Review on Photomicrography based Full Blood Count (FBC) Testing and Recent Advancements

With advancements in related sub-fields, research on photomicrography in life science is emerging and this is a review on its application towards human full blood count testing which is a primary test in medical practices. For a prolonged period of time, analysis of blood samples is the basis for bio medical observations of living creatures. Cell size, shape, constituents, count, ratios are few of the features identified using DIP based analysis and these features provide an overview of the state of human body which is important in identifying present medical conditions and indicating possible future complications. In addition, functionality of the immune system is observed using results of blood tests. In FBC tests, identification of different blood cell types and counting the number of cells of each type is required to obtain results. Literature discuss various techniques and methods and this article presents an insightful review on human blood cell morphology, photomicrography, digital image processing of photomicrographs, feature extraction and classification, and recent advances. Integration of emerging technologies such as microfluidics, micro-electromechanical systems, and artificial intelligence based image processing algorithms and classifiers with cell sensing have enabled exploration of novel research directions in blood testing applications.

In Vivo Fluorescence Imaging of E-Selectin: Quantitative Detection of Endothelial Activation in Arthritis

Rheumatoid arthritis (RA) is a chronic progressive systemic inflammatory disease, characterized by synovial inflammation and localized destruction of cartilage and bone. Heterogeneity in the clinical presentation of RA and uncertainty about which patients will respond to treatment makes diagnosis and management challenging. Fluorescent imaging in the near infrared (NIR) spectrum significantly decreases tissue autofluorescence offering unique potential to detect specific molecular targets in vivo. E-selectin or endothelial adhesion molecule-1 (ELAM-1), a 115kDa glycoprotein induced on endothelial cells in response to pro-inflammatory cytokines involved in RA, such as interleukin (IL)-1 beta and tumour necrosis factor alpha (TNF alpha). E-selectin has been well validated as a potential biomarker of disease activity. My study aimed to investigate whether E-selectin targeted optical imaging in vivo could be developed as a sensitive, specific and quantifiable preclinical molecular imaging technique, and also whether this approach could be used to delineate the molecular effects of novel therapies. I utilised anti-E-selectin antibody labelled with NIR fluorophore in a mouse model of paw swelling induced by intra-plantar injection of TNF alpha, and in acute collagen-induced arthritis (CIA) in DBA/1 mice, a widely used model of RA. E-selectin generated signal, localised to points of maximal clinical inflammation in the inflamed mouse paw in both models with significant differences to control antibody. Binding of anti-E-selectin antibody was also demonstrated by immunohistochemistry in both models. The ability of E-selectin targeted imaging to detect sub-clinical endothelial activation was also investigated, demonstrating that E-selectin may be an excellent way of determining subclinical vascular activation in CIA. Finally the effect of novel targeted therapy – RB200 which blocks epidermal growth factor (EGF) signalling was investigated. This demonstrated that E-selectin targeted signal could be absolutely abrogated to a level seen in unimmunised healthy control animals, following combination treatment with RB200 and the TNF alpha inhibitor etanercept. E-selectin targeted optical imaging is a viable in vivo imaging technique that can also be applied to quantify disease and investigate the effects of novel molecular therapies. It holds significant promise as a molecular imaging technique for future translation into the clinic for patients with rheumatoid arthritis and other inflammatory diseases

RXR ligands negatively regulate thrombosis and hemostasis

OBJECTIVE: Platelets have been found to express intracellular nuclear receptors including the Retinoid X receptors (RXRα and RXRβ). Treatment of platelets with ligands of RXR has been shown to inhibit platelet responses to ADP and thromboxane A2, however the effects on responses to other platelet agonists as well as the underlying mechanism has not been fully characterised. APPROACH AND RESULTS: The effect of 9-cis-retinoic acid (9-cis-RA), docosahexaenoic acid and synthetic ligand for RXR, methoprene acid on collagen receptor (GPVI) agonists and Thrombin stimulated platelet function; including aggregation, granule secretion, integrin activation, calcium mobilisation, integrin αIIbβ3 outside-in signalling and thrombus formation in vitro and in vivo were determined. Treatment of platelets with RXR ligands resulted in attenuation of platelet functional responses following stimulation by GPVI agonists and thrombin and inhibition of integrin αIIbβ3 outside-in signalling. Treatment with 9-cis-RA caused inhibition of thrombus formation in vitro and an impairment of thrombosis and haemostasis in vivo. Both RXR ligands stimulated protein kinase A activation, measured by VASP S157 phosphorylation, that was found to be dependent on both cAMP and NFκB activity. CONCLUSIONS: This study identifies a widespread, negative regulatory role for RXR in the regulation of platelet functional responses and thrombus formation and describes novel events that lead to the upregulation of PKA, a known negative regulator of many aspects of platelet function. This mechanism may offer a possible explanation for the cardioprotective effects described in vivo following treatment with RXR ligands

Multinode Acoustic Systems for High Throughput Cellular Analysis

For decades, flow cytometry is used as the gold standard for cellular analyses as it measures multiple properties of single cells. Traditional flow cytometry uses the hydrodynamic focusing technique where the sheath fluid focuses cells in the sample into a narrow stream. Although such precise focusing provides accurate optical measurements, high sheath fluid pressure and high linear velocities limit analysis rate to 50,000 particles/s. Such rates are too low for detecting rare events where one cell may have to be detected in a population of about a billion cells. Therefore, it is necessary to eliminate the sheath fluid and improve throughput by several orders of magnitude by using a suitable alternate focusing mechanism that allows focusing cells in high volume samples into multiple focused streams. Toward this aim, this dissertation presents the multinode acoustic technique that uses high frequency ultrasonic waves to focus particles and cells into highly parallel focused streams. One challenge, however, is that acoustic attenuation is significant at high frequencies. Therefore, to optimize acoustic energy density within multinode acoustic flow cells, a simple model is derived based on exhaustive literature review, suggesting that attenuation may be minimized by proper choice of fabrication material. Using such acoustically transparent materials, multinode acoustic flow cells were fabricated by three approaches, which include using machined aluminum frame and glass slides, disposable rectangular glass capillaries and etched silicon flow cells fabricated using standard photolithography and deep reactive ion etching techniques. Among these, flexibility in design using microfabrication approach has allowed fabricating etched flow cells having multiple parallel channels. Such parallelization improves acoustic energy density within each channel and precisely focuses particles at volumetric throughput of few tens of mL/min. Finally, this dissertation presents a proof-of-concept flow cytometry instrumentation using laser line generation optics and microscopy image sensors for imaging parallel focused streams in multinode acoustic flow cells. High throughput and precise focusing suggest that multinode focusing is a suitable alternative to conventional hydrodynamics, and multinode acoustic flow cells integrated with such optical imaging systems incorporating real-time signal processing circuitry will provide throughput matching that required for the detection of circulating tumor cells

The advancement of blood cell research by optical tweezers

Demonstration of the light radiation pressure on a microscopic level by A. Ashkin led to the invention of optical tweezers (OT). Applied in the studies of living systems, OT have become a preferable instrument for the noninvasive study of microobjects, allowing manipulation and measurement of the mechanical properties of molecules, organelles, and cells. In the present paper, we overview OT applications in hemorheological research, placing emphasis on red blood cells but also discussing OT applications for the investigation of the biomechanics of leukocytes and platelets. Blood properties have always served as a primary parameter in medical diagnostics due to the interconnection with the physiological state of an organism. Despite blood testing being a well-established procedure of conventional medicine, there are still many complex processes that must be unraveled to improve our understanding and contribute to future medicine. OT are advancing single-cell research, promising new insights into individual cell characteristics compared to the traditional approaches. We review the fundamental and practical findings revealed in blood research through the optical manipulation, stretching, guiding, immobilization, and inter-/intracellular force measurements of single blood cells

Development of Molecular Contrast-enhanced Imaging for Optical Coherence Tomography

Biological imaging techniques that are able to detect a contrast-enhanced signal from the target molecules have been widely applied to various techniques in the imaging field. The complex biological environment provides numerous and more efficient pathways along which the chromophores (light absorber) may release its energy. This energy can provide not only morphological information, but also specific molecular information such as a biochemical map of a sample. All diseases correlate with both morphological and biochemical changes. Optical coherence tomography (OCT) system is one of the biological imaging techniques. OCT has widely been applied to many medical/clinical fields, giving benefit from a penetration depth of a few millimeters while maintaining a spatial resolution on the order of a micron. Unfortunately, OCT lacks the straightforward functional molecular imaging extensions available for other technologies, e.g. confocal fluorescence microscopy and fluorescence diffuse optical tomography. This is largely because incoherent processes such as fluorescence emission and Raman scattering are not readily detectable with low coherence interferometry that is the central technique that underlies all OCT systems. Despite a drawback of molecular imaging with OCT, it is highly desirable to measure not only morphological, but also molecular information from either endogenous or exogenous molecules. In order to overcome the limitation of molecular contrast imaging for OCT, our group has been researched the hybrid OCT imaging technique and a new exogenous contrast agent. Our contrast-enhanced imaging technique integrates OCT with a well-researched and well-established technique: two-colored pump-probe absorption spectroscopy. Our novel imaging technique is called Pump-Probe OCT (PPOCT). Based upon current successful results, molecular imaging with OCT potentially gives us the ability to identify pathologies. In order to expand the capacity of PPOCT, this dissertation focuses on development of molecular contrast-enhanced imaging for optical coherence tomography (OCT). In the first phase of the research, we developed and optimized for sensitivity a two-color ground state recovery Pump-Probe Optical Coherence Tomography (gsrPPOCT) system and signal algorithm to measure the contrast-enhanced signal of endogenous and exogenous contrast agents such as Hemoglobin (Hb) and Methylene blue (MB) from in vivo samples. Depending on the absorption peak of a target molecule, the pump light sources for PPOCT used 532nm Q-switched laser or 663nm diode laser. Based on different experimental application, Ti:sapp or SLD of 830nm center wavelength were utilized. The PPCOT system was firstly used to image Hb of in vivo vasulature in a Xenopus laevis as the endogenous contrast agent and a larval stage zebrafish using MB as the exogenous contrast agent via transient changes in light absorption. Their morphological in addition to molecular specific information from a live animal was described. The incorporation of a pump laser in an otherwise typical spectrometer based OCT system is sufficient to enable molecular imaging with PPOCT. In the second phase of this research, based on endoscopic molecular contrast-enhanced applications for OCT, we invented an ultra-wideband lensless fiber optic rotary joint based on co-aligning two optical fibers has excellent performance (~0.38 dB insertion loss). The developed rotary joint can cover a wavelength range of at least 355- 1360 nm with single mode, multimode, and double clad fibers with rotational velocities up to 8800 rpm (146 Hz). In the third phase of this research, we developed and manufactured a microencapsulated methylene blue (MB) contrast agent for PPOCT. The poly lactic coglycolic acid (PLGA) microspheres loaded with MB offer several advantages over bare MB. The microsphere encapsulation improves the PPOCT signal both by enhancing the scattering and preventing the reduction of MB to leucomethylene blue. The surface of the microsphere can readily be functionalized to enable active targeting of the contrast agent without modifying the excited state dynamics of MB that enable PPOCT imaging. Both MB and PLGA are used clinically. PLGA is FDA approved and used in drug delivery and tissue engineering applications. 2.5 µm diameter microspheres were synthesized with an inner core containing 0.01% (w/v) aqueous MB. As an initial demonstration the MB microspheres were imaged in a 100 µm diameter capillary tube submerged in a 1% intralipid emulsion. By varying the oxygen concentration both 0% and 21%, we observed he lifetime of excited triple state using time-resolved Pump-Probe spectroscopy and also the relative phase shift between the pump and probe is a reliable indicator of the oxygen concentration. Furthermore, these results are in good agreement with our theoretical predictions. This development opens up the possibility of using MB for 3-D oxygen sensing with PPOCT

Microfluidics in Haemostasis: a Review

Haemostatic disorders are both complex and costly in relation to both their treatment and subsequent management. As leading causes of mortality worldwide, there is an ever-increasing drive to improve the diagnosis and prevention of haemostatic disorders. The field of microfluidic and Lab on a Chip (LOC) technologies is rapidly advancing and the important role of miniaturised diagnostics is becoming more evident in the healthcare system, with particular importance in near patient testing (NPT) and point of care (POC) settings. Microfluidic technologies present innovative solutions to diagnostic and clinical challenges which have the knock-on effect of improving health care and quality of life. In this review, both advanced microfluidic devices (R&D) and commercially available devices for the diagnosis and monitoring of haemostasis-related disorders and antithrombotic therapies, respectively, are discussed. Innovative design specifications, fabrication techniques, and modes of detection in addition to the materials used in developing micro-channels are reviewed in the context of application to the field of haemostasi

Development of Molecular Contrast-enhanced Imaging for Optical Coherence Tomography

Biological imaging techniques that are able to detect a contrast-enhanced signal from the target molecules have been widely applied to various techniques in the imaging field. The complex biological environment provides numerous and more efficient pathways along which the chromophores (light absorber) may release its energy. This energy can provide not only morphological information, but also specific molecular information such as a biochemical map of a sample. All diseases correlate with both morphological and biochemical changes. Optical coherence tomography (OCT) system is one of the biological imaging techniques. OCT has widely been applied to many medical/clinical fields, giving benefit from a penetration depth of a few millimeters while maintaining a spatial resolution on the order of a micron. Unfortunately, OCT lacks the straightforward functional molecular imaging extensions available for other technologies, e.g. confocal fluorescence microscopy and fluorescence diffuse optical tomography. This is largely because incoherent processes such as fluorescence emission and Raman scattering are not readily detectable with low coherence interferometry that is the central technique that underlies all OCT systems. Despite a drawback of molecular imaging with OCT, it is highly desirable to measure not only morphological, but also molecular information from either endogenous or exogenous molecules. In order to overcome the limitation of molecular contrast imaging for OCT, our group has been researched the hybrid OCT imaging technique and a new exogenous contrast agent. Our contrast-enhanced imaging technique integrates OCT with a well-researched and well-established technique: two-colored pump-probe absorption spectroscopy. Our novel imaging technique is called Pump-Probe OCT (PPOCT). Based upon current successful results, molecular imaging with OCT potentially gives us the ability to identify pathologies. In order to expand the capacity of PPOCT, this dissertation focuses on development of molecular contrast-enhanced imaging for optical coherence tomography (OCT). In the first phase of the research, we developed and optimized for sensitivity a two-color ground state recovery Pump-Probe Optical Coherence Tomography (gsrPPOCT) system and signal algorithm to measure the contrast-enhanced signal of endogenous and exogenous contrast agents such as Hemoglobin (Hb) and Methylene blue (MB) from in vivo samples. Depending on the absorption peak of a target molecule, the pump light sources for PPOCT used 532nm Q-switched laser or 663nm diode laser. Based on different experimental application, Ti:sapp or SLD of 830nm center wavelength were utilized. The PPCOT system was firstly used to image Hb of in vivo vasulature in a Xenopus laevis as the endogenous contrast agent and a larval stage zebrafish using MB as the exogenous contrast agent via transient changes in light absorption. Their morphological in addition to molecular specific information from a live animal was described. The incorporation of a pump laser in an otherwise typical spectrometer based OCT system is sufficient to enable molecular imaging with PPOCT. In the second phase of this research, based on endoscopic molecular contrast-enhanced applications for OCT, we invented an ultra-wideband lensless fiber optic rotary joint based on co-aligning two optical fibers has excellent performance (~0.38 dB insertion loss). The developed rotary joint can cover a wavelength range of at least 355- 1360 nm with single mode, multimode, and double clad fibers with rotational velocities up to 8800 rpm (146 Hz). In the third phase of this research, we developed and manufactured a microencapsulated methylene blue (MB) contrast agent for PPOCT. The poly lactic coglycolic acid (PLGA) microspheres loaded with MB offer several advantages over bare MB. The microsphere encapsulation improves the PPOCT signal both by enhancing the scattering and preventing the reduction of MB to leucomethylene blue. The surface of the microsphere can readily be functionalized to enable active targeting of the contrast agent without modifying the excited state dynamics of MB that enable PPOCT imaging. Both MB and PLGA are used clinically. PLGA is FDA approved and used in drug delivery and tissue engineering applications. 2.5 µm diameter microspheres were synthesized with an inner core containing 0.01% (w/v) aqueous MB. As an initial demonstration the MB microspheres were imaged in a 100 µm diameter capillary tube submerged in a 1% intralipid emulsion. By varying the oxygen concentration both 0% and 21%, we observed he lifetime of excited triple state using time-resolved Pump-Probe spectroscopy and also the relative phase shift between the pump and probe is a reliable indicator of the oxygen concentration. Furthermore, these results are in good agreement with our theoretical predictions. This development opens up the possibility of using MB for 3-D oxygen sensing with PPOCT