(586a) A Low-Cost Flow Cytometer for Blood Cell Differentiation.

A complete blood count is an essential diagnostic tool for most of the diseases involving immune response. Diagnostic centers are equipped with hematology analysers or flow cytometers that are used to provide a complete blood count, the technology based on flow cytometry. Flow cytometry is a powerful tool for measuring sub-microscopic or microscopic particles or biological cells. It is an essential analytical technique that enables measurement of biological, physical and chemical properties of cells, microorganisms, and/or particles. The system works on the Mie Scattering of light by the particles/ cells which enables their detection and enumeration, here the size of the scatterer is similar or larger than the wavelength of light. In these instruments, laser light is scattered from particles focused in a flow cell and collected by light sensors (such as photodiodes/photomultiplier tubes), where the intensity of the scattered light depends on the refractive index of the particle and surrounding medium, scattering angle, wavelength of light, and the size and the shape of particle. The particles or cells can be fluorescently tagged to give information based on the fluorescent signals which provide information on the complexity of the particles. The widely prevalent application is the five-part differential of white blood cells.

The detection of blood cells takes place in one of the critical parts of the cytometer which is the flow cell or flow chamber where the particle stream is constrained into a focused region using hydrodynamic focusing enabling single cell analysis. The conventional flow cells use thick quartz flow cells which are expensive and therefore not suitable for instruments targeted for resource-constrained settings. We have successfully designed and constructed a low-cost flow cell using micro-capillaries of the order of a few hundred microns in diameter, which delivers results close to the commercial cuvette-based flow cell in commercial flow cytometers (Fig 1(a)). We propose a low-cost sheath flow assembly design 3D-printed in polymer resin integrated to an assembly of capillary tubes for the passage of focussed flow of particles. The entire flow cell assembly is a simplified version of a sheath flow cuvette design that is able to focus and accelerate a stream of particles in a narrow region probed by the incident laser light. The flow cell is embedded in a 3D-printed module, which houses PMMA micro-lenses for scatter collection and photodetectors. The laboratory flow cytometer consists of a laser, focusing lenses and the flow cell, a set of mini-lenses for signal collection and focusing and a microcontroller for signal processing and analysis (Fig 1(b)). We show excellent agreement between the size distribution of model particles obtained via direct imaging and those obtained from light scattering. We have also proposed a low-cost alternative to flow focusing of particles inside the flow cell by the addition of a little amount of viscoelastic polymer in the sample. The method can result in the cost reduction of the enormous diluent used for flow focusing, by virtue of reducing the flow-rate of the sheath fluid (diluent).

In figure 1(c), we compare the size distribution of population of particles obtained from image analysis of microscope images and that obtained via measurement of forward scattering data. The figure shows (a) an overlap between the normalized distribution of the cross-sectional area of 14-20 μm particles, (d/d_m), and the normalized distribution of forward scattered intensity, I/I_m. Here, d_m is the mean value of the diameter of the particles in the mixture while, I_m is the mean intensity value in the scattered light intensity distribution. The forward scatter intensity is directly proportional to the square of the particle diameter or its cross-sectional area. We have provided a microscope image of the 14-20 μm particles in suspension in the inset for reference. We conducted experiments with human blood and were able to distinguish between the WBCs on the basis of size and granularity (Fig. 1(d)). We have been able to obtain a 3-part differential of WBCs (Granulocytes, Monocytes and Lymphocytes) based on the forward scatter measurement on our device from both the flow cell models (shown for two donors). Here, the granulocytes (G) scatter the highest intensity, and lymphocytes (L) being the smallest and agranular scatter the lowest intensity of laser in the forward direction. The monocytes (M) occupy the middle region, the results are confirmed with the available literature. We have been able to incorporate fluorescence detection using fluorescent dyes in the buffer solution and capture fluorescent signals from blood cells to obtain a complete blood count. With further modifications, the device may be used for disease detection, such as targeting viruses and bacteria and has various applications in immunology.