Leukocytes respond to toxic, infectious, and inflammatory processes to defend tissues and eliminate disease process or toxic challenge. Accurate and prompt counting and differentiation of leukocytes is critical for diagnoses of infection, leukemia, or allergy; monitoring bone marrow function; or monitoring the body’s response to various treatments. White blood cell (WBC), or leukocyte, differential count is a clinical analysis that numerates the total number of leukocytes in per volume blood, and classifies leukocytes into different types, such as lymphocytes, monocytes, neutrophils, eosinophils, and basophils.

Presently, automated leukocyte differential count is conducted in hematology analyzers or in flow cytometers by utilizing differences in cell morphologies or cellular contents. The analysis of cell morphologies uses detection methods such as light scattering and electrical impedance of individual cells. Alternatively, leukocytes can be analyzed based on their cellular contents, which can be selectively stained by fluorescent dyes and detected by correspondent fluoresce emissions.

A novel reagent comprises a mixture of three fluorescent dyes: a primary cationic dye carrying two or more positive charges such as propidium iodide, a secondary cationic dye carrying one positive charge such as Basic Orange 21, and either an anionic dye or a derivative of an anionic dye such as fluorescein isothiocyanate. This dye reagent can be used to selectively stain nucleus, acidic cytoplasm, and protein contents of leukocytes, and classify different types of leukocytes.

When leukocytes are stained by the present dye reagent, the intensities of the leukocytes’ fluorescence, including fluorescence of the nucleus stained by the primary cationic dye such as propidium iodide, fluorescence of the cytoplasm stained by the secondary cationic dye such as Basic Orange 21, and fluorescence of the protein contents stained by the anionic dye or a derivative of an anionic dye such as fluorescein isothiocyanate, can be measured to classify different types of leukocytes.

By staining the blood sample with the present dye reagent and flowing the stained sample through a microfluidic cytometer, the fluorescence emission of each leukocyte cell can be individually measured. In this way, the total number of leukocyte cells can be enumerated, and different types of leukocytes can be classified to achieve the leukocyte differerential count.

Unlike commercial bulk instruments and previous efforts by other groups, the requirement for dilution was completely eliminated by specifically staining leukocytes. Sensing and differentiation of leukocytes from fluorescent-labeled undiluted blood greatly simplify the system design for handheld hemacytometers. Fluorescent dye and anticoagulant can be coated on the device wall so no mixing and storage of dilution buffer are required. Blood cell sedimentation is no longer a concern, thanks to the high throughput. Enough counts can be collected for one sample in seconds.

This work was done by Yu-ChongTai, Wendian Shi, Harvey Kasdan, Siyang Zheng, and Jeffrey Chun-Lin of Caltech; and Harvey Kasdan of International Remote Imaging Systems for Johnson Space Center. MSC-25107-1/024-1

NASA Tech Briefs Magazine

This article first appeared in the October, 2015 issue of NASA Tech Briefs Magazine.

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