Four-part leukocyte differential count based on sheathless microflow cytometer and fluorescent dye assay

Abstract

Leukocyte differential count is one of the most frequently ordered clinical tests in hospitals. This paper reports a point-of-care test for the leukocyte count by using a microflow cytometer and a fluorescent dye assay. The dye assay relied on fluorescent detection alone to count leukocytes in blood and to identify leukocyte subtypes. By combining the fluorescent assay with a sheathless microflow design, the proposed method achieved a minimal sample volume by eliminating excessive dilution and sheath flow. In this paper, a four-part leukocyte differential count including lymphocyte, monocyte, neutrophil and eosinophil was demonstrated, and the whole test consumed only a small amount of blood (5 μL) and reagents (68 μL in total). The merits of minimal sample volume, long reagent shelf life and portable instrument made this method optimal for point-of-care applications.

Considerations for point-of-care diagnostics: evaluation of acridine orange staining and postprocessing methods for a three-part leukocyte differential test

“…In general, the method described here is specific to our image-based POC system and AO colorimetric classification; however, this could be extrapolated to other red to green colorimetric POC tests, for instance, image-based methods implementing color cameras and nonimage-based methods such as POC flow cytometry methods implementing microfluidics and fluorescence signal detection using photomultiplier tubes. 7,13 Additionally, future work could incorporate a five-part differential test to explore whether it can be accurately quantified using AO.…”

“…1 For this device and other similar devices, miniaturizing the system using the combination of an impedance technique (Coulter principle) and microfluidics (microchannels) is a common approach to reduce cost and complexity by simply minimizing optical components and reagents. 10,13,21,22 Some disadvantages to impedance-based measurements include electrolysis, gas formation, and the need for red blood cell (RBC) lysis and dilution reagents, all of which add design complexity and, potentially, operational cost. 10,[23][24][25] Alternatively, there are many different types of image-based methods that have become popular due to their reduced size and cost from the development of low-cost plastic optics, light-emitting diodes (LEDs), and other new optical technologies.…”

“…dimensions, a cellphone adaptor weighing ∼18 g) and have the advantage of providing test results at or near a patient. [7][8][9][10][11][12][13] In the United States, a Clinical Laboratory Improvement Amendments (CLIA)-waived test classification is required to use these devices in a clinical setting. It defines the test accuracy of a leukocyte differential to be within AE15% of a clinical hematology analyzer while being simple enough for an inexperienced user to operate.…”

Homogeneous agglutination assay based on micro-chip sheathless flow cytometry

“…A microflow cytometer without particle focusing has been reported for fluorescent dye assay. 21 This sheathless microflow cytometer only utilized fluorescence from target analytes and did not use scattered light, which could ignore the scattered light from the microchannel walls. By narrowing the microchannel down to the size fairly smaller than the laser spot, the light intensity of laser spot and induced fluorescence in the interrogation region was quite uniform.…”

Reduced-gravity Environment Hardware Demonstrations of a Prototype Miniaturized Flow Cytometer and Companion Microfluidic Mixing Technology

“…Flow cytometry is one attractive approach for in-space POC because of the broad utility of the technology, including toward cell counting and biomarker quantification, as well as significant miniaturization potential. Previous space-relevant flow cytometers include the 'nuclear packing efficiency' (NPE) instrument that utilized simultaneous arc-lamp induced fluorescence and electronic volume (Coulter volume) measurement [1][2][3][4] , a relatively small benchtop flow cytometer representing the 'first generation of real-time flow cytometry data during zero gravity' 5 , a 'sheathless microflow cytometer' capable of 4-and 5-part white blood cell (WBC) differential count using pretreated 5 µl whole blood samples [6][7][8][9] , and a 'fiber-optic-based' flow cytometer recently tested onboard in the International Space Station 10 .…”