Thrombospondin-1 induced programmed death-ligand 1-mediated immunosuppression by activating the STAT3 pathway in osteosarcoma.

Thrombospondin-1 (TSP1) is generally assumed to suppress the growth of osteosarcoma through inhibiting angiogenesis; however, it is unclear whether TSP1 could affect the antitumor immunity against osteosarcoma. We aimed to explore the immune-related tumor-promoting effects of TSP1 and decipher its underlying mechanism. First, we identified that TSP1 regulated programmed death-ligand 1 (PD-L1) expression, which was related to the CD8+ T cells anergy in osteosarcoma cells. The exact role of PD-L1 in the immunosuppressive effect of TSP1 was then further confirmed by the addition of the PD-L1 neutralizing Ab. With the addition of PD-L1 neutralizing Abs during cocultivation, the inhibition of CD8+ T cells was abolished to a certain extent. Further mechanistic investigations showed that TSP1-induced PD-L1 upregulation was achieved by activation of the signal transducer and activator of transcription 3 (STAT3) pathway. In vivo experiments also indicated that TSP1 overexpression could promote the growth of primary lesions, whereas TSP1 knockdown effectively inhibits the growth of the primary lesion as well as lung metastasis by restoring the antitumor immunity. Thrombospondin-1 knockdown combined with PD-L1 neutralizing Ab achieved a more pronounced antitumor effect. Taken together, our study showed that TSP1 upregulates PD-L1 by activating the STAT3 pathway and, therefore, impairs the antitumor immunity against osteosarcoma.

Super-resolved Raman imaging via galvo-painted structured line illumination.

Traditional line-scan Raman imaging features a rapid imaging speed while preserving complete spectral information, yet has diffraction-limited resolution. Sinusoidally structured line excitation can yield an improvement in the lateral resolution of the Raman image along the line's direction. However, given the need for the line and spectrometer slit to be aligned, the resolution in the perpendicular direction remains diffraction limited. To overcome this, we present here a galvo-modulated structured line imaging system, where a system of three galvos can arbitrarily orient the structured line on the sample plane, while keeping the beam aligned to the spectrometer slit in the detection plane. Thus, a two-fold isotropic improvement in the lateral resolution fold is possible. We demonstrate the feasibility using mixtures of microspheres as chemical and size standards. The results prove an improvement in the lateral resolution of 1.8-fold (limited by line contrast at higher frequencies), while preserving complete spectral information of the sample.

Multicomponent Raman spectral regression using complete and incomplete models and convolutional neural networks.

With the advent of hyperspectral Raman imaging technology, especially the rapid and high-resolution imaging schemes, datasets with thousands to millions of spectra are now commonplace. Standard preprocessing and regression methods such as least squares approaches are time consuming and require input from highly trained operators. Here we propose a solution to this analytic bottleneck through a convolutional neural network trained fully on synthetic data and then applied to experimental measurements, including cases where complete spectral information is missing (i.e. an underdetermined model). An advantage of the model is that it combines background correction and regression into a single step, and does not require user-selected parameters. We compare our results with traditional least squares methods, including the popular asymmetric least squares (AsLS) approach. Our results demonstrate that the proposed CNN model boasts less sensitivity to parameter selection, and with a rapid processing speed, with performance equal to or better than comparison methods. The performance is validated on synthetic spectral mixtures, as well as experimentally measured single-vesicle liposome data.

Lin28 enhances de novo fatty acid synthesis to promote cancer progression via SREBP-1.

Lin28 plays an important role in promoting tumor development, whereas its exact functions and underlying mechanisms are largely unknown. Here, we show that both human homologs of Lin28 accelerate de novo fatty acid synthesis and promote the conversion from saturated to unsaturated fatty acids via the regulation of SREBP-1. By directly binding to the mRNAs of both SREBP-1 and SCAP, Lin28A/B enhance the translation and maturation of SREBP-1, and protect cancer cells from lipotoxicity. Lin28A/B-stimulated tumor growth is abrogated by SREBP-1 inhibition and by the impairment of the RNA binding properties of Lin28A/B, respectively. Collectively, our findings uncover that post-transcriptional regulation by Lin28A/B enhances de novo fatty acid synthesis and metabolic conversion of saturated and unsaturated fatty acids via SREBP-1, which is critical for cancer progression.

Fast confocal Raman imaging via context-aware compressive sensing.

Raman hyperspectral imaging is a powerful method to obtain detailed chemical information about a wide variety of organic and inorganic samples noninvasively and without labels. However, due to the weak, nonresonant nature of spontaneous Raman scattering, acquiring a Raman imaging dataset is time-consuming and inefficient. In this paper we utilize a compressive imaging strategy coupled with a context-aware image prior to improve Raman imaging speed by 5- to 10-fold compared to classic point-scanning Raman imaging, while maintaining the traditional benefits of point scanning imaging, such as isotropic resolution and confocality. With faster data acquisition, large datasets can be acquired in reasonable timescales, leading to more reliable downstream analysis. On standard samples, context-aware Raman compressive imaging (CARCI) was able to reduce the number of measurements by ∼85% while maintaining high image quality (SSIM >0.85). Using CARCI, we obtained a large dataset of chemical images of fission yeast cells, showing that by collecting 5-fold more cells in a given experiment time, we were able to get more accurate chemical images, identification of rare cells, and improved biochemical modeling. For example, applying VCA to nearly 100 cells' data together, cellular organelles were resolved that were not faithfully reconstructed by a single cell's dataset.

Combined Morpho-Chemical Profiling of Individual Extracellular Vesicles and Functional Nanoparticles without Labels.

Biological nanoparticles are important targets of study, yet their small size and tendency to aggregate makes their heterogeneity difficult to profile on a truly single-particle basis. Here we present a label-free system called 'Raman-enabled nanoparticle trapping analysis' (R-NTA) that optically traps individual nanoparticles, records Raman spectra and tracks particle motion to identify chemical composition, size, and refractive index. R-NTA has the unique capacity to characterize aggregation status and absolute chemical concentration at the single-particle level. We validate the method on NIST standards and liposomes, demonstrating that R-NTA can accurately characterize size and chemical heterogeneity, including determining combined morpho-chemical properties such as the number of lamellae in individual liposomes. Applied to extracellular vesicles (EVs), we find distinct differences between EVs from cancerous and noncancerous cells, and that knockdown of the TRPP2 ion channel, which is pathologically highly expressed in laryngeal cancer cells, leads the EVs to more closely resemble EVs from normal epithelial cells. Intriguingly, the differences in EV content are found in small subpopulations of EVs, highlighting the importance of single-particle measurements. These experiments demonstrate the power of the R-NTA system to measure and characterize the morpho-chemical heterogeneity of bionanoparticles.

Low resolution Raman: the impact of spectral resolution on limit of detection and imaging speed in hyperspectral imaging.

The majority of problems in analytical Raman spectroscopy are mathematically over-determined, where many more spectral variables are measured than analytic outputs (such as chemical concentrations) are calculated. Thus, to improve spectral throughput and simplify system design, some researchers have explored the use of low resolution Raman systems for cell or tissue classification, achieving accuracy independent of spectral resolution. However, the tradeoffs inherent in this approach have not been systematically studied. Here, we theoretically and experimentally explore the relationship between spectral resolution and analytical error. We show that decreased spectral resolution leads to spectral signal-to-noise ratio and therefore more reliable results and lower limits of detection for equivalent integration times in blind unmixing of hyperspectral images. Our theoretical analysis demonstrates that the primary benefit of low resolution Raman spectroscopy is in overcoming detector noise (such as thermal or electronic noise). Therefore, the benefits are most pronounced when utilizing lower-grade, uncooled detectors. Therefore, using a low-cost CMOS camera we experimentally demonstrate the ability of low resolution Raman spectroscopy to achieve substantially improved imaging performance compared to fully-resolved Raman spectral imaging, paving the way for cost-effective, pervasive Raman spectroscopy.

Natural spider silk as a photonics component for humidity sensing.

Biological microfibers are remarkable materials with diversity in their chemistry, structure and functions that provide a range of solutions for photonic structures. Here we proposed and demonstrated a humidity detection technique for spectral tuning of whispering gallery modes (WGMs) in a cylindrical microresonator formed by a piece of spider egg sac silk (SpEss) from Araneus Ventricosus. We launched a supercontinuum laser into the SpEss via a tapered single-mode fiber to excite WGMs. When the ambient humidity changed, the profile diameter and effective refractive index of the SpEss changed, which caused the WGM resonant dips to shift. The experimental results showed that when the relative humidity (RH) changed from 20% to 75% RH, the average testing sensitivity of the proposed sensor was 389.1 pm/%RH and the maximum testing sensitivity was 606.7 pm/%RH in the range of 60% to 75% RH. Also, the proposed SpEss-based humidity sensor showed a fast response time of 494 ms and good repeatability with fluctuations less than 8% compared with the initial test values. The SpEss-based sensor expanded the application of spider silk as a biodegradable and biocompatible material in biochemical sensing.

Exosomal miR-675 from metastatic osteosarcoma promotes cell migration and invasion by targeting CALN1.

Exosomal microRNAs(miRNAs) transfer from tumor to stromal cells is reportedly associated with cancer progression and metastasis in various epithelial cancers. However, the role of exosomal miRNA in the metastasis of osteosarcoma(OS) -the most common bone malignancy-still largely remains unknown. In this study, we purified exosomes with a median size close to 100 nm from cell culture media as well as patient serum, and proved that exosomes derived from the metastatic, but not the non-metastatic OS cells increase the migration and invasion of non-malignant fibroblast cells (hFOB1.19) in vitro. Furthermore, the differential miRNA cargo between metastatic and non-metastatic OS is identified by small RNA sequencing and RT-PCR validation, we found a highly expression of exosomal, but not cellular miR-675 level in the metastatic OS cell-lines compared with non-metastatic counterparts. Meanwhile, we also found that exosomal miR-675 could down-regulate CALN1 expression in recipient cell, which may influence the invasion and migration of hFOB1.19. Finally, the up regulation serum exosomal miR-675 and down regulation of CALN1 in tumor specimen was also found to be associated with the metastatic phenotype in OS patients. Our findings indicate that the exosomal miR-675 is a gene associated with OS and serum exosomal miR-675 expression may serve as a novel biomarker for the metastasis of OS.

DownRegulated SOX4 Expression Suppresses Cell Proliferation, Migration, and Induces Apoptosis in Osteosarcoma In Vitro and In Vivo.

The SOX4 transcription factor is involved in various cellular processes, such as embryonic development and differentiation. Deregulated expression of Sox4 in several human cancers has been reported to date, but its biological functions in the progression of osteosarcoma remain unclear. In this study, we found that the expression levels of SOX4 protein were significantly higher in high-grade osteosarcoma tissues and metastatic osteosarcoma tissues. Its overexpression was associated with poor prognosis in osteosarcoma. Knockdown of the SOX4 gene in the osteosarcoma cell lines resulted in decreased cell proliferation, migration, invasion, and induced apoptosis. After SOX4 gene silencing, the protein expression levels of Bax, Caspase-3, and P53 in osteosarcoma cells were significantly elevated, while the protein expression levels of Bcl-2, MMP2, and MMP9 were obviously decreased. In vivo analysis in nude mice further confirmed that knockdown of SOX4 suppressed tumor growth. In conclusion, SOX4 appears to be an important tumor oncogene in the regulation of osteosarcoma cell proliferation, apoptosis, and invasion, and it may be a potential target for effective osteosarcoma therapy.

Design of a portable phantom device to simulate tissue oxygenation and blood perfusion.

We propose a portable phantom system for calibration and validation of medical optical devices in a clinical setting. The phantom system comprises a perfusion module and an exchangeable tissue-simulating phantom that simulates tissue oxygenation and blood perfusion. The perfusion module consists of a peristaltic pump, two liquid storage units, and two pressure suppressors. The tissue-simulating phantom is fabricated by a three-dimensional (3D) printing process with microchannels embedded to simulate blood vessels. Optical scattering and absorption properties of biologic tissue are simulated by mixing graphite powder and titanium dioxide powder with clear photoreactive resin at specific ratios. Tissue oxygen saturation (StO2) and blood perfusion are simulated by circulating the mixture of blood and intralipid at different oxygenation levels and flow rates. A house-made multimodal imaging system that combines multispectral imaging and laser speckle imaging are used for non-invasive detection of phantom oxygenation and perfusion, and the measurements are compared with those of a commercial Moor device as well as numerical simulation. By acquiring multimodal imaging data from one phantom and applying the calibration factors in different settings, we demonstrate the technical feasibility to calibrate optical devices for consistent measurements. By simulating retina tissue vasculature and acquiring functional images at different tissue oxygenation and blood perfusion levels, we demonstrate the clinical potential to simulate tissue anomalies. Our experiments imply the clinical potential of a portable, low-cost, and traceable phantom standard to calibrate and validate medical optical devices for improved performance.

Clinical and radiological outcome of Gustilo type III open distal tibial and tibial shaft fractures after staged treatment with posterolateral minimally invasive plate osteosynthesis (MIPO) technique.

OBJECTIVES: To evaluate the methods and the outcomes of Gustilo type III open distal tibial and tibial shaft fractures with severe anterior and medial soft-tissue injuries, treated with posterolateral minimally invasive plate osteosynthesis (MIPO) technique. METHODS: From May 2015 to May 2016, 10 patients with Gustilo type III open distal tibial and tibial shaft fractures with severe anterior and medial soft-tissue injuries (Gustilo-Anderson classification IIIA, 6; IIIB, 4) were treated with staged protocol using posterolateral minimally invasive plate osteosynthesis (MIPO) technique. The initial wound lavage, debridement, and application of a spanning external fixator were performed within 24 h and the mean interval from injury to definitive surgical treatment was 12.8 (range 4-21) days. An additional bone graft was performed in two patients when definitive internal fixation was performed. All patients were followed to union. Postoperative radiographs, postoperative complications, bone union, ankle joint motion, and limb functional outcome information of AOFAS ankle-hindfoot score were recorded. RESULTS: The mean follow-up period was 17.8 (range 12-26) months. The mean interval to bony union was 25.8 (range 20-40) weeks. Bone union was achieved in all cases. There were no complications, such as incision breakdown, deep infection, or impingement of the flexor hallucis longus tendon. The average AOFAS score was 90 (range 83-96). In ten patients, two patients had a superficial wound infection and another one patient showed a 6° varus deformity. CONCLUSIONS: Staged treatment using MIPO technique through a posterolateral approach is a reasonable and safe treatment option for open distal tibial and tibial shaft fractures, especially Gustilo type III with severe anterior and medial soft-tissue injuries. However, it should have a higher level of research evidence in more patients to confirm the safety of the clinical application of this technique.

Antitumor effect of focal adhesion kinase inhibitor PF562271 against human osteosarcoma in vitro and in vivo.

Focal adhesion kinase (FAK) overexpression is related to invasive and metastatic properties in different kinds of cancers. Target therapy by inhibiting FAK has achieved promising effect in some cancer treatments, but its effect in human osteosarcoma has not been well studied. In the present study, we analyzed the antitumor efficacy of PF562271, an FAK inhibitor, against osteosarcoma in vitro and in vivo. Phosphorylated FAK (Y397) was highly expressed in primary human osteosarcoma tumor samples and was associated with osteosarcoma prognosis and lung metastasis. PF562271 greatly suppressed proliferation and colony formation in human osteosarcoma cell lines. In addition, treatment of osteosarcoma cell lines with PF562271 induced apoptosis and downregulated the activity of the protein kinase B/mammalian target of rapamycin pathway. PF562271 also impaired the tube formation ability of endothelial cells in vitro. Finally, oral treatment with PF562271 in mice dramatically reduced tumor volume, weight, and angiogenesis of osteosarcoma xenografts in vivo. These results indicate that FAK inhibitor PF562271 can potentially be effectively used for the treatment of osteosarcoma.

Raman Plus X: Biomedical Applications of Multimodal Raman Spectroscopy.

Raman spectroscopy is a label-free method of obtaining detailed chemical information about samples. Its compatibility with living tissue makes it an attractive choice for biomedical analysis, yet its translation from a research tool to a clinical tool has been slow, hampered by fundamental Raman scattering issues such as long integration times and limited penetration depth. In this review we detail the how combining Raman spectroscopy with other techniques yields multimodal instruments that can help to surmount the translational barriers faced by Raman alone. We review Raman combined with several optical and non-optical methods, including fluorescence, elastic scattering, OCT, phase imaging, and mass spectrometry. In each section we highlight the power of each combination along with a brief history and presentation of representative results. Finally, we conclude with a perspective detailing both benefits and challenges for multimodal Raman measurements, and give thoughts on future directions in the field.

An Anatomical Study of the Nutrient Foramina of the Human Humeral Diaphysis.

BACKGROUND Understanding the nutrient foramina is critical to clinical practice. An insult to the nutrient foramina can be caused by trauma and/or surgical dissection and lead to devascularization and bad outcomes. Few studies have looked at the humerus, and no studies have described relative information of humeral nutrient foramen related to anatomical structures that might be located by palpable landmarks. In this study, we analyzed the anatomical features of the nutrient foramina of the diaphyseal humerus and provide a discussion of clinical relevance. MATERIAL AND METHODS We dissected 19 cadavers and analyzed the relative positions of the foramina and surrounding muscles, and the number, direction, diameter, and location of the nutrient foramina. Foramina index and a new landmark index were used to calculate the location. We compared the data from both sides and the relationships between transverse and longitudinal locations, diameter and total length, and foramina index and landmark index were also analyzed. RESULTS The humeri had one or two main nutrient foramina located in a small area between the coracobrachialis and brachial muscles and oriented toward the elbow. The mean diameter was 1.11±0.32 mm. The mean index and landmark index were 43.76±4.94% and 42.26±5.35%, respectively. There were no differences between sides in terms of diameter, length, or nutrient foramina index. There were no significant correlations between transverse and longitudinal locations or diameter and total length. The foramina index and landmark index showed strong positive correlation (r=0.994, p<0.0001). CONCLUSIONS Our study provides details about the nutrient foramina that will benefit clinicians who treat injuries and diseases of the humerus. Surgeons should be mindful of soft tissue in the foraminal area during surgical procedures.

Effects of different surgical techniques on mid-distal humeral shaft vascularity: open reduction and internal fixation versus minimally invasive plate osteosynthesis.

BACKGROUND: Humeral shaft fractures are generally managed with the conventional posterior open reduction and internal fixation (ORIF) or minimally invasive plate osteosynthesis (MIPO). This study was aimed at comparing the outcomes of these surgical techniques in terms of the vascular integrity of the mid-distal humeral shaft. METHODS: Twelve upper limbs were harvested from 6 fresh cadavers. ORIF or MIPO was randomly performed on either side of each pair of limbs. The axillary artery was perfused with a latex-lead tetraoxide red solution to visualize the vascular structures. The vascular integrity of the humerus was examined by plain radiography and dissection. The periosteal filling achieved with each technique was scored and the scores compared. RESULTS: In each limb, one main nutrient artery entering the mid-distal humeral shaft anteromedially (83.3 %) or medially (16.7 %) was first identified. No case of injury to the main nutrient artery was noted for either surgical technique. Injuries to the accessory nutrient arteries entering the mid-distal humeral shaft from the posterior aspect were absent in the MIPO cases, but occurred in 52.9 % of the ORIF cases. In addition, MIPO was also superior to the open plate technique showed superior periosteal filling than. CONCLUSIONS: Our results showed that the MIPO technique is superior to the ORIF in terms of preserving the vascular integrity of the mid-distal humeral shaft.

Metformin inhibits the proliferation, metastasis, and cancer stem-like sphere formation in osteosarcoma MG63 cells in vitro.

Metformin is an oral drug that has been widely used to treat type 2 diabetes mellitus. Interestingly, accumulated evidence indicate that metformin may reduce the risk of cancer in patients with type 2 diabetes and inhibit tumor cell growth and survival in numerous malignancies, including osteosarcoma (OS) cells. In the present study, we aimed to investigate the effects of metformin on the proliferation, migration, invasion, and sphere formation in OS MG63 cells in vitro. Metformin suppressed OS MG63 cell proliferation in a dose- and time-dependent manner and markedly blocked anti-metastatic potentials, migration, and invasion, by downregulating matrix metalloproteinase 2 (MMP2) and MMP9. Besides, we established OS cancer stem-like cell (CSC) model with sarcosphere formation assay and demonstrated that metformin posed damage on CSCs in OS by inhibiting sphere formation and by inducing their stemness loss. The stemness of CSCs in OS such as self-renewal and differentiation potentials was both impaired with a significant decrease of Oct-4 and Nanog activation. Consistent with this, the positive rates of CD90, CD133, and stage-specific embryonic antigen-4 (SSEA-4) were all observed with reductions in response to metformin exposure. In addition, Western blot showed that metformin activated AMPKα at Tyr172, followed by a downregulated phosphorylation of mammalian target of rapamycin (mTOR)/S6 and feedback activation of p-AKT Ser(473) in both OS MG63 cells and CSCs. This indicates that AMPK/mTOR/S6 signaling pathway might be involved in the growth inhibition of both OS MG63 cells and CSCs. These results suggest that metformin, a potential anti-neoplastic agent, might make it a novel therapeutic choice for the treatment of OS in the future.

Open reduction and internal fixation of Ideberg IV and V glenoid intra-articular fractures through a Judet approach: a retrospective analysis of 11 cases.

OBJECTIVES: To evaluate the methods and the outcomes of complex intra-articular glenoid fractures, treated by open reduction and internal fixations. METHODS: The outcomes of 11 cases of complex intra-articular glenoid scapular fractures were retrospectively analyzed. The fractures were classified as type IV in five cases, type Va in two and Vb in four cases, according to Ideberg classification system. The mean step or gap between the main articular fragments was 6.3 ± 6.2 (4-25) mm. The fractures were openly reduced through a Judet approach and fixed with reconstructive plates or bands placed on the lateral and medial side of affected scapula, respectively. The main articular fragments were strengthened with a 4.0-mm cannulated screw in five cases. The bone union, the anterior flexion, the external and internal rotation of the shoulders were checked and recorded. The functional outcomes were evaluated using DASH questionnaire, Constant and UCLA shoulder score systems, respectively. RESULTS: 11 patients were followed up with an average of 28.2 ± 12.6 (12-50) months. All the fractures were united smoothly without second intervention. At the latest visiting, the mean anterior flexion of affected shoulder was 157.3 ± 7.37° (range 150°-170°), the mean external rotation of the affected shoulder was 58.2 ± 7.5° (range 50°-70°). When the shoulder in the internal rotation, the extended thumb reached to L4 or L1 or T10 or T7 in one case, to T12 in two cases and to T8 in four cases, respectively, the mean Constant score was 91.7 ± 2.8 (86-96) points. The mean UCLA score was 32.7 ± 1.7 (30-35) points, leading to four cases of excellent and seven cases of good results. The mean DASH score was 7.4 ± 3.3 (3.4-13) points. CONCLUSION: Good outcomes could be obtained when Ideberg IV and V glenoid fractures were treated by open reduction and internal fixation through a Judet approach.

Targeting autophagy enhances atezolizumab-induced mitochondria-related apoptosis in osteosarcoma.

In this study, we identified the multifaceted effects of atezolizumab, a specific monoclonal antibody against PD-L1, in tumor suppression except for restoring antitumor immunity, and investigated the promising ways to improve its efficacy. Atezolizumab could inhibit the proliferation and induce immune-independent apoptosis of osteosarcoma cells. With further exploration, we found that atezolizumab could impair mitochondria of osteosarcoma cells, resulting in increased release of reactive oxygen species and cytochrome-c, eventually leading to mitochondrial-related apoptosis via activating JNK pathway. Nevertheless, the excessive release of reactive oxygen species also activated the protective autophagy of osteosarcoma cells. Therefore, when we combined atezolizumab with autophagy inhibitors, the cytotoxic effect of atezolizumab on osteosarcoma cells was significantly enhanced in vitro. Further in vivo experiments also confirmed that atezolizumab combined with chloroquine achieved the most significant antitumor effect. Taken together, our study indicates that atezolizumab can induce mitochondrial-related apoptosis and protective autophagy independently of the immune system, and targeting autophagy is a promising combinatorial approach to amplify its cytotoxicity.

Nanometer precise red blood cell sizing using a cost-effective quantitative dark field imaging system.

Because of the bulk, complexity, calibration requirements, and need for operator training, most current flow-based blood counting devices are not appropriate for field use. Standard imaging methods could be much more compact, inexpensive, and with minimal calibration requirements. However, due to the diffraction limit, imaging lacks the nanometer precision required to measure red blood cell volumes. To address this challenge, we utilize Mie scattering, which can measure nanometer-scale morphological information from cells, in a dark-field imaging geometry. The approach consists of a custom-built dark-field scattering microscope with symmetrically oblique illumination at a precisely defined angle to record wide-field images of diluted and sphered blood samples. Scattering intensities of each cell under three wavelengths are obtained by segmenting images via digital image processing. These scattering intensities are then used to determine size and hemoglobin information via Mie theory and machine learning. Validation on 90 clinical blood samples confirmed the ability to obtain mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW) with high accuracy. Simulations based on historical data suggest that an instrument with the accuracy achieved in this study could be used for widespread anemia screening.