Clinical training and validation of the LeukoScope: a low-cost, point-of-care device to perform white blood cell and neutrophil counts.

A white blood cell (WBC) count with partial differential is an important clinical laboratory test. However, current methods to perform a WBC count and differential are difficult to use at the point of care or too expensive for use in low-resource settings. To meet this need, we developed the LeukoScope: a low-cost system to measure a WBC and neutrophil count from a single drop of blood at the point of care. The LeukoScope is battery powered and has a sample-to-answer time of <5 minutes. A drop of blood from a finger stick is added to a LeukoScope sample cartridge where pre-dried acridine orange fluorescently stains WBCs. The cartridge is then inserted into the LeukoScope reader where a portable fluorescence microscope captures a color image of the sample, which is analyzed to report results to the user. The LeukoScope system was tested at the point of care using fingerprick samples collected from 105 general oncology patients in Houston, TX. Performance of the LeukoScope was compared to that of a HemoCue WBC DIFF performed using the same fingerprick sample; clinical laboratory analysis of a venous blood draw was used as the gold standard in all cases. Bland-Altman analysis showed that the LeukoScope and HemoCue WBC DIFF had similar accuracy for measurement of WBC and neutrophil counts as compared to the gold standard. Seven out of eight patients with abnormal WBC count values were correctly identified using the LeukoScope, while six out of eight were correctly identified using the HemoCue WBC DIFF. Five out of six patients with abnormal neutrophil counts were correctly identified using the LeukoScope, while six of six were correctly identified using the HemoCue WBC DIFF.

Optical systems for point-of-care diagnostic instrumentation: analysis of imaging performance and cost.

One of the key elements in point-of-care (POC) diagnostic test instrumentation is the optical system required for signal detection and/or imaging. Many tests which use fluorescence, absorbance, or colorimetric optical signals are under development for management of infectious diseases in resource limited settings, where the overall size and cost of the device is of critical importance. At present, high-performance lenses are expensive to fabricate and difficult to obtain commercially, presenting barriers for developers of in vitro POC tests or microscopic image-based diagnostics. We recently described a compact "hybrid" objective lens incorporating both glass and plastic optical elements, with a numerical aperture of 1.0 and field-of-view of 250 µm. This design concept may potentially enable mass-production of high-performance, low-cost optical systems which can be easily incorporated in the readout path of existing and emerging POC diagnostic assays. In this paper, we evaluate the biological imaging performance of these lens systems in three broad POC diagnostic application areas; (1) bright field microscopy of histopathology slides, (2) cytologic examination of blood smears, and (3) immunofluorescence imaging. We also break down the fabrication costs and draw comparisons with other miniature optical systems. The hybrid lenses provided images with quality comparable to conventional microscopy, enabling examination of neoplastic pathology and infectious parasites including malaria and cryptosporidium. We describe how these components can be produced at below $10 per unit in full-scale production quantities, making these systems well suited for use within POC diagnostic instrumentation.

Chromatography paper as a low-cost medium for accurate spectrophotometric assessment of blood hemoglobin concentration.

Anemia affects a quarter of the world's population, and a lack of appropriate diagnostic tools often prevents treatment in low-resource settings. Though the HemoCue 201+ is an appropriate device for diagnosing anemia in low-resource settings, the high cost of disposables ($0.99 per test in Malawi) limits its availability. We investigated using spectrophotometric measurement of blood spotted on chromatography paper as a low-cost (<$0.01 per test) alternative to HemoCue cuvettes. For this evaluation, donor blood was diluted with plasma to simulate anemia, a micropipette spotted blood on paper, and a bench-top spectrophotometer validated the approach before the development of a low-cost reader. We optimized impregnating paper with chemicals to lyse red blood cells, paper type, drying time, wavelengths measured, and sensitivity to variations in volume of blood, and we validated our approach using patient samples. Lysing the blood cells with sodium deoxycholate dried in Whatman Chr4 chromatography paper gave repeatable results, and the absorbance difference between 528 nm and 656 nm was stable over time in measurements taken up to 10 min after sample preparation. The method was insensitive to the amount of blood spotted on the paper over the range of 5 µL to 25 µL. We created a low-cost, handheld reader to measure the transmission of paper cuvettes at these optimal wavelengths. Training and validating our method with patient samples on both the spectrometer and the handheld reader showed that both devices are accurate to within 2 g dL(-1) of the HemoCue device for 98% and 95% of samples, respectively.

Low cost, high performance, self-aligning miniature optical systems.

The most expensive aspects in producing high quality miniature optical systems are the component costs and long assembly process. A new approach for fabricating these systems that reduces both aspects through the implementation of self-aligning LIGA (German acronym for lithographie, galvanoformung, abformung, or x-ray lithography, electroplating, and molding) optomechanics with high volume plastic injection molded and off-the-shelf glass optics is presented. This zero alignment strategy has been incorporated into a miniature high numerical aperture (NA = 1.0 W) microscope objective for a fiber confocal reflectance microscope. Tight alignment tolerances of less than 10 microm are maintained for all components that reside inside of a small 9 gauge diameter hypodermic tubing. A prototype system has been tested using the slanted edge modulation transfer function technique and demonstrated to have a Strehl ratio of 0.71. This universal technology is now being developed for smaller, needle-sized imaging systems and other portable point-of-care diagnostic instruments.