick and healthy astronauts. Blood analysis can also be a powerful technique to monitor bone loss and radiation effects. Therefore, NASA should have an in-space, real-time blood analysis capability. However, NASA still doesn't have blood analysis capability other than blood gas and electrolyte analysis. This proposal is specifically to develop an in-box blood analysis technology for NASA. As a whole, we believe that the lab-on-a-chip technology is the best choice for multiple blood analysis in space. Therefore, our long-term objective is to develop blood analysis in-a-box using lab-on-a-chip technology specifically for space applications, emphasizing small form factor, lightweight, and autonomous operation to accommodate Crew Exploration Vehicle (CEV) and International Space Station (ISS) size requirement for medical kits.
The specific aims for this project period are to develop space technologies for (a) 5-part WBC (white blood cell) differential, (b) analysis of WBC subtypes (e.g., CD4+ T helper and natural killer cells). Our approach to achieve the goal is to develop the capability of minimally diluted micro flow cytometer to enable a comprehensive WBC differential, and allow detection of fluorescent labels attached to ligands used for cell surface marker for WBC subtype analysis. Embedded in the two specific aims is a research component on the data analysis software. This software has been developed in Matlab to facilitate both quantitative assessment of fluorescence detection and cell and analyte recognition and quantitation.
For the last funding years, we worked extensively on searching for a new staining method and optimizing the previously proposed Acridine Orange staining. We successfully developed a series of assays including a 4-part differential assay (i.e., Lymphocyte, Monocyte, Neutrophil, and Eosinophil) with a cocktail staining of fluorescent dyes fluorescein isothiocyanate (FITC) and propidium iodide (PI), a 5-part differential assay (i.e., Lymphocyte, Monocyte, Neutrophil, Eosinophil, and Basophil) with a cocktail staining of fluorescent dyes fluorescein isothiocyanate (FITC), propidium iodide (PI), and Basic Orange 21, and a specific assay for the rare cell type basophil differential using fluorescent dye Basic Orange 21. The differential assays were investigated in a correlation study with the commercial hematology analyzer, and further verified with the purified WBC types. For the Acridine Orange assay, the differential capability is also extended from 2-part (Lymphocyte and Neutrophil) into 4-part (Lymphocyte, Monocyte, Neutrophil, and Eosinophil). The time and temperature dependence of the Acridine Orange staining are also investigated.
Within the project period, we have also explored the possibility of improving the (box) platform in terms of spectroscopic detection. Two different approaches have been implemented; one uses a commercial mini-spectrometer and the other approach uses a 8-channel PMT (photomultiplier) module. Measurement of fluorescent emission spectrum from blood cells stained with the dye assay has been successfully demonstrated on the spectroscopic approach. Single cell fluorescence emission spectrum has been measured on the mini-spectrometer prototype. Distinct spectrums were measured from lymphocyte, neutrophil, and eosinophil cells. Besides, multicolor fluorescent beads have been successfully measured on the 8-color reader. Those two approaches enable detection of multiple fluorophore simultaneously from WBC subtype immune-staining. The additional spectral information should provide better discrimination between multiple fluorophores used simultaneously. It may also provide additional information about the intracellular environment in which Acridine Orange fluorescence occurs, leading to efficient WBC subtype discrimination. For WBC subtypes analysis, we have also successfully demonstrated assays that identified and counted CD4 and CD8 WBCs. In addition, we also developed synthesized peptides specifically targeted for leukocyte cells. The binding peptides were custom-synthesized with a fluorescein fluorophore attached to their n-terminus for binding quantification. A library of 72 potential peptide candidates has been tested using a modified protocol for leukocytes utilized in our studies. Among the results, 4 peptides from our initial library exhibited a 2-3x higher binding strength to the B-cells compared to the other peptides, which confirmed the capability of this approach for WBC subtype analysis.
|Research Impact/Earth Benefits:
|| This project developed assays and instrumentation (i.e., hardwares, and softwares) that provide new ways of WBC count and subtype analysis. This project also proved that these new methods are as good as, if not better, as currently available commercial methods on Earth. Therefore, for the first time, this project provided the capability for NASA to do blood cell analysis in space, although further improvement needs to be done over our prototype for space qualification. In addition, both the developed assays and instrument can be used on Earth, too, and the technology has been licensed to a company, i.e., LeukoDx Inc., for the development of a point of care sepsis monitoring system initially targeted for the detection and monitoring of neonatal sepsis.