MICROFLUIDIC CD-BASED SOMATIC CELL COUNTER FOR THE EARLY DETECTION OF

BOVINE MASTITIS J.L. Garcia-Cordero1, L. Kent1, I.K. Dimov1,

C. Viguier2, L.P. Lee1,3 and A.J. Ricco1 1Biomedical Diagnostics Institute, Dublin City University, IRELAND

2Enfer Diagnostics, Kildare, IRELAND 3Biomolecular Nanotechnology Center, Berkeley Sensor & Actuator Center,

Department of Bioengineering, University of California, Berkeley, USA

ABSTRACT We report a rapid, low-cost microfluidic CD-based assay for the early detection

of bovine mastitis. The assay separates and concentrates somatic (white) cells in milk by sedimentation in a centrifugal field. Centrifugation is performed with a con-ventional CD player; a low-cost microscope images centrifuged cell volume, provid-ing a sufficiently accurate cell count to diagnose infection. KEYWORDS: Centrifugal Microfludics, Milk, Somatic Cells, Bovine Mastitis, Point-of-Care Diagnostics

INTRODUCTION

Somatic cell count in milk has become the gold standard for diagnosing early signs of mastitis (infections of the udder) in cows and for estimating milk’s mone-tary value and quality [1]. Common assay procedures to diagnose bovine mastitis either offer low levels of confidence (e.g., an electrical conductivity test), are time-consuming (direct microscopy), require toxic reagents, or remain too expensive (flow cytometry) to be used by a farmer or dairyman. Although various cytometric microfluidic devices have been developed recently [2], these assays remain difficult for a layperson to set up and use, and are typically not suited for barnyard testing. Recently, a lab-on-a-disc was reported to determine hematocrit (red blood cell con-tent) of human blood with high precision [3]. The geometry of the device reported here has been adapted for the lower number of cells per milliliter in milk: they com-prise less than 0.01% of its volume.

Milk is a complex matrix composed in its majority of water, fat, and proteins.

Cells and fat globules are the largest particles in milk and have dissimilar densitites. Our CD-based assay separates cells based on their densities (sedimentation princi-ple), concentrating white cells in a defined area for the estimation of total “cell pel-let” volume. The assay is accomplished using a rotating CD platform to effect cell separation. Because milk from healthy cows contains relatively few cells of any type, the volume of the pellet of cells, which are denser than any other milk compo-nents, is proportional to somatic cell count for an infected cow. Normal milk con-tains less than 200,000 cells per mL, whereas infected milk cell counts range from 200,000 to over 3,000,000 cells per mL.

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DEVICE STRUCTURE AND FABRICATION An on-disc funnel structure with a narrow, blind channel at its end was chosen as

the geometry for the assay. The V-shaped region of the funnel has tapered walls and surfaces to direct cells more quickly and reliably into the blind channel, which facili-tates packing of the cells into a column whose length is proportional to cell count. The device has a single inlet and holds 150 µL of milk. The blind channel can ac-commodate up to 450,000 cells. Initial prototypes were fabricated by precision mi-cromilling of planar CDs (CD blanks: Åmic AB, Sweden). A pressure-sensitive ad-hesive layer bonds the machined disc to an acrylic CD blank to form the device.

Figure 1. View of one funnel-shaped device (left), of which there are 9 on each

CD. Cells are separated and concentrated in the blind channel (right). Schematic of the materials used in the fabrication of the device. Patterns were micromilled

into a cyclo olefin polymer (COP) disk and bonded to a layer of poly(methylmethacrylate) (PMMA) with a layer of pressure-sensitive adhesive.

EXPERIMENTS AND RESULTS

Fresh raw milk samples were collected from a local Irish farmer through Enfer Diagnostics. CD-based assay devices were loaded with samples of raw milk, mounted in an ordinary CD player, and spun for 5 min at 3,000 rpm. Images of the channel tips were recorded with a low-cost microscope (MIC-D, Olympus, USA) and analyzed with customized software (MATLAB, Mathworks, USA). Image processing results were compared to a commercial enzyme-based somatic cell counter (PortaSCC, Portacheck, USA), revealing the correlation shown in Figure 4.

Figure 2. Image of the fabricated CD-based somatic cell counter (right). The assay is carried out in a conventional CD player (left).

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Twelfth International Conference on Miniaturized Systems for Chemistry and Life SciencesOctober 12 - 16, 2008, San Diego, California, USA

Figure 3. Images of compacted cell “pellets” from several milk sam-ples compacted in the blind channel at the end of each funnel (bottom).

Figure 4. Image analysis software detects and measures the area occupied by

the pellet, highlighting it with false color (darkened area at the end of the channel, at left). Volume of milk somatic cell pellet compared to readings from the same milk

samples using a commercial enzyme-based cellular assay, Portacheck (right). CONCLUSIONS

Unlike existing cell-counting techniques, this assay is easy to set up, requires no reagents, and the design can measure up to nine samples at once. Made of thermo-plastic, the custom CD is amenable to mass fabrication at low cost, e.g. by injection molding. We are currently developing an optical scanner based on a conventional CD player optical read head so that assay results can be measured using the same device that centrifuges, providing a simple, robust, barnyard-ready point-of-care as-say of cell count in milk.

ACKNOWLEDGEMENTS

We thank Jimmy Baker (Åmic) for providing the planar CDs and Aideen O’Neill (Adhesives Research, Ireland) for helpful discussions. This work was supported by the Science Foundation Ireland under Grant No. 05/CE3/B754. REFERENCES [1] S. Pyörälä. Indicators of inflammation in the diagnosis of mastitis. Veterinary

Research, 34, 565 (2003). [2] D. Huh, W. Gu, J. B. Grotberg, S. Takayama. Microfluidics for flow cytometric

analysis of cells and particles. Physiological Measurement, 26, R73 (2005). [3] L. Riegger et al. Single-step centrifugal hematocrit determination on a 10-$

processing device. Biomedical Microdevices, 9, 795 (2007).

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