Optimization of Formaldehyde Fixative Concentration for Individual Blood Cells to Develop a Stabilized Blood Control for Automated Hematology Analyzers.

In a modern haematology laboratory, the complete and differential counts of blood are performed using complex haematology auto analyzers. In order to ensure the accuracy and reliability of test results, various regulatory authorities have prescribed the use of stabilized blood controls. The major pitfalls of these blood controls are their short shelf life. This could be due to the fact that they are prepared by a common cocktail of fixatives which acts on the discrete cells in various ways and would result in either under-fixation or over-fixation of various cells. Thus, in the present study, we have explored and optimized fixative and buffering for individual cells to achieve stable blood control. Blood cells were isolated using the centrifugation technique and were fixed individually with different concentrations of formaldehyde. After fixation, cells were pooled. Analysis of cell count was done till six months. Cells were also analysed morphologically to see the effect of fixation and storage on cell morphology. In the present study we compared the effect of the concentration of formaldehyde fixative for individual cells in the blood and their role in enhancing the shelf life and maintaining the morphology of the cells when suspended in plasma or suitable buffers post-fixation. It was observed that WBCs can be better fixed with 3 and 3.5% formaldehyde in a buffered solution, whereas RBCs and Platelets can be optimally fixed with 2.5% formaldehyde in a buffered solution.

Physico chemical characterisation of pectin incorporated gelatin sponge and its functional evaluation in modulating contact activation haemostasis.

Even though it is a common occurrence in practice, maintaining haemostasis can sometimes become a challenging issue in case of trauma, perioperative period, coagulation disorders, cancers, etc. Hemostatic materials are extensively used to assist in the cessation of bleeding. However, the definition of efficiency of haemostasis varies between intended procedures. This paper explores the feasibility of incorporating agents to increase the efficiency of local haemostasis. Pectin or ß -D galacto hexopyranuronic acid/ß Gal A, a structural polysaccharide widely present in terrestrial plants having an intrinsic hemostatic potential, is blended with gelatin and is explored in modulating passive haemostasis. The sponges are physico chemically characterized, and their hemostatic efficiency is evaluated in vitro using various assays. Biocompatibility evaluation is done by in vitro cytotoxicity assay. The results suggest that this biopolymer combination is a promising candidate for hemostatic control.

To evaluate the feasibility of cadmium/tellurium (Cd/Te) quantum dots for developing N-terminal Natriuretic Peptide (NT-proBNP) in-vitro diagnostics.

Quantum dots have been widely used for biomedical applications like imaging, targeted drug delivery, and in-vitro diagnostics for better sensitivity. In-vitro diagnostic, lateral flow-based assay systems are gaining attention in the field of biomarker analysis mainly due to ease of test and quick availability of results. In the study, the potential of water-soluble carboxylic (-COOH) functionalized photoluminescent Cadmium Telluride Quantum Dots (CdTe) nanoparticles for lateral flow-based detection of N-terminal Natriuretic Peptide (NT-proBNP) biomarker (for heart failure) detection has been evaluated. Monoclonal antibodies were conjugated with COOH functionalized CdTe with EDC-NHS coupling chemistry, and conjugation was confirmed using FTIR. The CdTe nanoparticle exhibited an emission maximum at 715 nm when it is excited with 375 nm. The COOH functionalized CdTe showed an antigen concentration-dependent linearity in the lateral flow applications when the dye was prepared freshly and used. However, a relative reduction in CdTe quantum dot fluorescence intensity with time was observed. Factors such as low stability could be due to the quenching of the fluorescence of CdTe. This limits its commercial viability as an in-vitro diagnostic tool; thus, modifications of the quantum dots are required to have a stable preparation for its commercial potential for quantifications.

Development and evaluation of a multicomponent bioink consisting of alginate, gelatin, diethylaminoethyl cellulose and collagen peptide for 3D bioprinting of tissue construct for drug screening application.

Three dimensional (3D) bioprinting technology has been making a progressive advancement in the field of tissue engineering to produce tissue constructs that mimic the shape, framework, and microenvironment of an organ. The technology has not only paved the way to organ development but has been widely studied for its application in drug and cosmetic testing using 3D bioprinted constructs. However, not much has been explored on the utilization of bioprinting technology for the development of tumor models to test anti-cancer drug efficacy. The conventional methodology involves a two dimensional (2D) monolayer model to test cellular drug response which has multiple limitations owing to its inability to mimic the natural tissue environment. The choice of bioink for 3D bioprinting is critical as cell morphology and proliferation depend greatly on the property of bioink. In this study, we developed a multicomponent bioink composed of alginate, diethylaminoethyl cellulose, gelatin, and collagen peptide to generate a 3D bioprinted construct. The bioink has been characterised and validated for its printability, shape fidelity and biocompatibility to be used for generating tumor models. Further, a bioprinted tumor model was developed using lung cancer cell line and the efficacy of 3D printed construct for drug screening application was established.

Biofabrication of skin tissue constructs using alginate, gelatin and diethylaminoethyl cellulose bioink.

INTRODUCTION: Biofabrication of skin tissue equivalents using 3D bioprinting technology has gained much attention in recent times due to the simplicity, the versatility of the technology and its ability in bioengineering biomimetic tissue histology. The key component being the bioink, several groups are actively working on the development of various bioink formulations for optimal skin tissue construction. METHODS: Here, we present alginate (ALG), gelatin (GEL) and diethylaminoethyl cellulose (DCEL) based bioink formulation and its application in bioprinting and biofabrication of skin tissue equivalents. Briefly, DEAE cellulose powder was dispersed in alginate solution with constant stirring at 60 °C to obtain a uniform distribution of cellulose fibers; this was then mixed with GEL solution to prepare the bioink. The formulation was systematically characterized for its morphological, physical, chemical, rheological, biodegradation and biocompatibility properties. The printability, shape fidelity and cell-laden printing were assessed using the CellInk bioprinter. RESULTS: The bioink proved to be a good printable, non-cytotoxic and stable hydrogel formulation. The primary human fibroblast and keratinocyte-loaded 3D bioprinted constructs showed excellent cell viability, collagen synthesis, skin-specific marker and biomimetic tissue histology. CONCLUSION: The results demonstrated the successful formulation of ALG-GEL-DCEL bioink and its application in the development of human skin tissue equivalents with distinct epidermal-dermal histological features.

Evaluation of diagnostic accuracy of developed rapid SARS-COV-2 IgG antibody test kit using novel diluent system.

Immunochromatographic assay kits are used in primary diagnostics which is based on the principle of antigen and antibody interaction. These kits play pivotal role in rapid surveillance of infectious diseases at early stages as well as for the surveillance of the contagious diseases. The immunochromatographic test kits lacks sensitivity and specificity with certain diseases. In this study, our intention was to develop a rapid test kit for SARS-COV-2 with a novel diluent system to enhance the efficacy of antigen-antibody binding and thereby the improvement in the sensitivity outlined. Finally, IgG antibodies against SARS-COV-2 virus peptides were analyzed using 25 positive and 25 negative confirmed clinical samples. The sensitivity of the clinical studies showed 91% sensitivity and 100% specificity. Therefore, the authors propose that this assay will be a potential tool for efficient community or sentinel surveillance of SARS-COV-2 infection and additionally, for effective monitoring of convalescent sera therapy.

Scaffold for liver tissue engineering: Exploring the potential of fibrin incorporated alginate dialdehyde-gelatin hydrogel.

INTRODUCTION: Development of a tissue-engineered construct for hepatic regeneration remains a challenging task due to the lack of an optimum environment that support the growth of hepatocytes. Hydrogel systems possess many similarities with tissues and have the potential to provide the microenvironment essential for the cells to grow, proliferate, and remain functionally active. METHODS: In this work, fibrin (FIB) incorporated injectable alginate dialdehyde (ADA) - gelatin (G) hydrogel was explored as a matrix for liver tissue engineering. ADA was prepared by periodate oxidation of sodium alginate. An injectable formulation of ADA-G-FIB hydrogel was prepared and characterized by FTIR spectroscopy, Scanning Electron Microscopy, and Micro-Computed Tomography. HepG2 cells were cultured on the hydrogel system; cellular growth and functions were analyzed using various functional markers. RESULTS: FTIR spectra of ADA-G-FIB depicted the formation of Schiff's base at 1608.53 cm-1 with a gelation time of 3 min. ADA-G-FIB depicted a 3D surface topography with a pore size in the range of 100-200 µm. The non-cytotoxic nature of the scaffold was demonstrated using L929 cells and more than 80 % cell viability was observed. Functional analysis of cultured HepG2 cells demonstrated ICG uptake, albumin synthesis, CYP-P450 expression, and ammonia clearance. CONCLUSION: ADA-G-FIB hydrogel can be used as an effective 3D scaffold system for liver tissue engineering.

Effect of membrane parameters and filter structure on the efficiency of leukocyte removal by electrospun poly(ethylene-co-vinyl alcohol) membranes.

Electrospinning technology is an experimentally verified tool for the generation of membranes with competing efficiency useful for leukodepletion filter, customarily used in blood banks to lessen the risks with blood transfusions. Even though a few electrospun polymers have been reported to be efficient leukodepletion filter membranes, poly(ethylene-co-vinyl alcohol) (EVAL) being one among, owe to their exceptional advantage of being electrospun from a non-toxic solvent like rubbing alcohol. Besides their excellent blood compatibility, EVAL membranes proffer a leukodepletion performance nearly as same as that of commercial leukodepletion filters. However, the role of various membrane parameters on the leukodepletion efficiency of electrospun EVAL membranes need to be disclosed in advance to their commercialization. Hence this study is an attempt to disclose the ability of electrospinning to being tuned towards the fabrication of filters with different membrane parameters including fiber diameter and pore diameter, with a typical example of EVAL. In addition, the impact of filter design upon the leukodepletion performance was also unveiled by comparing a symmetric filter where all the membranes were of uniform pores with that of an asymmetric filter where the pore sizes of upper and lower layer in the filter was different. The results of blood filtration experiments through the developed prototype filters underline the superiority of asymmetric filters over to symmetric one and, the asymmetric filters wherever the fiber diameter was 1.8 and 3 µm, offered rapid and excellent leukodepletion. Membranes with thinner fibers showed an increased flow resistance. The pore sizes of the membranes being 9 - 29 µm, larger than the size of blood cells, alludes to the direct adhesion of leukocytes to filter membranes as the major mechanism of leukocyte removal. Hence it is concluded that despite the suitability of electrospinning for fabrication of leukodepletion filter media, the use of nano-dimension fibers is not preferred for leukocyte adhesion filter when the material of choice is such a polymer with ideal surface chemistry, wettability and inherent ability for leukocyte binding.

Formulation and Characterization of Alginate Dialdehyde, Gelatin, and Platelet-Rich Plasma-Based Bioink for Bioprinting Applications.

Layer-by-layer additive manufacturing process has evolved into three-dimensional (3D) "bio-printing" as a means of constructing cell-laden functional tissue equivalents. The process typically involves the mixing of cells of interest with an appropriate hydrogel, termed as "bioink", followed by printing and tissue maturation. An ideal bioink should have adequate mechanical, rheological, and biological features of the target tissues. However, native extracellular matrix (ECM) is made of an intricate milieu of soluble and non-soluble extracellular factors, and mimicking such a composition is challenging. To this end, here we report the formulation of a multi-component bioink composed of gelatin and alginate -based scaffolding material, as well as a platelet-rich plasma (PRP) suspension, which mimics the insoluble and soluble factors of native ECM respectively. Briefly, sodium alginate was subjected to controlled oxidation to yield alginate dialdehyde (ADA), and was mixed with gelatin and PRP in various volume ratios in the presence of borax. The formulation was systematically characterized for its gelation time, swelling, and water uptake, as well as its morphological, chemical, and rheological properties; furthermore, blood- and cytocompatibility were assessed as per ISO 10993 (International Organization for Standardization). Printability, shape fidelity, and cell-laden printing was evaluated using the RegenHU 3D Discovery bioprinter. The results indicated the successful development of ADA-gelatin-PRP based bioink for 3D bioprinting and biofabrication applications.

Glycine integrated zwitterionic hemocompatible electrospun poly(ethylene-co-vinyl alcohol) membranes for leukodepletion.

This paper describes a novel strategy for the hemocompatibility improvement of poly(ethylene-co-vinyl alcohol) (EVAL) membranes by incorporation of a naturally occurring zwitterion, glycine. Crystalline glycine was directly integrated to the EVAL fibers via electrospinning. The membranes were characterized by Attenuated Total Reflection-Fourier Transform Infrared spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM), Water Contact Angle measurements (WCA) and measurement of Critical Wetting Surface Tension (CWST). The impact of glycine integration on the membrane parameters was assessed by variations in fiber diameter, pore size and percentage porosity. The release of glycine from the membranes was also quantitatively evaluated by ninhydrin assay. The interplay of zwitterion structural features on the blood compatibility was studied by in vitro hemocompatibility evaluation and blood filtration studies. The outcomes of these investigations highlight that glycine incorporated membranes offer greater hemocompatibility than virgin EVAL membranes in terms of reduced hemolysis, increased RBC retention, decreased adhesion and activation of platelets. The type of membrane modification can be considered in future for the development of leukodepletion filter membranes.

Circulating Thrombotic Risk Factors in Young Patients with Coronary Artery Disease Who Are on Statins and Anti-platelet Drugs.

Thrombotic risk factors may contribute to premature coronary artery disease (CAD), in addition to the conventional risk factors. There is paucity of data on studies evaluating the role of thrombotic factors in premature CAD in Indian patients. Thus a case-control study was performed to evaluate the role of thrombotic and atherogenic factors in young patients with angiographically proven CAD who are on treatment with statins and anti-platelet drugs. 152 patients (≤55 years) with angiographically proven CAD and 102 asymptomatic controls were recruited. Clinical and biochemical data were obtained in both groups. Blood levels of thrombotic factors-fibrinogen, antithrombin-III, tissue-plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), von-Willebrand factor (v-WF), lipoprotein(a) [Lp(a)] and homocysteine were analyzed. Patients had high levels of conventional CAD risk factors (diabetes mellitus, smoking, hypertension, dyslipidemia and positive family history) compared to controls. Logistic regression analysis revealed that low antithrombin-III (odds ratio/OR 11.2; 95 % confidence interval/CI 2.29-54.01), high fibrinogen (OR 6.04; 95 % CI 1.09-33.21) and high Lp(a) (OR 4.54; 95 % CI 0.92-22.56), as important, independent risk factors in patients. PAI-1(OR 0.15; 95 % CI 0.03-0.69) levels were significantly lower in patients. But other thrombotic risk factors studied (t-PA, v-WF and homocysteine) were comparable among patients and controls. The treatment using statins and anti-platelet drugs might be contributing to the control of some of the thrombotic risk factors. The strategies aiming at lowering the levels of thrombotic risk factors along with conventional risk factors may be useful in primary and secondary prevention of CAD.

Enhanced P-selectin expression on platelet-a marker of platelet activation, in young patients with angiographically proven coronary artery disease.

P-selectin (CD62p) exposure is an established marker for platelet activation. P-selectin exposure can trigger variety of thrombotic and inflammatory reactions. In patients with coronary artery disease (CAD), platelets are activated, and hence, there is increased P-selectin exposure. The role of P-selectin exposure in patients on treatment with statins and anti-platelets is conflicting. A case-control study was performed to determine P-selectin exposure in consecutively recruited 142 patients (age ≤ 55 years) with angiographically proven CAD on treatment and 92 asymptomatic controls. P-selectin exposure was determined by flow cytometry. Data on conventional risk factors were obtained along with estimation of levels of thrombotic [fibrinogen, lipoprotein (a), tissue plasminogen activator, plasminogen activator inhibitor-1, homocysteine and von Willebrand factor] and anti-thrombotic factors (antithrombin III). The P-selectin exposure was compared among patient groups who had different modes of presentation of CAD and categories of CAD disease severity. The patients were followed up for a period of 26 months. The results indicate that P-selectin exposure was significantly elevated in patients (mean ± SD 9.24 ± 11.81) compared to controls (mean ± SD 1.48 ± 2.85) with p < 0.0001. Similarly, conventional risk factors were significantly elevated in patients. P-selectin exposure showed significant negative correlation with antithrombin III levels. P-selectin exposure was higher in patients who presented with acute coronary syndromes than those who presented with effort angina. Cardiovascular event rate was 6 % on follow-up. The study establishes that thrombotic-inflammatory pathways enhancing P-selectin exposure unrelated to treatment might be activated in patients, while the event rate remained lowered, and hence, treatment strategies should be inclusive to control these factors.

Effect of photografting 2-hydroxyethyl acrylate on the hemocompatibility of electrospun poly(ethylene-co-vinyl alcohol) fibroporous mats.

Poly(ethylene-co-vinyl alcohol) (EVAL) has been recommended as a material suitable for blood contacting applications. Effect of ethylene content and associated hydrophobicity of EVAL on the blood-material interactions have been documented in the literature. In this work, surface chemistry of EVAL substrate was altered by photografting a hydrophilic monomer, 2-hydroxyethyl acrylate (HEA) with the aid of a photoinitiator, benzophenone (BP), and the effect of surface modification on the blood-material interactions was investigated. Since the modified material was intended to be used as leukodepletion filters, a solution containing EVAL, HEA and BP was electrospun into fibroporous mats and UV treated to induce grafting. Degree of grafting, bonding between fibers and fiber diameter increased with increase in UV exposure time whereas mechanical properties showed a decreasing trend. Decreased water contact angle indicated improved wetting characteristics. In vitro hemocompatibility tests revealed that the modified EVAL surface exhibited significantly lower hemolytic activity, protein adsorption and platelet adhesion than neat EVAL. The modification did not have any substantial effect on the activation of the complement system and coagulation parameters. Photografting led to significant reduction in the adhesion of red blood cells (RBC) whereas white blood cell (WBC) consumption remained above 90%. The results implied that photografting HEA on EVAL substantially improves hemocompatibility of EVAL and when it is used as a filter, it selectively removes leukocytes and allows easy passage of other blood components.

Physicochemical and in vitro biocompatibility evaluation of water-soluble CdSe/ZnS core/shell.

Group II-VI semiconductor quantum dots (Q-dots) have found various applications in biomedical field during last decade. In this study, we have synthesized CdSe Q-dots and CdSe/ZnS core/shell (CS) by wet chemical route and characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier-transform infrared spectroscopy (FT-IR) and Photoluminescence (PL) spectroscopy. CS formation was confirmed by red shift as well as enhancement in the luminescence peak compared to bare Q-dots. Processing parameters such as core and sulfur concentrations were optimized at maximum luminescence efficiency during the shell preparation. Effects of dialysis, aging and cell culture medium on the properties of the Q-dots and CS were also studied by luminescence and DLS techniques. DLS data showed Q-dots and CS to be stable, and there was no effect on the integrity of the Q-dots and CS after various modifications. CS was found to be hemocompatible and cytocompatible for human umbilical vein endothelial cells even at a high concentration of 0.1 mg/ml up to 48 h indicating high potential for CS in various biomedical applications.

Mercaptoethanol capped CdSe quantum dots and CdSe/ZnS core/shell: synthesis, characterization and cytotoxicity evaluation.

CdSe Quantum dots (Q-dots) and CdSe/ZnS core/shell have been synthesized by wet chemical route using mercaptoethanol (ME) as cappant. The synthesized Q-dots and core/shell were characterized using X-ray diffraction (XRD), Transmission electron microscopy (TEM), Energy dispersive X-ray analysis (EDS), Dynamic Light Scattering (DLS), Optical absorption and luminescence spectroscopy. The core/shell formation was confirmed by both XRD and TEM analysis. The luminescence was shown to be considerably enhanced in the core/shell sample. Effect of dialysis process on the optical properties of the Q-dots and core/shell has also been discussed. Cytotoxicity studies have been carried out for Q-dots and core/shell. CdSe/ZnS core/shell was found to be non-cytotoxic as compared to CdSe Q-dots up to a certain concentration range. Polyethylene glycol (PEG) coating enhances the non-cytotoxic nature of CdSe/ZnS core/shell when compared with bare core/shell.