Development and evaluation of a candidate reference measurement procedure for detecting 17α-hydroxyprogesterone in dried blood spots using isotope dilution liquid chromatography tandem mass spectrometry.

17α-Hydroxyprogesterone (17α-OHP) quantification in dried blood spots (DBS) is essential for newborn screening for congenital adrenal hyperplasia (CAH), which is challenging due to its low physiological concentration. The high false-positive rates of immunoassays necessitate the development of more accurate methods. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers increased specificity and sensitivity, yet standardized procedures for 17α-OHP measurement are required for clinical application. A candidate reference measurement procedure (cRMP) using isotope dilution LC-MS/MS was developed for 17α-OHP quantification in DBS. By utilizing stable isotope-labeled D8-17α-OHP as an internal standard, the cRMP was optimized, covering sample preparation, calibration, and LC-MS/MS analysis. The method performance was validated across several parameters, including precision, accuracy, specificity, detection limits, and matrix effects. Clinical applicability was further assessed through the establishment of reference intervals for healthy newborns. The developed cRMP exhibited a linear range of 1.00 to 80.00 ng/mL for 17α-OHP, with detection and quantification limits of 0.14 ng/mL and 0.52 ng/mL, respectively. Inter- and intraday precision demonstrated coefficients of variation within 1.27 to 5.69%. The recovery rates and matrix effects were well within acceptable limits, ensuring method reliability. Clinical application showed distinct reference intervals for healthy newborns that were unaffected by sex but influenced by weight and gestational age. This method significantly enhances CAH diagnostic accuracy in newborns, providing a valuable tool for clinical laboratories and improving newborn screening program standardization and traceability.

Evaluation and clinical application of a bracketing calibration-based isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for serum theophylline.

A candidate reference measurement procedure (RMP) for serum theophylline via isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. With a single-step precipitation pretreatment and a 6-min gradient elution, the method achieved baseline separation of theophylline and its analogs on a C18-packed column. A bracketing calibration method was used to ensure repeatable signal intensity and high measurement precision. The intra-assay and inter-assay imprecisions were 1.06%, 0.84%, 0.72% and 0.47%, 0.41%, 0.25% at concentrations of 4.22 µg/mL (23.40 µmol/L), 8.45 µg/mL (46.90 µmol/L), and 15.21 µg/mL (84.43 µmol/L), respectively. Recoveries ranged from 99.35 to 102.34%. The limit of detection (LoD) was 2 ng/mL, and the lowest limit of quantification (LLoQ) was 5 ng/mL. The linearity range extended from 0.47 to 60 µg/mL (2.61-333.04 µmol/L). No ion suppression and carry-over (< 0.68%) were observed. The relative bias for this candidate RMP that participated in 2023 External Quality Control for Reference Laboratories (RELA) conducted by the International Federation of Clinical Chemistry (IFCC) was within a range of 0.17 to 0.93%. Furthermore, two clinical immunoassay systems were compared with this candidate RMP, demonstrating good correlations. The results of the Trueness Verification Plan indicate significant differences among routine systems, highlighting the need for standardization efforts. The developed candidate RMP for serum theophylline serves as a precise reference baseline for standardizing clinical systems and assigning values to reference materials.

Stable isotope dilution assay and HS-SPME-GCMS quantification of key aroma volatiles of Australian pineapple (Ananas comosus) cultivars.

Pineapple aroma is one of the most important sensory quality traits that influences consumer purchasing patterns. Reported in this paper is a high throughput method to quantify in a single analysis the key volatile organic compounds that contribute to the aroma of pineapple cultivars grown in Australia. The method constituted stable isotope dilution analysis in conjunction with headspace solid-phase microextraction coupled with gas-chromatography mass spectrometry. Deuterium labelled analogues of the target analytes purchased commercially were used as internal standards. Twenty-six volatile organic compounds were targeted for quantification and the resulting calibration functions of the matrix -matched validated method had determination coefficients (R2) ranging from 0.9772 to 0.9999. The method was applied to identify the key aroma volatile compounds produced by popular pineapple cultivars such as 'Aus Carnival', 'Aus Festival', 'Aus Jubilee', 'Aus Smooth (Smooth Cayenne)' and 'Aussie Gold (73-50)', grown in Queensland, Australia. Pineapple cultivars varied in its content and composition of free volatile components, which were predominantly comprised of esters, followed by terpenes, alcohols, aldehydes, and ketones.

Development and validation of stable isotope dilution LC-MS/MS method for simultaneous quantification of four Alternaria toxins in 15 food commodities.

Alternaria toxins (ATs) are produced from Alternaria species that result in crop losses and harmful impacts on human health. A stable isotope dilution LC-MS/MS method was established to quantify four ATs in 15 food commodities: alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), and tenuazonic acid (TeA). Based on systematically optimization of detection conditions and pre-processing steps, the limits of detection and limits of quantification of the four ATs ranged from 0.1 to 10 µg/kg and 0.2 to 30 µg/kg, respectively. The results showed that the recoveries of the four ATs were 72.0%-119.1%. The intra-precision and inter-precision ranged from 0.7% to 11.1% and 1.1% to 13.1%, respectively. The method was successfully applied to the determination of four ATs in 35 food samples, suggesting that this method could provide meaningful occurrence data to support the assessment of emerging ATs in food commodities.

The patterns in urine excretion and transvascular fluid exchange in human subjects during intravenous fluid infusion: A quantitative analysis.

INTRODUCTION: Investigations of responses of animals and humans to changes of plasma volume are usually reported as average responses of groups of individuals. This ignores considerable quantitative variation between individuals. We examined the hypothesis that individual responses follow a common temporal pattern with variations reflecting different parameters describing that pattern. METHODS: We illustrate this approach using data of Hahn, Lindahl and Drobin (Acta Anaesthesiol Scand.2011, 55:987-94) who measured urine volume and haemoglobin dilution of 10 female subjects during intravenous Ringer infusions for 30 min and subsequent 3.5 h. The published time courses were digitised and analysed to determine if a family of mathematical functions accounted for the variation in individual responses. RESULTS: Urine excretion was characterised by a time delay (Td) before urine flow increased and a time course of cumulative urine excretion described by a logarithmic function. This logarithmic relation forms the theoretical basis of a family of linear relations describing urine excretion as a function of Td. Measurement of Td enables estimation of subsequent values of urine excretion and thereby the fraction of infused fluid retained in the body. CONCLUSION: The approach might be useful for physiologists and clinical investigators to compare the response to infusion protocols when both test and control responses can be described by linear relations between cumulative urine volume at specific times and Td. The approach may also be useful for clinicians by complementing strategies to guide fluid therapy by enabling the later responses of an individual to be predicted from their earlier response.

Determination of ileal endogenous nitrogen losses and true ileal nitrogen digestibility during non-steady-state conditions of the 15N-isotope dilution technique.

The aim was to determine ileal endogenous nitrogen losses (ENL) and true ileal N-digestibility (TD-N) under non-steady-state conditions of the 15N-isotope dilution technique (15N-IDT), using diets generating low and high ENL and compare results to those obtained under steady-state conditions. Twelve growing pigs (mean LW 22.4 kg) fitted with a post-valve T-caecum cannula were fed an enzyme-hydrolysed casein (EHC)-based diet or an EHC diet + 4% quebracho tannins (QT) and were labelled via continuous 15N-leucine i.v. infusion or twice daily oral 15N-leucine administration. Digesta were collected daily over three consecutive hours with blood plasma sampled on the four consecutive days after cessation of 15N-labelling. There was a significant effect of sampling day on the dilution factor. Endogenous N losses were significantly lower for the EHC than the EHC+QT diet (2.41 vs. 8.69 g/kg DMI), while no significant effect of sampling day was observed. The TD-N of the EHC+QT diet did not differ from the TD-N of the EHC diet (95.1 vs. 92.0%). A significant effect of sampling day was observed for TD-N with day 1 and 2, being higher than day 4. Non-steady-state conditions overestimated ENL by 25-28% as compared to 3 h collections in steady-state conditions, but the relative overestimation was similar for the EHC diet as for the EHC+QT diet. TD-N did not differ significantly compared to 12 h steady-state measurements, but comparison to 3 h steady-state measurements showed that non-steady-state conditions overestimated TD-N for the EHC+QT diet by 9%. However, on day 4 this overestimation disappeared. Using the 15N-IDT during non-steady-state conditions can provide valuable additional data on endogenous N losses and TD-N.

Development of an SI-traceable N-terminal pro-B-type natriuretic peptide certified reference material using structure-based impurity-corrected isotope dilution mass spectrometry approaches.

N-Terminal pro-B-type natriuretic peptide (NT-proBNP) is a pivotal biomarker for the diagnosis and prognosis of heart failure (HF). However, no SI-traceable certified reference material (CRM) or reference measurement procedure (RMP) is available for this biomarker, and so clinical testing results obtained in different laboratories cannot be traced to a higher-order standard, leading to incomparable measurements. Protein hydrolysis and protein cleavage isotope dilution mass spectrometry (AAA-IDMS and PepA-IDMS) were used to develop a CRM. Structurally related impurities were identified by high-resolution mass spectrometry. The quantitative AAA-IDMS results were corrected according to the amino acid compositions of the impurities. Using PepA-IDMS, two peptides from the proteolyzed product were confirmed as signature peptides. To obtain traceable and accurate results, the signature peptides were quantified using impurity-corrected AAA-IDMS. The candidate NT-proBNP solution was denatured and enzymatically digested using the Glu-C endoproteinase. The released signature peptides were measured using an isotopic dilution approach. The homogeneity and stability of the candidate CRM were characterized, and their uncertainties were combined with the value assignment process. The developed CRM can be considered a unique SI-traceable NT-proBNP reference material and is expected to be used as a primary calibrator for matrix NT-proBNP CRM development.

Determination of calcium, iron, and selenium in human serum by isotope dilution analysis using nitrogen microwave inductively coupled atmospheric pressure plasma mass spectrometry (MICAP-MS).

In this study, we demonstrate the applicability of nitrogen microwave inductively coupled atmospheric pressure mass spectrometry (MICAP-MS) for Ca, Fe, and Se quantification in human serum using isotope dilution (ID) analysis. The matrix tolerance of MICAP-MS in Na matrix was investigated, revealing that high Na levels can suppress the signal intensity. This suppression is likely due to the plasma loading and the space charge effect. Moreover, 40Ca and 44Ca isotopic fractionation was noted at elevated Na concentration. Nine certified serum samples were analyzed using both external calibration and ID analysis. Overestimation of Cr, Zn, As, and Se was found in the results of external calibration, which might result from C-induced polyatomic interference and signal enhancement, respectively. Further investigations performed with methanol showed a similar enhancement effect for Zn, As, and Se, potentially supporting this assumption. The mass concentrations determined with ID analysis show metrological compatibility with the reference values, indicating that MICAP-MS combined with ID analysis can be a promising method for precise Ca, Fe, and Se determination. Moreover, this combination reduces the influence of matrix effects, broadening the applicability of MICAP-MS for samples with complex matrixes.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of primidone in human serum and plasma.

OBJECTIVES: Primidone is an anticonvulsive drug used in the treatment of epilepsy and essential tremor. It offers beneficial effects in controlling seizures, but its usage is also associated with possible side effects. To ensure optimal therapy, it is crucial to measure its concentration through accurate quantification methods. Therefore, our main goal was to develop and validate a new reference measurement procedure (RMP) for accurately measuring primidone levels in human serum and plasma. METHODS: In our study, we focused on the separation of primidone from both known and unknown interferences using a C18 column. To achieve accurate sample preparation, we developed a protocol involving protein precipitation followed by a high dilution step. The validation of the assay and determination of measurement uncertainty were carried out following guidelines from organizations such as the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. These rigorous validation processes ensure the reliability and accuracy of our method for quantifying primidone levels in human serum and plasma samples. RESULTS: The RMP was shown to be highly selective and specific, with no evidence of matrix interference. It can be used to quantify primidone in the range of 0.150-30.0 µg/mL. Intermediate precision was less than 4.0 %, and repeatability CV ranged from 1.0 to 3.3 % across all concentration levels. The relative mean bias ranged from 0.1 to 3.9 % for native serum levels, and from -2.6 to 2.8 % for lithium-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were 1.5-4.1 % and 0.9-1.0 %, respectively. CONCLUSIONS: In this study, we introduce an innovative LC-MS/MS-based candidate RMP specifically designed for primidone in human serum and plasma. Our RMP offers a traceable platform, facilitating the standardization of routine assays and enabling the evaluation of clinically relevant samples. With this novel approach, we aim to enhance the accuracy and reliability of primidone measurements, ultimately benefiting the field of clinical research and patient care.

Enhancing LOD determination in gas chromatography: Validating the Hubaux-Vos method for gas concentration measurement.

The limit of detection (LOD) is a crucial measure in analytical methods, representing the smallest amount of a substance that can be distinguished from background noise. In the realm of gas chromatography (GC), however, determining LOD can be quite subjective, leading to significant variability among researchers. In this study, we validate the Hubaux-Vos method, an International Standards Organization(ISO)-approved approach for determining LOD in gas concentration measurements, using a GC equipped with a discharge ionization detector (DID) and a dynamic dilution system. We employ a gas mixture certified reference material (CRM) of CO, CH4, and CO2 at various concentrations to generate calibration curves for each gas. Subsequently, we estimate the LODs for each gas using the Hubaux-Vos method. Surprisingly, our findings indicate a notable difference between the LODs calculated using the Hubaux-Vos method and those confirmed through experiments. This highlights the importance of critically examining the theoretical foundations of LOD determination. We strongly recommend researchers to scrutinize the principles guiding LOD determination. The method proposed in this study offers an effective way to rigorously validate theoretical approaches for estimating LODs in gas concentration measurements using GC.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine-10,11-epoxide in human serum and plasma.

OBJECTIVES: A reference measurement procedure (RMP) using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated with the aim of accurately measuring carbamazepine-10,11-epoxide concentrations in human serum and plasma. METHODS: To establish traceability to SI units, the absolute content of the reference material was determined using quantitative nuclear magnetic resonance (qNMR) spectroscopy. As sample preparation a protein precipitation protocol followed by a high dilution step was established. Chromatographic separation from carbamazepine and potential metabolites was achieved using a C18 stationary phase. Selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement were evaluated based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. RESULTS: The RMP demonstrated very good selectivity and specificity, showing no evidence of a matrix effect. This enabled accurate quantification of carbamazepine-epoxide in the concentration range of 0.0400-12.0 µg/mL. The intermediate precision was found to be less than 2.1 %, and the repeatability coefficient of variation (CV) ranged from 1.2 to 1.8 % across all concentration levels. Regarding accuracy, the relative mean bias varied from 1.4 to 2.5 % for native serum levels and from 1.4 to 3.5 % for Li-heparin plasma levels. The measurement uncertainty for single measurements ranged from 1.6 to 2.1 %. CONCLUSIONS: In this study, we introduce a new LC-MS/MS-based candidate RMP for accurately measuring carbamazepine-10,11-epoxide in human serum and plasma. This novel method offers a traceable and dependable platform, making it suitable for standardizing routine assays and assessing clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of phenobarbital in human serum and plasma.

OBJECTIVES: Phenobarbital serves as an antiepileptic drug (AED) and finds application in the treatment of epilepsy either as monotherapy or adjunctive therapy. This drug exhibits various pharmacodynamic properties that account for its beneficial effects as well as potential side effects. Accurate measurement of its concentration is critical for optimizing AED therapy through appropriate dose adjustments. Therefore, our objective was to develop and validate a new reference measurement procedure (RMP) for the accurate quantification of phenobarbital levels in human serum and plasma. METHODS: A sample preparation protocol based on protein precipitation followed by a high dilution step was established in combination with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a C8 column to separate target analytes from known and unknown interferences. Assay validation and determination of measurement uncertainty were performed based on current guidelines. Selectivity and Specificity were assessed using spiked serum and plasma samples; to investigate possible matrix effects (MEs) a post-column infusion experiment and a comparison of standard line slopes was performed. Precision and accuracy were determined within a multiday precision experiment. RESULTS: The RMP was shown to be highly selective and specific, with no evidence of matrix interferences. It can be used to quantify phenobarbital in the range of 1.92 to 72.0 µg/mL. Intermediate precision was less than 3.2 %, and repeatability coefficient of variation (CV) ranged from 1.3 to 2.0 % across all concentration levels. The relative mean bias ranged from -3.0 to -0.7 % for native serum levels, and from -2.8 to 0.8 % for Li-heparin plasma levels. The measurement uncertainties (k=1) for single measurements and target value assignment were 1.9 to 3.3 % and 0.9 to 1.6 %, respectively. CONCLUSIONS: A novel LC-MS/MS-based candidate RMP for the quantification of phenobarbital in human serum and plasma is presented which can be used for the standardization of routine assays and the evaluation of clinically relevant samples.

Omicron-specific and bivalent omicron-containing vaccine candidates elicit potent virus neutralisation in the animal model.

Omicron variant (B.1.1.529) is able to escape from naturally acquired and vaccine-induced immunity, which mandates updating the current COVID-19 vaccines. Here, we investigated and compared the neutralising antibody induction of the ancestral variant-based BIV1-CovIran vaccine, the Omicron variant-based BIV1-CovIran Plus vaccine, and the novel bivalent vaccine candidate, BBIV1-CovIran, against the Omicron and ancestral Wuhan variants on the rat model. After inactivating the viral particles, the viruses were purified and formulated. Bivalent vaccines were a composition of 2.5 µg (5 µg total) or 5 µg (10 µg total) doses of each ansectral-based and Omicron-based monovalent vaccine. Subsequently, the potency of the monovalent and bivalent vaccines was investigated using the virus neutralisation test (VNT). The group that received three doses of the Omicron-specific vaccine demonstrated neutralisation activity against the Omicron variant with a geometric mean titer of 337.8. However, three doses of the Wuhan variant-specific vaccine could neutralise the Omicron variant at a maximum of 1/32 serum dilution. The neutralisation activity of the Omicron-specific vaccine, when administered as the booster dose after two doses of the Wuhan variant-specific vaccine, was 100% against the Omicron variant and the Wuhan variant at 1/64 and 1/128 serum dilution, respectively. Three doses of 5 µg bivalent vaccine could effectively neutralise both variants at the minimum of 1/128 serum dilution. The 10 µg bivalent vaccine at three doses showed even higher neutralisation titers: the geometric mean of 388 (95% CI 242.2-621.7) against Omicron and 445.7 (95% CI 303.3-655.0) against Wuhan. It is shown that the candidate bivalent and Omicron-specific vaccines could elicit a potent immune response against both Wuhan-Hu-1 and Omicron BA.1 variants.

Molecular Signal Transfer of Highly Diluted Antibodies to Interferon-Gamma Regarding Kind, Time, and Distance of Exposition.

Physicochemical examinations of very high dilution (UHD) solutions subjected to certain physical factors (such as shaking) are becoming more frequent and are increasingly producing conclusive results. A much less studied phenomenon is the transfer of molecular information (i.e., UHD signals of dilute substances) from one liquid to another without an intermediate liquid phase. The aim of this study was to investigate the possibility of such a transfer of the UHD signal from the UHD solutions to the receiver solution, in particular, if the molecular source used in the donor solutions was the biologically active antibodies to interferon-gamma molecule. We were especially interested in how the transfer of the UHD signal is affected by the time of exposure of the receiver to the donor, the distance between the two, and how the transfer is affected by activation (striking) versus exposure alone. Signal transfer was evaluated by differential measurements of electrical conductivity, ORP, pH, and UV/VIS spectroscopy of the exposed liquid. The results showed that activation strongly influences signal transfer and that this can be compensated to some extent by prolonged direct exposure. In principle, exposure time has a positive effect on signal transfer. Interestingly, the results of different distances between the donor and receiver showed similar changes in the parameters in the range of 0-4 cm, as estimated in this study. While the study mainly confirms the two hypotheses, it also raises a number of new questions and provides clues for further research.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of zonisamide in human serum and plasma.

OBJECTIVES: To describe and validate an isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure (RMP) for zonisamide to accurately measure serum and plasma concentrations. METHODS: Quantitative nuclear magnetic resonance (qNMR) spectroscopy was employed to determine the absolute content of the reference material used in order to establish traceability to SI units. Separation of zonisamide from known or unknown interferences was performed on a C8 column. For sample preparation a protocol based on protein precipitation in combination with a high dilution step was established. Assay validation and determination of measurement uncertainty were performed based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. RESULTS: The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of zonisamide within the range of 1.50-60.0 µg/mL. Intermediate precision was <1.4 % and repeatability CV ranged from 0.7 to 1.2 % over all concentration levels. The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment ranged from 1.1 to 1.4 % and 0.8-1.0 %, respectively. CONCLUSIONS: We present a novel LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Estimating Muscle Mass Using D3-Creatine Dilution: A Narrative Review of Clinical Implications and Comparison With Other Methods.

BACKGROUND: The D3-creatine (D3-Cr) dilution method is of emerging interest for estimating total-body skeletal muscle mass. This review explores the association of muscle mass estimated via D3-Cr with various clinical outcomes and provides a summary of the literature comparing D3-Cr with other body composition techniques. METHODS: A literature search was conducted on PubMed/MEDLINE and Web of Science for studies using D3-Cr to measure muscle in adult populations (ie, ≥18 years old) from inception until September 2023. RESULTS: Out of the 23 included studies, 15 investigated the correlation between D3-Cr and clinical outcomes. More consistent associations were reported for mortality (100%, n = 2), mobility disability (100%; n = 5), falls and fractures (100%; n = 3), physical performance (63.3%; n = 11), muscle strength (44.4%; n = 9), and muscle composition (33.3%; n = 3). However, conflicting findings were also reported for such correlations. Among the 23 studies, 14 compared D3-Cr-estimated muscle with other body composition techniques, including magnetic resonance imaging (MRI) as a reference method. Strong and positive correlations were found between D3-Cr and MRI. Nonetheless, variations in muscle measurements were noted, with differences in D3-Cr values ranging from 0.62 kg lower to 13.47 kg higher compared to MRI. CONCLUSIONS: D3-Cr-estimated muscle mass may be a valuable predictor of clinical outcomes showing consistent associations with falls and fractures, mobility disability, and mortality. However, less consistent associations were found with muscle strength and composition, and physical performance. Although a strong correlation exists between D3-Cr-estimated muscle mass and MRI measurements, under- or overestimation may occur.

Real-time and accurate estimation of surgical hemoglobin loss using deep learning-based medical sponges image analysis.

Real-time and accurate estimation of surgical hemoglobin (Hb) loss is essential for fluid resuscitation management and evaluation of surgical techniques. In this study, we aimed to explore a novel surgical Hb loss estimation method using deep learning-based medical sponges image analysis. Whole blood samples of pre-measured Hb concentration were collected, and normal saline was added to simulate varying levels of Hb concentration. These blood samples were distributed across blank medical sponges to generate blood-soaked sponges. Eight hundred fifty-one blood-soaked sponges representing a wide range of blood dilutions were randomly divided 7:3 into a training group (n = 595) and a testing group (n = 256). A deep learning model based on the YOLOv5 network was used as the target region extraction and detection, and the three models (Feature extraction technology, ResNet-50, and SE-ResNet50) were trained to predict surgical Hb loss. Mean absolute error (MAE), mean absolute percentage error (MAPE), coefficient (R2) value, and the Bland-Altman analysis were calculated to evaluate the predictive performance in the testing group. The deep learning model based on SE-ResNet50 could predict surgical Hb loss with the best performance (R2 = 0.99, MAE = 11.09 mg, MAPE = 8.6%) compared with other predictive models, and Bland-Altman analysis also showed a bias of 1.343 mg with narrow limits of agreement (- 29.81 to 32.5 mg) between predictive and actual Hb loss. The interactive interface was also designed to display the real-time prediction of surgical Hb loss more intuitively. Thus, it is feasible for real-time estimation of surgical Hb loss using deep learning-based medical sponges image analysis, which was helpful for clinical decisions and technical evaluation.

Indocyanine Green (ICG) Fluorescence Is Dependent on Monomer with Planar and Twisted Structures and Inhibited by H-Aggregation.

The optical properties of indocyanine green (ICG) as a near-infrared (NIR) fluorescence dye depend on the nature of the solvent medium and the dye concentration. In the ICG absorption spectra of water, at high concentrations, there were absorption maxima at 700 nm, implying H-aggregates. With ICG dilution, the main absorption peak was at 780 nm, implying monomers. However, in ethanol, the absorption maximum was 780 nm, and the shapes of the absorption spectra were identical regardless of the ICG concentration, indicating that ICG in ethanol exists only as a monomer without H-aggregates. We found that emission was due to the monomer form and decreased with H-aggregate formation. In the fluorescence spectra, the 820 nm emission band was dominant at low concentrations, whereas at high concentrations, we found that the emission peaks were converted to 880 nm, suggesting a new form via the twisted intramolecular charge transfer (TICT) process of ICG. The NIR fluorescence intensity of ICG in ethanol was approximately 12- and 9-times brighter than in water in the NIR-I and -II regions, respectively. We propose an energy diagram of ICG to describe absorptive and emissive transitions through the ICG structures such as the monomer, H-aggregated, and TICT monomer forms.

[Optimization of three-dimensional culture conditions of L02 cells with response surface methodology based on VitroGel system].

OBJECTIVE: This study optimizes three-dimensional(3D)culture conditions of L02 cells using response surface methodology(RSM) based on the VitroGel system to construct the hepatocytes model in vitro. METHODS: L02 cells were 3D cultured by the VitroGel system. The appropriate level of three key factors(concentration of inoculated cells, culture time and dilution degree of the hydrogel) was determined by single-factor experiment, and the optimal conditions of 3D culture of L02 cells based on the VitroGel system were determined by RSM. During the detection process, the optical density(OD) value of cell viability was used as the detection index, and the cell viability was detected using the cell counting kit-8(CCK-8) assay. The proliferative performance and viability of L02 cells was measured by fluorescent staining assay. RESULTS: The selected optimal culture conditions by RSM were as follows: concentration of inoculated cells was 1.1 × 10~5/mL, culture time was 9.5 days, and dilution degree of hydrogel was 1∶3.7. The result shows that under optimal conditions, the predicted OD value of cell viability was 2.17 and measured 2.13 with a relative error of 1.84%, indicating that the condition was suitable and reliable. The fluorescent staining and dead and live cells detection results showed the 3D hepatocytes model was successfully constructed. CONCLUSION: The optimal conditions for 3D culture of L02 cell based on the VitroGel system were determined by RSM, and a hepatocytes model with high cellular activity was successfully constructed.