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Background: Von Willebrand factor (VWF) is a critical glycoprotein in hemostasis and is an important factor in diagnosing bleeding disorders. Albeit the analysis of VWF is often compromised by inconsistent methodologies and challenges quantifying multimeric size. Current VWF multimer analysis methods are costly, time-consuming, and often inconsistent; thus, demanding skilled professionals. This study aimed to streamline and optimize the VWF multimer analysis technique, making it more efficient and reproducible, particularly for identifying or predicting mechanical circulatory support (MCS) induced bleeding disorders., Methods: Blood samples from healthy volunteers were exposed to high shear forces via a Medtronic HeartWare ventricular assist device. VWF multimers were analyzed using vertical-gel agarose electrophoresis and Western blotting. Differences in VWF distribution were determined using densitometry, and two methods of densitometric analysis were compared: proprietary software against open-source software., Results: Using the developed method: (i) protocol duration was accelerated from three days (in classical methods) to ~ eight hours; (ii) the resolution of the high molecular weight (HMW) VWF multimers were substantially improved; and (iii) densitometric analysis tools were validated. Additionally, the densitometry analysis using two software types showed a strong correlation between results, with the proprietary software reporting slightly higher HMW VWF percentages., Conclusion: This methodology is recommended for affordable, accurate, and reproducible VWF multimer evaluations during MCS use and testing. Further research comparing this method with semi-automated methods would provide additional insight and improve inter-laboratory comparisons., (© 2024 The Author(s). Artificial Organs published by International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.)

The aim of this study was to develop and validate a High-Performance Thin Layer Chromatographic (HPTLC) method for simultaneous determination of ceftriaxone and ceftriaxone e-isomer in powder for injection formulation. Ceftriaxone sodium injection is an antibiotic that used globally. It has Z/E geometrical conformation, in which ceftriaxone sodium and 3 ene-isomer have Z- conformation while (E)-isomer has E- conformation and the potential toxicity of ceftriaxone (E)-isomer has been reported. Thus, to safeguard the public health, a simple and easy to use, rapid and reliable method was developed for qualitative and quantitative determination of ceftriaxone sodium and its (E)-isomer. Samples were applied on HPTLC glass plates precoated with silica gel 60F254 by using Linomat semi-auto sampler. Separation was carried out using acetone, triethyl amine, water, chloroform and ethyl acetate as a mobile phase in different ratios. The R f values of separated compounds were 0.51 ± 0.01 and 0.62 ± 0.01 for ceftriaxone sodium and ceftriaxone (E)-isomer respectively. The method was validated by studying Specificity, Linearity, Accuracy, Precision, Robustness, Limit of Detection (LOD) and Limit of Quantification (LOQ) and Solution stability. The developed method was successfully, sensitive, simple, precise, accurate, robust and applicable for the simultaneous determination of ceftriaxone sodium and ceftriaxone (E)-isomer in powder for injection formulation., (© 2024. The Author(s).)

Ophthalmic pharmaceutical preparation containing antazoline (ANT) and tetryzoline (TET) is prescribed widely as an over the counter medication for allergic conjunctivitis treatment. Development of a selective, simple and environmentally friendly thin-layer chromatographic method established to determine both ANT and TET in their pure forms, pharmaceutical formulation and spiked aqueous humor samples. By using silica gel plates and means of a developing system consists of ethyl acetate:ethanol (5:5, by volume), the studied drugs separation was achieved, and scanning was carried out at 220.0 nm for the separated bands with a 0.2-18.0 μg/band concentration range for each of ANT and TET. Standard addition technique application was carried out to determine the proposed method validity. Statistical comparison was made between the proposed method and the official methods ANT and TET showing no significant difference concerning accuracy and precision. Furthermore, greenness profile assessment was accomplished by means of four metric tools, namely, analytical greenness, green analytical procedure index, analytical eco-scale and national environmental method index., Highlights., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Purpose: To evaluate and compare the repeatability of corneal densitometry (CD) measurements obtained using both an anterior-segment optical coherence tomography (AS-OCT) device and a Scheimpflug camera system, while also assessing the level of agreement. The study also sought to investigate the correlation of CD with age, gender, and central corneal thickness (CCT) in normal eyes., Methods: CD measurements were obtained using the Casia 2 and the Pentacam AXL Wave. Data were collected on Total Corneal Densitometry and 4 concentric corneal annular areas, these are referred to as zone 1, denoting the central area, through to zone 4, designating the outermost peripheral region. Repeatability was assessed using intra-session test-retest variability, coefficient of variation (CoV), and intraclass correlation coefficient (ICC). The agreement was evaluated using Bland-Altman plots. Correlation analysis was performed between CD, age, gender, and CCT., Results: The study included 96 healthy volunteers. The Casia 2 demonstrated high repeatability with ICC values exceeding 0.9 in all the corneal zones and lower CoV values compared to the Pentacam AXL Wave (ranging from 1.07% to 2.25% for Casia 2 and from 1.91% to 6.89% for Pentacam).95% LoA were within ± 2 standard deviation from the average mean except from zone 1 (± 2.42).However, the measurements showed a consistent bias among all the corneal zones. CD values were positively correlated with age, except for zone 1 with the Pentacam (p = 0.083)., Conclusions: The findings suggest that the Casia 2 can be a reliable tool for assessing corneal transparency in healthy individuals, however its measurements are not interchangeable with those provided by the Pentacam. The AS-OCT device may be more sensitive in detecting subtle age-related changes in CD within the central zone., (© 2024. The Author(s).)

The purpose of this study was to evaluate the quantitative computed tomography (CT) volumetry and densitometry and in pediatric patients with pectus excavatum (PE). We measured pectus index (PI) and separated inspiratory and expiratory lung volumes and densities. We obtained the total lung volume (TLV) and mean lung density (MLD) during inspiration and expiration, and the ratio of end expiratory to inspiratory volume (E/I volume) and MLD (E/I density) were calculated. The difference between inspiratory and end expiratory volume (I-E volume) and MLD (I-E density) were also calculated. A total of 199 patients, including 164 PE patients and 35 controls, were included in this study. The result shows that the PE group had lower inspiratory TLV (mean, 2670.76±1364.22 ml) than the control group (3219.57±1313.87 ml; p = 0.027). In the PE group, the inspiratory (-787.21±52.27 HU vs. -804.94±63.3 HU) and expiratory MLD (-704.51±55.41 HU vs. -675.83±64.62 HU) were significantly lower than the indices obtained from the control group (p = 0.006). In addition, significantly lower values of TLV and MLD difference and higher value of TLV and MLD ratio were found in the PE group (p <0.0001). PE patients were divided into severe vs. mild groups based on the PI cutoff value of 3.5. The inspiratory MLD and TLV ratio in the severe PE group were lower than those in the mild PE group, respectively (p <0.05). In conclusion, quantitative pulmonary evaluation through CT in pediatric PE patients may provide further information in assessing the functional changes in lung parenchyma as a result of chest wall deformity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Song, Im. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: To report the clinical course and compare the utility of Scheimpflug tomography (ST) and anterior segment optical coherence tomography (AS-OCT) for central corneal thickness (CCT) and corneal densitometry (CD) assessment in patients with corneal crystals owing to nephropathic cystinosis., Methods: A retrospective chart analysis of three patients with nephropathic cystinosis and the presence of corneal cystine crystals in both eyes was performed. All patients underwent clinical examination and anterior segment photography, ST, and AS-OCT scans. Corneal densitometry was exported from built-in proprietary software for ST and from custom-made validated software for AS-OCT. Anterior segment optical coherence tomography images were rescaled to grayscale units from 0 (maximum transparency) to 100 (minimum transparency) to match built-in ST densitometry readings. Furthermore, the mean pixel intensity, representative of CD, was calculated from the pixels corresponding to the segmented cornea., Results: All three patients had pathognomonic cystine crystals deposits in the cornea and were treated with cysteamine medications that resulted in clinical improvement. The CCT measured using ST exhibited a range from 560 to 958 μm. Conversely, when assessed with AS-OCT, the CCT varied within the range of 548 to 610 μm. Both examinations could be performed, but in the more severe cases, AS-OCT showed far greater utility to estimate CD. In four of six eyes examined, ST showed disproportionate CCT values, compared with the AS-OCT, whereas reliable CD measurements were only available in AS-OCT., Conclusion: The AS-OCT could be considered a baseline ocular measurement in cystinosis and in the evaluation of disease progression and treatment efficacy., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Contact Lens Association of Ophthalmologists.)

Objective: To evaluate the effects of rosacea on ocular surface changes such as alterations in dry eye parameters, corneal densitometry, and aberrations, in comparison with healthy controls., Methods: A total of 88 eyes of 44 patients diagnosed with rosacea and 88 eyes of 44 healthy controls were enrolled in this cross-sectional study. All participants underwent a comprehensive dermatologic and ophthalmic examination and Tear Break-Up Time (TBUT) and Schirmer-1 tests were performed. The rosacea subtype and Demodex count and OSDI scores of all participants were recorded. Corneal topographic, densitometric, and aberrometric measurements were obtained using the Scheimpflug imaging system., Results: The mean age of the 44 patients was 41.2 ± 11.0 years of whom 31 (70.5%) were female. The mean TBUT and Schirmer-1 test values were significantly decreased and OSDI scores were significantly increased in the rosacea group compared to healthy controls (p < 0.01 for all). The most common subtype of rosacea was erythematotelangiectatic rosacea (70.4%). The severity grading of rosacea revealed that 18 (40.9%) patients had moderate erythema. The median (min-max) Demodex count was 14.0 (0-120) and the disease duration was 24.0 (5-360) months. The comparison of the corneal densitometry values revealed that the densitometry measurements in all concentric zones, especially in central and posterior zones were higher in rosacea patients. Corneal aberrometric values in the posterior surface were also lower in the rosacea group compared to healthy controls. The topographic anterior chamber values were significantly lower in the rosacea group., Study Limitations: Relatively small sample size, variable time interval to hospital admission, and lack of follow-up data are among the limitations of the study. Future studies with larger sample sizes may also enlighten the mechanisms of controversial anterior segment findings by evaluating rosacea patients who have uveitis and those who do not., Conclusion: Given the fact that ocular signs may precede cutaneous disease, rosacea is frequently underrecognized by ophthalmologists. Therefore, a comprehensive examination of the ocular surface and assessment of the anterior segment is essential. The main priority of the ophthalmologist is to treat meibomian gland dysfunction and Demodex infection to prevent undesired ocular outcomes., (Copyright © 2024. Published by Elsevier España, S.L.U.)

Purpose: To compare corneal haze between active ulcer and healed scarring using a Scheimpflug densitometry., Materials and Methods: A prospective longitudinal study enrolled 30 patients (30 eyes) with ulcerative keratitis (UK). Each subject's corneal optical density (COD) was measured with a Scheimpflug corneal densitometry, Pentacam® AXL (Oculus GmbH, Wetzlar, Germany), at the active ulcerative and complete scarring stage. The COD data were analyzed through distinct methods (inbuilt, sorted annular partitions, and ulcer-matching densitometric maps). We compared different CODs to select the better index for clinically monitoring the transition from corneal ulceration to healed scar., Results: The CODs of the periphery (P = 0.0024) and outside of the active ulcer (P = 0.0002) significantly decreased after scarring. Partitioning the cornea into different depths and annular zones, the anterior layer, center layer, and the 2-6 mm annular zone had a more remarkable COD decrease after scar formation. The 3rd-sorted COD in the anterior layer revealed the highest area under the receiver-operating characteristic curves (0.709), in which 90% of subjects had COD reduction during the ulcer-to-scar transition., Conclusions: Aside from subjective judgment based on clinical signs, the Scheimpflug tomography-based densitometry could provide objective and efficient monitoring of the corneal opacity evolution in UK patients. Because the 3rd-sorted annular COD is a better index than the inbuilt or mapping CODs in differentiating active ulcers from healed scars, this COD could be a clinically promising parameter to monitor the progression of UK patients., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A novel and highly sensitive high-performance thin-layer chromatographic (HPTLC) method was developed and validated to quantify a combination of five pharmaceutical mixtures spiked to human plasma. The compounds comprised Amlodipine (AML) along with five angiotensin II receptor antagonist drugs (AIIRAs), namely Olmesartan (OLM), Telmisartan (TLM), Candesartan (CAN), Losartan (LOS), and Irbesartan (IRB). HPTLC was performed on silica gel 60 F254 plates using a mobile phase of Toluene: ethyl acetate: methanol: acetone: acetic acid (6:1.5:1:0.5:1, v/v/v/v/v). In a pioneering move, a reflectance/fluorescence detection mode was employed to identify two concurrently administered drugs at different pH levels for the first time. This method utilized the same chromatographic system, incorporating a specific measurement for AML at a neutral medium to achieve its maximum fluorescence at a 360 nm excitation wavelength, and measuring emission using a 540 nm optical filter. The process involved obtaining a very low fluorescence response from AIIRA. Subsequently, to enhance AIIRA's fluorescence, the plate was sprayed with perchloric acid to transition to a strong acidic medium, ultimately attaining the maximum fluorescence of AIIRA using various excitation wavelengths and a 400 nm emission filter. Through this strategic process, we could optimize the fluorescence signals of both drugs, thereby elevating the sensitivity of detection for this drug combination. AML demonstrated a linear range of 18-300 ng/band, while AIIRAs drugs exhibited a linear range of 6-150 ng/band. The method satisfied the International Conference on Harmonization (ICH) criteria for recovery, precision, repeatability, and robustness, showcasing exceptional sensitivity. The approach was successfully applied to quantify AML and AIIRAs drugs in both bulk drug and plasma samples, achieving high recovery percentages and minimal standard deviations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Purpose: To investigate the clinical significance of the correlation between optical densitometry and both biomechanical and morphological parameters in keratoconus (KC) and to verify the diagnostic value of optical densitometry in KC., Method: This cross-sectional study included 436 eyes of 295 patients with KC. Corneal optical densitometry, morphological parameters and biomechanical parameters were measured. Spearman's correlation analysis was employed to investigate the association between optical densitometry and both biomechanical and morphological parameters., Result: Optical densitometry of the anterior (0-2 mm and 2-6 mm), central (0-2 mm), posterior (2-6 mm) and total (2-6 mm) layers correlated positively with SPA1, while the posterior layer (0-2 mm) correlated negatively. Optical densitometry of the anterior layers 2-6 mm, 6-10 mm, and the central layer 6-10 mm negatively affected AL1, while the posterior layer 0-2 mm positively affected it. Optical densitometry of the anterior, central, and posterior layers 0-2 mm and 2-6 mm positively influenced the morphological parameters K 1 F , K 2 F , K m F and the absolute values of K 1 B , K 2 B , K m B . Optical densitometry of the center (0-2 mm) and posterior (2-6 mm) layers negatively influenced TCT. Optical densitometry of the anterior (0-2 mm and 2-6 mm), center (0-2 mm), posterior (2-6 mm) and total (2-6 mm) layers correlated positively with ACE and PCE, whereas the posterior layer (0-2 mm) correlated negatively., Conclusion: Optical densitometry was correlated with biomechanical and morphological parameters in keratoconus, suggesting its potential as a diagnostic indicator for assessing keratoconus progression and treatment efficacy., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Examination of corneal densitometry measurements using the Pentacam Scheimpflug imaging system in cases of pseudoexfoliative glaucoma (PEXG) and ocular hypertension (OHT)., Methods: The study included 50 eyes of 29 PEXG patients, 25 eyes of 16 OHT patients, and 76 eyes of 38 healthy control subjects followed in the glaucoma clinic. Corneal densitometry values of all cases were measured using the Scheimpflug imaging system (Pentacam, Oculus, Germany). Corneal densitometry was assessed based on 4 concentric radial zones (0-2 mm, 2-6 mm, 6-10 mm, and 10-12 mm) and depths (anterior, central, posterior, and total) within the Scheimpflug imaging system. The results were statistically analyzed., Results: Corneal densitometry values examined between the OHT and control groups were higher in OHT and statistically significant (p < 0.05). Corneal densitometry values examined between the PEXG and control groups were higher in PEXG and statistically significant (p < 0.05). In comparison between the PEXG and OHT groups, corneal densitometry values in the central 0-2 mm, 2-6 mm, and 6-10 mm; posterior 0-2 mm and 2-6 mm radial zones were higher in PEXG and statistically significant (p < 0.05)., Conclusions: It was observed that elevated intraocular pressure levels in OHT cases could lead to changes in the cornea, consequently increasing corneal densitometry values. The higher corneal densitometry values in PEXG cases compared to OHT were attributed to the accumulation of pseudoexfoliative material in the cornea. Based on our study, corneal densitometry could serve as a potential biomarker for early glaucoma detection in OHT cases and could be employed to assess corneal transparency during the follow-up of PEXG cases., Competing Interests: Declaration of competing interest No external funding was received for this study, (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Objective: To justify the optimal method for determining indocyanine green plasma disappearance rate (PDR ICG )., Material and Methods: We analyzed PDR ICG in intensive care units. Indocyanine green was administered intravenously at a dose of 0.25 mg/kg. PDR ICG was analyzed simultaneously by using of three methods: 1) PDD (PiCCO2 LiMON device), 2) SBS with analysis of plasma samples on precise spectrophotometer, 3) SBS with analysis of plasma samples on simple experimental photometer., Results: PDD method was used for 346 PDR ICG tests in 256 patients. Of these, 14.3% of measurements were erroneous. Paired tests using PDD and SBS methods were performed in 299 cases. SBS method resulted erroneous data in 0.6% of cases. Certain correlation ( r =0.79, p <0.001) was found between the reference method (SBS with spectrophotometry) and the PDD method. Bland-Altman plot for these two methods showed that proportional bias of mean difference was caused by extremely high PDR ICG of the PDD method (for example, more than 30%/min). Comparison of two SBS variants (spectrophotometer and experimental photometer) revealed good correlation ( r =0.91, p <0.001)., Conclusion: SBS method for measuring PDR ICG ensures accurate results under mechanical interferences in patients with impaired capillary blood flow. This eliminates the need for redo measurement. Duplication of the PDD and SBS methods is recommended when repeating the test is not possible (organ donors).

The common antihypertensive drugs are B-blockers and diuretics. For the determination of beta-blocker medicines (bisoprolol fumarate and carvedilol) and diuretic drug (Furosemide), new and accurate chromatographic method has been developed. The separation was achieved using a developing system that includes chloroform:methanol:ethyl acetate:ammonia (6:2:2:0.2 by volume) as a mobile phase and the bands were detected at 240 nm. The concentration ranges were 5-25, 1-7, and 1-3.5 μg/band for bisoprolol fumarate, carvedilol, and furosemide, respectively. This chromatographic approach is the first methodology for simultaneously determining bisoprolol fumarate, carvedilol, and furosemide in their pure forms and in their pharmaceutical dosage forms. The advantages of using known analytical procedures are their simplicity, speed, cost effectiveness, lack of laboriousness, and ability to save time as the three tablets are determined in one step and can be used for routine analysis of the investigated combinations in quality control laboratories. According to International Conference of Harmonization guidelines, the established procedures have been validated, and the results were statistically compared to those obtained by the reported reversed-phase-high-performance liquid chromatography methods using Student's t-test and F-test, with no significant difference between them, indicating that the proposed methods can be used for routine drug quality control analysis., (© 2023 Wiley-VCH GmbH.)

Recently, the aim of analytical community is to reduce the usage of hazardous chemicals; so eco-friendly, rapid, selective and cost-effective methods were developed for simultaneous determination of montelukast sodium (MKT) and loratadine (LRT). The first method was based on chromatographic separation performed on precoated silica gel 60 GF254 plates with ethyl acetate-ethanol 9: 1 (v/v) as the mobile phase. The developed plates were scanned and quantified at 260 nm. The method gives linear correlation over concentration ranges of 0.3-3.6 μg/spot and 0.2-4.0 μg/spot for MKT and LRT, respectively. It was also successfully applied to analysis of both drugs in their pharmaceutical preparation and human plasma. The other methods are UV-spectrophotometric methods based on smart spectra manipulating to zero order spectrum of each drug. These methods are named response correlation (RC), a-centering and ratio derivative methods. RC and a-centering methods were dependent on the presence of an isosbestic point between the overlapped spectra of both drugs. While ratio derivative method based on manipulation of the ratio spectra of both drugs. The two drugs obey Beer-Lambert law over the concentration ranges of 3.0-30.0 μg/mL in the three spectrophotometric methods. Moreover, the greenness of the developed methods is assessed using suitable analytical Eco-Scale and Green Analytical Procedure Index., (Published by Oxford University Press 2023.)

Purpose: By comparing the densitometry findings of patients with type 1 diabetes mellitus (T1DM) and the healthy group, we aimed to understand the possible changes that may occur in the cornea and lens in those with diabetes mellitus (DM)., Design: Prospective, cross-sectional study., Methods: A total of 60 eyes of 60 patients with T1DM and 101 eyes of 101 healthy subjects were included in this study. A complete ophthalmologic evaluation was performed in all participants. Scheimpflug tomography was performed to record the corneal and lens densitometry and other tomographic data. Mean glycosylated hemoglobin (HbA1c) and mean DM duration were recorded., Results: The mean age of the patients with T1DM and patients in the control group was 29.93 ± 8.56 years and 27.27 ± 1 4.96 years, respectively. The mean HbA1c was 8.43 ± 1.92, and the mean DM duration was 14.10 ± 7.77 years. Corneal densitometry (CD) values were significantly higher in the 0- to 2-mm zone in all layers and in the anterior and central 6- to 10-mm zone in the diabetic group (P = .03, P = .018, P = .001, P = .000, P = .004). The mean crystalline lens densitometry was higher in the T1DM group (P = .129). There were positive correlations between DM duration and CD in the anterior 0- to 2-mm (P = .043), central 6- to 10-mm (P = .016), posterior 6- to 10-mm (P = .022), and posterior 10- to 12-mm zone (P = .043)., Conclusions: CD values were significantly higher in the diabetic group. Diabetes duration and HbA1c values showed correlations with densitometry especially in the 6- to 10-mm corneal zone. Evaluation of the cornea with optical densitometry will be useful in the early diagnosis and follow-up of clinical structural and functional changes in the cornea., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The healthy cornea is transparent, however, disease can affect its structure, rendering it more or less opaque. The ability to assess the clarity of the cornea objectively could thus be of considerable interest for keratoconus patients. It has previously been suggested that densitometry can be used to diagnose early keratoconus, and that the values of densitometry variables increase with increasing disease severity, indicating that densitometry could also be used to assess progressive keratoconus. Previous studies have only assessed the repeatability of corneal densitometry measurements on the same day, which does not reflect the clinical setting in which changes are evaluated over time. We have therefore evaluated the inter-day repeatability of densitometry measurements in both patients with keratoconus and healthy controls. Measurements in the middle layer of the 2-6 mm zone of the cornea showed the best repeatability. Although an objective measure of the corneal transparency could be interesting, the generally poor repeatability of densitometry measurements limits their use. The repeatability of corneal clarity measurements could be improved by using other approaches such as optical coherence tomography, but this remains to be investigated. Such improvements would allow the more widespread use of corneal densitometry in clinical practice., (© 2023. The Author(s).)

Purpose: To evaluate the long-term effects of uncomplicated traumatic hyphema on endothelial morphology, anterior segment structure, and corneal and lenticular densitometry., Methods: In this retrospective comparative study, eyes with a history of uncomplicated traumatic hyphema were compared with the healthy contralateral unaffected eyes. The corneal endothelial cell properties were captured using specular microscopy. Anterior segment analysis, corneal densitometry (12-mm corneal diameter), and lens densitometry measurements were performed using the Pentacam imaging system., Results: Measurements were obtained at a mean follow-up of 49.5 ± 15.8 months after injury. The average endothelial cell density was significantly lower in the study group than in the control group (2,506.6 ± 294.0 cells/mm² vs. 2,665.7 ± 195.0 cells/mm², p = 0.020). There was no difference between the groups in respect of polymegathism and pleomorphism (p = 0.061 and p = 0.558, respectively). All the investigated corneal tomographic and angle parameters were similar in both groups (all p > 0.05). The corneal densitometry values in all concentric zones and layers showed no statistically significant difference between the groups (p > 0.05 for all). The lens zone 1 densitometry value was significantly higher in the study group than in the control group (9.6% ± 1.1% vs. 8.9% ± 1.2%, p = 0.031). No difference was observed in zone 2 and 3 (p = 0.170 and p = 0.322, respectively). The degree of hyphema was not correlated with endothelial cell and lenticular clarity loss (p = 0.087 and p = 0.294, respectively)., Conclusions: Even if traumatic hyphema is not complicated, long-term outcomes indicate endothelial cell loss and increased lenticular density.

From evolution, thin-layer chromatography (TLC) attracts attention as a versatile technique for efficient separation and identification of many drug substances and chemicals. Owing to its simplicity and other outstanding advantages, TLC is extensively used by chromatographers in quantification and purity profiling objectives. In the present study two TLC-Densitometric methods are established and validated for the synchronous estimation of Cinnarizine (Cinn) and Acefyline Heptaminol (Acef) in the presence of Cinn/Acef reported degradation products and Thoephylline (Theo) as Acef potential impurity. The proposed methods are based on densitometric measurements of the spots of Cinn and Acef after separation from their degradation products. Separation is attained on silica gel sheet with dichloromethane: methanol: formic acid as a developing system in ratio: (15, 1, 0.5, by volume) and (15, 0.75, 0.4, by volume) for Cinn (method 1) and Acef (method 2) degradation, consecutively. Quantification is done at 254 nm over concentration ranges of 0.2-1.8 and 2-18 μg/spot for Cinn and Acef; respectively, with mean percentage recoveries of 99.18 ± 0.60/99.84 ± 0.53 and 99.19 ± 0.93/99.66 ± 0.58 for method 1 and method 2; consecutively. The two methods are fully validated and proven to be selective, robust and retained their accuracy in up to 50% of Cinn/Acef reported degradation products and Theo. Moreover, the two methods are applied to a coformulated drug product comprising Cinn and Acef showing satisfactory results. Comparison of the obtained results by the proposed methods with that of the reference ones statistically shows no significant differences., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Skin thickness, including the adipose layer, which varies from individual to individual, affects the bone density measurement using light. In this study, we proposed a method to measure skin thickness using light and to correct the bias caused by differences in skin thickness and verified the proposed method by experiments using a phantom. We measured simulated skin of different thicknesses and bovine trabecular bone of different bone mineral densities (BMDs) using an optical system consisting of lasers of 850 and 515 nm wavelengths, lenses, and slits. Although the slope of the light intensity distribution formed on the surface of the material when irradiated by the 850 nm laser is affected by the thickness of the skin phantom. The difference of the intensity distribution peaks (δ y ) between the 850 and 515 nm lasers was strongly correlated with the thickness of the skin phantom. The coefficient of determination between the measurements and the BMD was improved by correcting the 850 nm laser measurements with δ y . This result suggests that the method is applicable to optical bone densitometry, which is insensitive to differences in skin thickness., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Two sensitive, selective and precise chromatographic methods have been established for concomitant quantification of ciprofloxacin HCl (CIP), fluocinolone acetonide (FLU) along with ciprofloxacin impurity A (CIP-imp A). The first method was thin-layer chromatography (TLC-densitometry) where separation was accomplished using TLC silica plates 60 G.F254 as a stationary phase and chloroform-methanol-33%ammonia (4.6:4.4:1, by volume) as a developing system. The obtained plates were scanned at 260 nm over concentration ranges of 1.0-40.0, 0.6-20.0 and 1.0-40.0 μg band-1 for CIP, FLU and CIP-imp A, respectively. The second method was based on high-performance liquid chromatography using a Zorbax ODS column (5 μm, 150 × 4.6 mm i.d.) where adequate separation was achieved through a mobile phase composed of phosphate buffer pH 3.6-acetonitrile (45:55, v/v) at flow rate 1.0 mL min-1 with ultraviolet detection at 254 nm. Linear regressions were obtained in the range of 1.0-40.0 μg mL-1 for CIP, 0.6-20.0 μg mL-1 for FLU and 1.0-40.0 μg mL-1 for CIP-imp A. The suggested methods were validated in compliance with the International Conference on Harmonization guidelines and were successfully applied for determination of CIP and FLU in bulk powder and newly marketed otic solution., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: To investigate whether Pentacam densitometry readings are affected by corneal tilt., Methods: In a prospective study, the right eyes of 86 healthy participants aged 42.8 ± 20.0 years (range 18-79 years) were imaged using Scheimpflug tomography. Elevation maps were exported to calculate corneal tilt using custom-made software, and densitometry readings were acquired directly from the corneal densitometry analysis add-on to the standard software Oculus Pentacam HR. Simple mediation analysis was applied to study age as a confounding factor in the correlation between corneal tilt and corneal densitometry., Results: Corneal tilt and corneal densitometry are not independent from one another because age is significantly correlated with both corneal tilt (r = 0.50, p < 0.001) and corneal densitometry (r = 0.91, p < 0.001). Only 3.8% of the correlation between tilt and densitometry operates directly, while the remaining 96.2% depends on age., Conclusions: Corneal tilt plays a role in corneal densitometry readings, even though the interaction is strongly influenced by age. Age is a well-known factor in densitometry readings that should be taken into consideration when interpreting Scheimpflug densitometry., (© 2022 The Authors. Ophthalmic and Physiological Optics published by John Wiley & Sons Ltd on behalf of College of Optometrists.)

Background: This study aimed to investigate the presence of subclinical inflammation in the eye by examining corneal and lens changes in children with familial Mediterranean fever (FMF) and provide the regulation for follow-up and treatment protocols according to the presence of signs of inflammation., Design: This is a cross-sectional, case-control study., Methods: Topographic parameters and corneal and lens densitometry of 48 patients with FMF (10.12 ± 3.84 years [range: 5-19 years]) and 33 healthy volunteers (10.94 ± 3.78 years [range: 5-19 years]; p > 0.05) were evaluated with a Scheimpflug camera. For corneal densitometric measurements, the cornea was divided into four concentric radial zones and anterior, central, and posterior layers according to corneal thickness. The mean densitometry value for the crystalline lens was calculated in three zones around the center of the pupil., Results: Corneal light backscattering in all layers and zones of the cornea were similar between the patient and control groups. Lens densitometry values in the three zones did not differ between the two groups (p > 0.05). Maximum lens densitometry values were found to be significantly higher in the patient group (22.14 vs 19.11; p = 0.011)., Conclusion: Monitoring the cornea and lens density in patients with FMF using Pentacam may help to show the presence of subclinical inflammation and regulate the follow-up and treatment protocols. Larger sample sizes and prospective design studies are needed to reach more conclusive results., Competing Interests: Declaration of Competing Interest The authors report there are no competing interests to declare., (Copyright © 2022. Published by Elsevier B.V.)

Purpose: The purpose of this study is to analyze the course of corneal densitometry (CD), endothelial cell density (ECD), best-corrected visual acuity (BCVA), and central corneal thickness (CCT) 5 years after uneventful Descemet membrane endothelial keratoplasty (DMEK)., Methods: Sixty uneventful cases (51 patients) with a minimum follow-up of 5 years were included. CD of various corneal layers (anterior, central, posterior, and total layer) and zones (0-2 mm, 2-6 mm, and 6-10 mm) were measured with Scheimpflug tomography. ECD, BCVA, and CCT were also evaluated., Results: Total CD at 0 to 2 mm and 2 to 6 mm zones significantly decreased from 33 ± 10 and 27.8 ± 8 grayscale units (GSU) preoperatively to 21.8 ± 3.1 and 22.2 ± 4.2 GSU at 5 years, respectively ( P < 0.001). On the contrary, total CD at the 6 to 10 mm zone significantly increased from 30 ± 8.3 GSU preoperatively to 34.6 ± 7.8 GSU at 5 years ( P < 0.001). ECD significantly decreased from 2496 ± 267 cells/mm 2 preoperatively to 1063 ± 470 cells/mm 2 at 5 years ( P < 0.001). Similarly, CCT significantly decreased from 686 ± 109 μm preoperatively to 557 ± 37 μm at 5 years ( P < 0.001). Postoperative BCVA was significantly better after DMEK for every examination time point., Conclusions: Despite a slight CD increase at all layers of all corneal zones from the second to the fifth postoperative year, the excellent visual outcome was maintained throughout 5-year follow-up. Thus, DMEK seems to effectively treat corneal endothelial disease in the long term., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: To describe a new algorithm to measure corneal densitometry based on images obtained by swept source anterior segment ocular coherence tomography (SS-AS-OCT) and establish standard densitometry values in a group of normal eyes., Methods: A total of 111 healthy participants (195 eyes) were enrolled in this study. Using a MATLAB designed algorithm, the cornea was segmented into three layers: anterior, posterior and mid-stroma, and it was divided into two concentric areas, 0-2 and 2-4 mm, resulting in nine areas for the analysis. The mean corneal densitometry values were calculated and expressed as grayscale units (GSU)., Results: The mean age was 57 years (range 22-87), with 100 (51.3%) right eyes and 95 (48.7%) left eyes. The total corneal densitometry was 86.9 ± 12.1 GSU. The mid-stroma layer had the highest densitometry values, 87.4 ± 12.1 GSU, and the anterior layer had the lowest values, 81.9 ± 14.2 GSU. Densitometry differences between the anterior layer and the mid-stroma layer (P < 0.001), as well as the anterior layer and the posterior layer (P < 0.05) were statistically significant. The 0-2 mm concentric area had higher mean densitometry values, 97.8 ± 12.7 GSU, and the differences were significant compared to the 2-4 mm concentric area (P < 0.001). No correlation was found between the corneal densitometry values and gender or age., Conclusions: The new MATLAB segmentation algorithm for the analysis of corneal SS-AS-OCT images is capable to objectively assess corneal densitometry. We provide standard and normal data for better clinical and research approach., (© 2021. The Author(s), under exclusive licence to The Royal College of Ophthalmologists.)

Sofosbuvir and daclatasvir are co-formulated as directly acting antiviral agents used for treatment of hepatitis C virus. Two chromatographic methods were developed for their determination; the first one is an Reversed phase-high performance liquid chromatography (RP-HPLC) method, in which the separation was performed on C8 Zorbax® SB column (4.6 × 250 mm, 5 μm) using acetonitrile:water:0.05 M phosphate buffer, pH = 8 (50:45:5, v/v/v) as a mobile phase, and ultraviolet detection was performed at 280 nm. Good resolution was obtained, and linearity was confirmed in the range of 10-100 μg/mL for both drugs. The second method is Thin layer chromatography (TLC)-densitometric one, in which sofosbuvir and daclatasvir were separated on silica gel plates using ethyl acetate:hexane:methanol (9:0.5:0.5, v/v/v) as a developing system and the scanning wavelength was 280 nm. Linearity was confirmed over a concentration range of 0.4-25.4 μg/band for sofosbuvir, whereas for daclatasvir linearity scanning was in the range of 0.4-12.8 μg/band. Both antiviral agents can be quantified simultaneously in one analytical run, which is a great time- and cost-saving valor of the developed methods. This valor is even more important in the case of the combined dosage form (Darvoni® tablets) to the pharmaceutical market., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Introduction: Thymoquinone (TQ) is a naturally derived bioactive compound with several therapeutic effects., Objective: The highly sensitive, rapid and green normal-phase (NP)/reversed-phase (RP) high-performance thin-layer chromatography (HPTLC) densitometry technique was developed for the determination of TQ in various plant extracts of different geographical regions, commercial capsules, creams and essential oils., Methodology: The NP densitometry estimation of TQ was performed using a cyclohexane-ethyl acetate (90:10, v/v) green solvent system, while, the RP-densitometry estimation of TQ was performed using an ethanol-water (80:20, v/v) green solvent system. The estimation of TQ was conducted at 259 nm., Results: The NP and RP densitometry techniques were observed linear in the range of 25-1000 and 50-600 ng/band, respectively. All validation parameters such as accuracy, precision, robustness and sensitivity of NP/RP densitometry were observed within the limit of regulatory requirements and hence found to be suitable for the determination of TQ. The TQ contents were found to be highest in the Saudi Arabian extract followed by the Syrian extract, Indian extract, commercial capsules, commercial creams, Jordanian extract, Egyptian extract, Palestinian extract and commercial essential oils using NP densitometry. The TQ contents were found in same order using RP densitometry, but they were much lower than those recorded using NP densitometry. The Analytical GREEnness (AGREE) scores of NP and RP densitometry were found to be 0.82 and 0.84, respectively, suggesting an excellent greenness profile., Conclusions: Based on these results, NP/RP densitometry was found to be suitable for the pharmaceutical assay of TQ., (© 2021 John Wiley & Sons, Ltd.)

Purpose: To quantify changes in corneal densitometry after long-term orthokeratology treatment in myopic children and to analyze the reversibility one month after discontinuation., Methods: Seventy-four myopic subjects aged 8-16 years, who wore orthokeratology lenses for two years, were divided into relatively steep- (lens movement within 1.0-1.5 mm, thirty-six participants) and flat-fitting groups (lens movement within 1.5-2.0 mm, thirty-eight participants). Based on refractive errors, they were divided into low and moderate myopia groups (thirty-seven participants in each group). Corneal densitometry was performed using Pentacam (Oculus Optikgeräte GmbH, Wetzlar, Germany) at each follow-up timepoint. Repeated-measures analysis of variance was used to compare the parameters before and after orthokeratology., Results: The corneal densitometry values over the 0-10 mm diameter area increased from 12.84±1.38 grayscale units (GSU) at baseline to 13.59±1.42 GSU after three-month orthokeratology (P = .001) and reached 14.92±1.45 GSU at two years (P < .001). An increase in densitometry began at one month (P = .001) over the 0-2 mm annulus compared with that at three months over the 2-6 mm and 6-10 mm zones (P = .002,.014). The densitometry values significantly increased at three months in the relatively steep-fitting group (P = .003) and at one year in the relatively flat-fitting group (P = .001). After discontinuation of orthokeratology for one month, the values showed no significant decrease., Conclusions: Long-term orthokeratology treatment causes a small but statistically significant increase in corneal densitometry values. During the first year, the onset of these changes was related to the fitting mode. Corneal densitometry values showed no significant reduction after one-month discontinuation., Competing Interests: The authors have declared that no competing interests exist.

Determination of valproic acid in the drug was carried out on the aluminum silica gel 60F 254 plates and using acetone-water-chloroform-ethanol-ammonia at a volume ratio of 30:1:8:5:11 as the mobile phase, respectively. Two methods of detection of valproic acid were used. The first was a 2% aqueous CuSO 4 ×5H 2 O solution, and the second was a 2',7'-dichlorofluorescein-aluminum chloride-iron (III) chloride system. The applied TLC-densitometric method is selective, linear, accurate, precise, and robust, regardless of the visualizing reagent used for the determination of valproic acid in Convulex capsules. It has low limits of detection (LOD) and limits of quantification (LOQ), which are equal to 5.8 μg/spot and 17.4 μg/spot using a 2% aqueous CuSO 4 ×5H 2 O solution as visualizing agent and also 0.32 μg/spot and 0.97 μg/spot using a 2',7'-dichlorofluorescein-aluminum chloride-iron (III) chloride system as visualizing reagent, respectively. The described analytical method can additionally be used to study the identity of valproic acid in a pharmaceutical preparation. The linearity range was found to be 20.00-80.00 μg/spot and 1.00-2.00 μg/spot for valproic acid detected on chromatographic plates using a 2% aqueous CuSO 4 ×5H 2 O solution and the 2',7'-dichlorofluorescein-aluminum chloride-iron (III) chloride system, respectively. A coefficient of variation that was less than 3% confirms the satisfactory accuracy and precision of the proposed method. The results of the assay of valproic acid equal 96.2% and 97.0% in relation to the label claim that valproic acid fulfill pharmacopoeial requirements. The developed TLC-densitometric method can be suitable for the routine analysis of valproic acid in pharmaceutical formulations. The proposed TLC-densitometry may be an alternative method to the modern high-performance liquid chromatography and square wave voltammetry in the control of above-mentioned substances, and it can be applied when other analytical techniques is not affordable in the laboratory.

Purpose: To investigate correlations between specular microscopy endothelial parameters and age with corneal densitometry values, as they are presented from a Scheimpflug device, in different levels of the cornea., Methods: Two hundred eighty-four eyes of 142 healthy subjects were included in this observational, prospective study. Corneal densitometry was evaluated with Scheimpflug imaging system in the central 0- to 2-mm annular zone of the cornea, whereas the endothelial cell properties were assessed with the use of a noncontact specular microscope., Results: Corneal densitometry values of all corneal layers were statistically significant and positively correlated with age. In univariate linear regression analysis among corneal densitometry values and the endothelial parameters, only endothelial cell density (CD) was statistically significant and inversely correlated with densitometry values in all corneal layers. In stepwise multivariate linear regression analysis, after adjustment for age, hexagonality was statistically significant and inversely correlated with posterior densitometry values, whereas coefficient of variation was positively and significantly correlated with the anterior densitometry values. When repeating stepwise multivariate linear regression analysis without adjusting for age, CD was negatively and significantly correlated with corneal densitometry values of all layers, whereas coefficient of variation was positively and significantly correlated with anterior and total corneal densitometry values., Conclusions: Corneal densitometry increases with age. It is also inversely correlated with CD, and this might be used as an indirect way to assess the status of the corneal endothelium., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

An epidemiological study was conducted in North-East India (part of Indo-Burma biodiversity hotspot) to better understand the distribution, diversity, and transmission of Clostridium perfringens among livestock, pets, wild animals (captive), and humans. A total of 160 C. perfringens isolates were recovered from 642 diarrhoeic faecal samples with an isolation rate of 24.92%. Isolation rate was the highest among captive wild animals (37.5%) followed by dog (34.6%), human (33.8%), pig (32.7%), cattle (20.8%), goat (18.3%) and poultry (9.3%). Isolates were toxin typed using a seven gene multiplex PCR designed for simultaneous detection of cpa, cpb, cpb2, etx, iap, cpe and netB. The majority of isolates, 128 (80%) were of type A, followed by 17 (10.62%), 5 (3.12%), 4 (2.5%), 3 (1.87%), 2 (1.25%) and 1 (0.63%) isolates of type C, D, E, G, F and B, respectively. Beta 2 toxin gene was present in 65 (50%) of type A isolates, followed by 7 (41.2%), 4 (80%), 1(25%), and 1 (100%) of type C, D, G and B isolates, respectively. Beta 2 toxin has a high prevalence among dogs (28.6%), cattle (27.3%), and pig (20.8%) compared to humans, goat, wild animals, and poultry (1.2-14.3%). The prevalence of CPE and NetB toxin-positive strains was low, with only 3 (1.8%) and 5 (3.1%) isolates, respectively. Association of C. perfringens with diarrhoea in Civet Cat, Golden Langur, and Gray Langur has been reported for the first time. The genetic diversity and transmission of isolates were investigated using automated rep-PCR (Diversilab®, bioMérieux) using two densitometry-based matrices: modified Kullback-Leibler (KL) and Pearson's correlation (PC). The PC and modified KL matrices formed three distinct clusters with 59% and 27.2% similarity, respectively. C. perfringens diversity and transmission were best studied using modified KL matrix that placed more emphasis on the presence of bands rather than intensity. However, the PC method was found to be more suitable for differentiating strains within a toxin type, with slightly higher D-values., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Metronidazole is a drug widely used in the prevention and treatment of bacterial infections. Due to its possibility of the formation of stable metal complexes, it was decided to broaden its activity spectrum by introducing the silver(I) coordination compounds i.e., [Ag(MTZ) 2 NO 3 ] and [(Ag(MTZ) 2 ) 2 ]SO 4 , which have significant antibacterial properties. The paper presents a description of a new qualitative and quantitative analysis of metronidazole in bulk and possible pharmaceutical preparations by thin-layer chromatography with densitometric detection. Optimal separation conditions were selected, and the analytical procedure was validated according to the ICH guidelines. The obtained data indicate that the method is sufficiently sensitive, precise, and accurate. The stability of the metronidazole solutions obtained from tablets, pure metronidazole, and its silver(I) complexes was tested. The research was carried out in various environments, at different temperatures, in H 2 O 2 solution, and during exposure to radiation (UV, sunlight). The greatest degradation was found in the alkaline environment and at higher temperatures. The silver(I) complexes exhibited relatively high stability under analyzed conditions that are higher than standard metronidazole solutions and tablets. The observations were confirmed by the kinetic and thermodynamic analysis. The described studies of new metronidazole silver(I) complexes increase the potential for their application in infections both in humans and animals.

Background: The aim of this pragmatic intervention study was to investigate changes in cardiometabolic outcomes, irisin plasma concentration, and body composition during a 4-month intervention in unselected obese individuals., Materials and Methods: In 111 obese women aged 36.73 ± 7.2 years, we measured changes in weight, lipid profiles, glucose, insulin, Homeostatic Model Assessment-Insulin Resistance Index (HOMA-IR), uric acid, aminotransferases, and irisin. Body composition including lean mass (LM) and total (TF), gynoid (GF), android (AF), and visceral fat (VF) was assessed using densitometry. Physical activity was assessed using the International Physical Activity Questionnaire (IPAQ). The participants received tailored written advice targeting lifestyle according to current guidelines. At follow-up, patients rated their adherence in the self-administered questionnaire., Results: Mean weight loss in the whole group was 3.12 kg (- 3.3%); 26% of the women achieved the desired target of weight loss (> 5% of the initial weight), whereas weight decreased moderately in 50% and increased in 14%. In 86 women with weight loss, there were significant changes in HOMA-IR (- 13.8%), insulin (- 11.2%), alanine aminotransferase (- 8.0%), VF (- 7.0%), AF (- 5.4%), TF (- 4.7%), GF (- 2.8%) and LM (- 1.5%), whereas irisin and HDL-C levels and the mean IPAQ score did not change., Conclusions: In this real-world evidence study, a successful weight loss achieved only 26% of patients, with overall much better adherence to diet restriction than to exercise. However, even mild to moderate weight loss resulted in significant improvements in cardiometabolic health. Weight loss was associated with a modest LM decrease but did not influence plasma irisin.

A high-performance thin-layer chromatographic method was developed and validated for the concurrent determination of simeprevir (SMV) and sofosbuvir (SOF). The chromatographic separation was attained on silica gel 60 F254 as stationary phase and ethyl acetate-hexane-methanol (5.0:4.0:1.0, v/v/v) as developing solvent with UV detection at 273 nm. The RF values were 0.67±0.02 and 0.43±0.02 for SMV and SOF, respectively. The method has been validated in respect to the guidelines of the International Conference on Harmonization. Linearity was maintained between 60-1,000 and 70-1,200 ng/band for SMV and SOF, respectively, with good correlation coefficients (0.9993-0.9997) for both drugs. The suggested method was highly sensitive as the calculated detection limits were 15 and 22 ng/band, while the quantitation limits were 44 and 66 ng/ band for SMV and SOF, respectively. The suggested methodology has been effectively employed for the determination of the mentioned drugs in their pure forms and their pharmaceutical dosage forms as well as human plasma without significant interference of the pharmaceutical excipients or plasma components., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: To compare densitometry distribution analysis (DDA), a platform-independent method to assess corneal transparency, with traditional corneal densitometry., Methods: A total of 196 healthy participants aged 43.3 ± 18.0 years (range 18-79 years) were recruited for assessment. All participants were assessed using the corneal densitometry analysis add-on to the standard software of the Oculus Pentacam HR. In addition, the Scheimpflug image corresponding to the horizontal meridian of each participant was exported for further analysis. For each image, corneal pixel intensities were statistically modeled. The estimated output parameters, α and β, were compared with the corresponding densitometry values. The analysis was performed considering 3 concentric areas and 3 layers defined at fixed corneal depths. To demonstrate the platform independence of the DDA method, a randomly selected subset of 80 participants also had their eye measured with Oculus Corvis ST., Results: α and β were found to be well correlated with densitometry, especially α (overall cornea; r = 0.89, P < 0.001), independent of the corneal region investigated. Changes in α, β, and corneal densitometry were correlated with age., Conclusions: In this work, we presented the relationship of DDA with age and traditional corneal densitometry. The α and β parameters, the output of DDA, are platform independent and can be used to investigate corneal clarity objectively., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc.)

Purpose: To report an observation made while performing Scheimpflug densitometry analysis on the corneal region affected in keratoconus (KC) that seems to delineate the base of the cone., Methods: Scheimpflug densitometries of 20 healthy subjects and 90 patients with KC were examined. Corneal densitometry was analyzed using both "1-layer" and "2-layer" approaches. The first considers the corneal transparency layer by layer at different depths, whereas the second averages densitometry between 2 corneal layers selected by the examiner. Fixed layers, 120 μm depth, and endothelium were selected. Repeated same-day scans and longitudinal series of scans were also evaluated to see whether the findings evolved over time., Results: Eighty-eight of 90 KC cases displayed a bright area on the densitometry map that corresponded to the cone location. The area's characteristics, such as its brightness, contrast, and the presence of a delimiting arc correlated with KC severity and was more noticeable in advanced cases. No similar marks were found in any of the normal subjects. The shape, location, and extent of the mark were consistent over consecutive measures taken on the same day. Changes over time were also seen in eyes with known clinical progression but was also seen in eyes considered clinically stable., Conclusions: The densitometry mark seems to correspond with the zone most affected by KC and could be a supplementary tool for documenting KC stage, alongside conventional parameters. Further studies are required to ascertain whether it could prove useful in KC detection, to determine progression, and to relate it to corneal biomechanical behavior., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Two chromatographic methods were validated for the determination of the widely prescribed analgesic and antipyretic drug combination of paracetamol (PC) (recently integrated into the supportive treatment of COVID-19), propyphenazone (PZ) and caffeine (CF) in the presence of two PC impurities, namely 4-aminophenol and 4-nitrophenol. A "dual-mode" gradient high-performance liquid chromatography method was developed, where the separation was achieved via "dual-mode" gradient by changing both the ternary mobile phase composition (acetonitrile: methanol: water) and the flow rate. This enables a good resolution within a relatively shorter analysis time. The analysis was realized using Zorbax Eclipse XDB column C18, 5 μm (250 × 4.6 mm) and the UV detector was set at 220 nm. The other method is a thin-layer chromatography densitometry method, where the separation was achieved using a mobile phase composed of chloroform: toluene: ethyl acetate: methanol: acetic acid (6: 6: 1: 2: 0.1, by volume). Densitometric detection was performed at 220 nm on silica gel 60 F254 plates. The developed methods were fully validated as per the ICH guidelines and proved to be accurate, robust, specific and suitable for application as purity indicating methods for routine analysis of PC in pure form or in pharmaceuticals with PZ and CF in quality control laboratories., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Purpose: To describe the normative data of corneal densitometry in photorefractive Keratectomy (PRK) candidates by using a Scheimpflug camera., Methods: Five hundred twenty-two eyes of 261 PRK candidates that underwent comprehensive optometric and ophthalmic examinations were enrolled in this study. Corneal densitometry values were obtained using a Scheimpflug camera (Pentacam HR; Oculus Optikgeräte GmbH, Wetzlar, Germany) and displayed in grayscale units (GSU)., Results: Of 261 participants, 187 (71.6%) were women. The mean age of the participants was 28.79 ± 5.02 years (range: 21-40 years). The mean entire corneal densitometry was 14.86 ± 2.37 GSU. According to corneal depth, maximum and minimum densitometry values were seen in the anterior (30.28 ± 7.42 GSU) and posterior (9.86 ± 2.08 GSU) layers, respectively (P < 0.001). Based on the annulus over the cornea, the highest and lowest densitometry values were seen in the 10- to 12-mm (21.31 ± 4.26 GSU) and 2- to 6-mm zones (13.26 ± 2.34 GSU), respectively. However, this difference was not seen after 35 years of age. Individuals aged 30 to 35 years showed higher corneal densitometry values compared with subjects aged 20 to 25 years old (P < 0.001). In generalized linear models, the corneal thickness was associated with the densitometry value in the central and posterior corneal layers. There were no significant differences in sex and refractive errors among participants., Conclusions: This study provided normal corneal densitometry data in PRK candidates. Corneal densitometry increased with age until 35 years of age and then decreased thereafter. This finding should be considered in patient selection for PRK and their postoperative examinations.

Purpose: To quantitate corneal haze and analyze the postoperative time course of corneal haze after transepithelial corneal collagen cross-linking (TECXL) in patients with keratoconus., Methods: Patients underwent TECXL and were randomized into 2 groups. One group received intraoperative riboflavin 0.10% every minute, and the second group received riboflavin 0.10% every 2 minutes during ultraviolet exposure. Scheimpflug densitometry was measured preoperatively, and at 1, 3, 6, and 12 months to assess the postoperative time course. Densitometry measurements were also correlated with visual acuity, pachymetry, and topography outcomes., Results: Fifty-nine eyes of 43 patients with keratoconus were analyzed. Preoperative mean corneal densitometry was 20.45 ± 2.79. Mean densitometry increased at 1 month (22.58 ± 3.79; P < 0.001), did not significantly change between 1 and 3 months (22.64 ± 3.83; P = 0.8), and significantly improved between 3 and 12 months postoperatively (mean6 21.59 ± 3.39; P = 0.002, mean12 20.80 ± 3.27; P = 0.002). There was no difference between preoperative and 1-year densitometry measurements (P = 0.21). There was no significant difference between the 1-minute and 2-minute subgroups. In addition, corneal densitometry at either 3 months or 1 year did not correlate with uncorrected distance visual acuity (P = 0.4), corrected distance visual acuity (P = 0.1), or maximum keratometry (P = 0.5), 1 year after corneal collagen cross-linking (CXL)., Conclusions: After TECXL, corneal haze increased slightly at 1 month, plateaued between 1 and 3 months, and returned to baseline between 3 and 12 months. In general, corneal haze in this study was substantially less than the haze previously reported for the standard cross-linking procedure. CXL-associated corneal haze did not correlate with the postoperative visual or topographic outcomes 1 year after CXL.

Purpose: The origin of blood in glaucoma-related disc hemorrhages (DH) remains unknown. A prior clinic-based study of primary open-angle glaucoma (POAG)-related DH showed that they had grayscale pixel intensities more similar to blood from retinal macroaneurysms and adjacent retinal arterioles than to blood from retinal vein occlusions or adjacent retinal venules, suggesting an arterial source. Here we assessed the densitometric profile of DH from fundus photographs in the Ocular Hypertension Treatment Study (OHTS)., Design: Retrospective cross-sectional study of prospectively collected images., Methods: Stereo disc photographs of 161 DH events from 83 OHTS participants (mean age [standard deviation (SD)]: 65.6 [9.2] years; 46.6% female; 13.0% black race) were imported into ImageJ to measure densitometry differences (adjacent arterioles minus DH [ΔA] or venules minus DH [ΔV]). Their size as percentage of disc area, ratio of length to midpoint width, and location relative to the disc margin were also analyzed. We performed t tests to compare ΔA and ΔV, analysis of variance to compare ΔA and ΔV across DH recurrent events, and multivariable linear regression to identify determinants of ΔA and ΔV., Results: Mean (SD) ΔA and ΔV were -2.2 (8.7) and -11.4 (9.7) pixel intensity units, respectively (P < .001). ΔA and ΔV each did not differ significantly across recurrence of DH (P ≥ .92) or between DH events with and without POAG (P ≥ .26)., Conclusions: OHTS DH had densitometric measurements more similar in magnitude to adjacent arterioles than venules, supporting an arterial origin for DH. Vascular dysregulation may contribute to disc hemorrhage formation in ocular hypertension., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Histamine poisoning is a significant public health problem. Therefore, the monitoring of histamine content in fish and fishery products is considered to be a crucial measure in the seafood industry. In the present study, a simple and rapid densitometric thin-layer chromatographic (TLC) method for histamine determination in fish samples was developed and validated. The samples were homogenized with 10% trichloroacetic acid and histamine was efficiently extracted. Then, an appropriate derivatization procedure was adopted with dansyl chloride. Once the derivatization was carried out, the samples were applied to silica gel TLC plates and developed by ascending chromatography with chloroform-triethylamine (6:4, v / v ) as the mobile phase. The intensity of the histamine-dansyl derivative spots was measured by densitometry at 365 nm, and the quantitation was performed by BIO-1D image processing software. The validation of this method revealed good linearity and specificity over a concentration range from 6.25 to 100 mg/kg. Adequate precision was shown by relative standard deviations (RSD) smaller than 4.82%, accuracy ranged from -6.88% to 5.28%, and satisfactory recoveries ranging from 93% to 105% were obtained. The Limit of Detection and the Limit of Quantification were calculated at 4.4 mg/kg and 10.5 mg/kg, respectively. In addition, the effectiveness of the proposed method was assessed by the analysis of various samples, and the obtained results were confirmed with those achieved by the HPLC-UV method. Moreover, the developed method was found to be simple, cheap, and suitable for application to analyze several samples simultaneously.

Cassia siamea leaf has been proven in vitro and in vivo to have a strong antimalarial activity with Cassiarin A as its active compound. To obtain a source of C. siamea medicinal plant with high level of active antimalarial compound (Cassiarin A), a valid method for determining Cassiarin A level is needed. For this reason, this research conducts the validation of the Cassiarin A content with determination method using thin-layer chromatography (TLC) densitometry which includes the determination of selectivity ( R s), linearity (r), accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ). Cassiarin A was chromatographed on silica gel 60 F 254 TLC plate using chloroform : ethanol (85 : 15 v/v) as a mobile phase. Cassiarin A was quantified by densitometric analysis at 368 nm. The linear regression analysis data for the calibration plots showed good linear relationship with r  = 0.9995. The method was validated for precision, recovery, repeatability. The minimum detectable amount was found to be 0.0027  μ g/spot, whereas the limit of quantitation was found to be 0.008  μ g/spot. The results of this validation are then used to determine the Cassiarin A level of C. siamea leaf from various regions in Indonesia. Based on the results of the study, it can be concluded that the TLC-densitometry method can be used to determine level of the Cassiarin A compound with the advantages of being fast, easy, accurate, and inexpensive. In addition, it showed that C. siamea leaves from Pacitan have the highest level of Cassiarin A compared to other areas studied., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2020 Wiwied Ekasari et al.)

Purpose: Chronic hyperglycemia induces morphological and functional changes of the cornea. Corneal clarity is essential for visual function, and the measurement of corneal optical density (COD) might provide further information on diabetes mellitus (DM)-induced alterations., Methods: COD of patients with DM and age-matched healthy subjects was measured using the Pentacam HR. Furthermore, central and thinnest corneal thickness and peripheral pachymetry of concentric circles around thinnest corneal thickness were investigated. In DM, information on disease duration, type, presence of diabetic retinopathy and maculopathy, and HbA1c value was recorded., Results: In this study, 76 patients with DM and 65 healthy subjects were included. In patients with DM, the COD values of nearly all corneal layers and zones were reduced in comparison with healthy subjects (P < 0.05). Furthermore, the COD measurements were inversely correlated with the HbA1c value (total COD central layer: r = -0.424, P = 0.044) and stage of diabetic retinopathy (total COD: r = -0.271, P = 0.019). Diabetic patients with maculopathy revealed lower total COD values than patients without maculopathy (16.5 ± 5.6 vs. 21 ± 7.6, P = 0.031), and COD was lower in DM type 1 than in type 2 (16.1 ± 5.1 vs. 20.8 vs. 7.5, P = 0.035). In both groups, the COD values increased with age (patients with DM: r = 0.336, P = 0.003; healthy subjects: r = 0.679, P < 0.001) and decreased with peripheral corneal thickness increase., Conclusions: In patients with DM, COD was significantly reduced in comparison with healthy subjects. These changes were associated to disease-specific factors and measurements of peripheral corneal thickness profiles.

A promising combination of tamsulosin HCl and tadalafil has recently been introduced for treating two prevalent and associated urological disorders: benign prostate hyperplasia and erectile dysfunction. Novel HPTLC methods were designed and validated for assaying the cited drugs in their challenging combined formulation. Separation was achieved using HPTLC silica gel 60 F 254 plates as a stationary phase with a densitometric measurement at 280 nm. The proposed methods with two different chromatographic systems were successfully applied: a conventional mixture (method I) of ethyl acetate-toluene-methanol-ammonia (5:3:2:0.5, by volume) and a greener one (method II) with ethyl acetate-ethanol-ammonia (8:2:0.1, by volume). The two methods were evaluated through a comparative study in terms of selectivity, tailing factor, developing time and concentration ranges. The greenness profile for each method was then appraised with several green guides, namely GlaxoSmithKline solvent sustainability guide, Environmental, Health and Safety (EHS) tool, National Environmental Method Index (NEMI) and Eco-scale. Moreover, method specificity and peak homogeneity were evaluated by peak purity assessment using the winCATS ® software spectral correlation tool. The methods have potential for being simple, fast, economic and selective, and the greener one could be a good option for sustainable analysis of the drugs., (© 2020 John Wiley & Sons, Ltd.)

Objective: Acute subdural hematoma (ASDH) is a major cause of mortality and morbidity after traumatic brain injury (TBI). Surgical evacuation is the mainstay of treatment in patients with altered neurological status or significant mass effect. Nevertheless, concerns regarding surgical indication still persist. Given that clinicians often make therapeutic decisions on the basis of their prognosis assessment, to accurately evaluate the prognosis is of great significance. Unfortunately, there is a lack of specific and reliable prognostic models. In addition, the interdependence of certain well-known predictive variables usually employed to guide surgical decision-making in ASDH has been proven. Because gray matter and white matter are highly susceptible to secondary insults during the early phase after TBI, the authors aimed to assess the extent of these secondary insults with a brain parenchyma densitometric quantitative CT analysis and to evaluate its prognostic capacity., Methods: The authors performed a retrospective analysis among their prospectively collected cohort of patients with moderate to severe TBI. Patients with surgically evacuated, isolated, unilateral ASDH admitted between 2010 and 2017 were selected. Thirty-nine patients were included. For each patient, brain parenchyma density in Hounsfield units (HUs) was measured in 10 selected slices from the supratentorial region. In each slice, different regions of interest (ROIs), including and excluding the cortical parenchyma, were defined. The injured hemisphere, the contralateral hemisphere, and the absolute differences between them were analyzed. The outcome was evaluated using the Glasgow Outcome Scale-Extended at 1 year after TBI., Results: Fifteen patients (38.5%) had a favorable outcome. Collected demographic, clinical, and radiographic data did not show significant differences between favorable and unfavorable outcomes. In contrast, the densitometric analysis demonstrated that greater absolute differences between both hemispheres were associated with poor outcome. These differences were detected along the supratentorial region, but were greater at the high convexity level. Moreover, these HU differences were far more marked at the cortical parenchyma. It was also detected that these differences were more prone to ischemic and/or edematous insults than to hyperemic changes. Age was significantly correlated with the side-to-side HU differences in patients with unfavorable outcome., Conclusions: The densitometric analysis is a promising prognostic tool in patients diagnosed with ASDH. The supplementary prognostic information provided by the densitometric analysis should be evaluated in future studies.

Purpose: To compare the degree of corneal light scatter as measured by densitometry in ultrathin Descemet Stripping Automated Endothelial Keratoplasty (UT-DSAEK) and Descemet Membrane Endothelial Keratoplasty (DMEK) in the Descemet endothelial thickness comparison trial., Methods: This was a prespecified secondary analysis of the Descemet endothelial thickness comparison trial, which was a prospective, randomized controlled trial. Subjects with isolated endothelial dysfunction were enrolled and were randomized to either UT-DSAEK or DMEK. Corneal opacity was quantitatively measured by Pentacam densitometry (OCULUS) at 3, 6, and 12 months., Results: Fifty eyes of 38 patients were enrolled at the Casey Eye Institute at Oregon Health & Science University and the Byers Eye Institute at Stanford University. Corneal densitometry for the anterior and posterior layers improved in both UT-DSAEK and DMEK after surgery. The decrease was more pronounced in the posterior layer for both groups. However, there was no difference in the degree of corneal light scatter between UT-DSAEK and DMEK at postoperative month 12, and no difference in change in densitometry was observed between the 2 arms from baseline to month 12., Conclusions: Both UT-DSAEK and DMEK experience an improvement in the degree of corneal light scatter after surgery. However, there was no difference in densitometry between the 2 groups at month 12. Therefore, other factors such as higher order aberrations in the posterior cornea rather than stromal-stromal interface haze mediate the superior visual outcomes in DMEK compared with UT-DSAEK.

The protein purity is generally checked using SDS-PAGE, where densitometry could be used to quantify the protein bands. In literature, few studies have been reported using image analysis for the quantification of protein in SDS-PAGE: that is, imaged with Stain-Free™ technology. This study presents a protocol of image analysis for electrophoresis gels that allows the quantification of unknown proteins using the molecular weight markers as protein standards. Escherichia coli WK6/pHEN6 encoding the bispecific nanobody CH10-12 engineered by the Pasteur Institute of Tunisia was cultured in a bioreactor and induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) at 28°C for 12 hr. Periplasmic proteins extracted by osmotic shock were purified by immobilized metal affinity chromatography (IMAC). Images of the SDS-PAGE gels were analyzed using ImageJ, and the lane profiles were obtained in grayscale and uncalibrated optical density. Protein load and peak area were linearly correlated, and optimal image processing was then performed by background subtraction using the rolling ball algorithm with radius size 250 pixels. No brightness and contrast adjustment was applied. The production of the nanobody CH10-12 was obtained through a fed-batch strategy and quantified using the band of 50 kDa in the marker as reference for 750 ng of recombinant protein. The molecular weight marker was used as a sole protein standard for protein quantification in SDS-PAGE gel images., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Purpose: To investigate the changes in corneal backward scattering using Scheimpflug tomography (densitometry) 6 months after intrastromal corneal ring segments (Keraring) implant for keratoconus and to correlate with clinical results., Methods: Along with standard ophthalmic examination, Scheimpflug tomography using Pentacam HR was performed to document corneal densitometry and corneal shape. A densitometry map was displayed using grayscale units (GSUs) ranging from 0 to 100 (minimum to maximum light scattering) in 4 concentric annuluses: central 2-, 2- to 6-, 6- to 10-, 10- to 12-, and total 12-mm corneal diameter and at 3 different depths: anterior 120 μm, posterior 60 μm, middle (varying accordingly to the total thickness), and total thickness., Results: The changes in mean corneal densitometry values for anterior, central, and posterior layers were 3.82 ± 3.13, 2.70 ± 1.78, and 1.68 ± 1.52 GSU, respectively, and for annuluses of 0 to 2, 2 to 6, 6 to 10, and 10 to 12 mm were 2.96 ± 2.60, 4.53 ± 2.28, 2.09 ± 1.74, and 0.45 ± 3.93 GSU, respectively. The mean values increased for all corneal layers (P < 0.05), except in the peripheral 10- to 12-mm annulus (P> 0.05). The highest increase was in the annulus 2 to 6 mm of the anterior layer (5.72 ± 3.70 GSU). Changes in the mean densitometry showed a significant correlation with the mean of average keratometry changes only for the front corneal surface and changes in the Q-values for both corneal surfaces in the 2- to 6-mm annulus for all layers., Conclusions: Significant increase in the corneal densitometry mainly in the anterior 2- to 6-mm annulus of the cornea is associated with the position of the ring segment implant. More studies are needed to elucidate the relevance of such findings.

Background: Protein concentration of monoclonal immunoglobulin in plasma-cell myeloma/multiple myeloma provides an estimate of the tumor mass and allows for monitoring of the response to treatment. Accurate and reproducible estimates of the monoclonal immunoglobulin concentration are important for patient care., Objective: To address the optimum method for estimation of the concentration of monoclonal immunoglobulins., Methods: Serum protein electrophoresis and immunofixation electrophoresis were conducted by using the Helena SPIFE Touch instrument. Estimation of the protein concentration of monoclonal immunoglobulin in the gamma region by computer-assisted reading was compared with the reading by technologists and pathology residents, in 300 gels. The data were compared using t-testing and analysis of variance., Results: Computer-generated readings had a consistent positive bias. The correlation coefficient of the average reading by technologists and residents with the computer generated value was 0.997. The average positive bias by the computer reading was 0.29 g per dL. The intercept on the regression analysis was 0.22 g per dL. The reading by the computer was significantly higher than each of the human-interpreted readings. The readings by the 3 human groups were not significantly different amongst them. The main reason for the higher reading by the computer was inclusion of a greater area on the anodal size of the peak on the densitometric scan., Conclusions: Human- and computer-interpreted readings of the protein concentration of monoclonal immunoglobulin have a high degree of correlation. The consistent positive bias by the computer reading occurred due to inclusion of a greater area of the densitometric scan on the anodal side of the peak. We suggest that vendors should adjust such computer programs to provide readings comparable to those generated by expert humans. We recommend manual delineation of the monoclonal peaks for measuring the concentration of monoclonal immunoglobulins., (© American Society for Clinical Pathology 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Three chromatographic methods were developed, optimized and validated for Paracetamol (PAR), Orphenadrine citrate (Or.cit) and Caffeine (CAF) determination in their mixture and in presence of PAR toxic impurity; P-aminophenol (PAP) in tablets. The first method is based on a thin layer chromatography combined with densitometry. Separation was achieved using silica gel 60 F254 TLC plates and dichloromethane:methanol:acetone:glacial acetic acid (9:1:0.5:0.3, v/v/v) as a developing system at 230 nm. The second method is based on high-performance liquid chromatography with diode array detection. The proposed compounds are separated on a reversed phase C18 analytical column using phosphate buffer (pH 9; 0.05 M) and methanol (80:20, v/v) at 1.2 mL/min. Linear regressions are obtained in the range of 1-500 μg/mL, 25-1000 μg/mL and 1-400 μg/mL for PAR, Or.cit and CAF, respectively. Quantification of the toxic PAP is carried out using LC-MS-MS by electrospray ionization in the positive mode using triple quadrupole mass spectrometry. The limit of quantification for PAP is 1 ng/mL. All methods are validated according to the ICH guidelines and successfully applied to determine PAR, Or.cit, CAF and PAP in pure powder and in combined tablets dosage form without interference from excipients., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)