Laser Sources for Traditional and Spectral Flow Cytometry.

Lasers for light scattering measurement and fluorescence excitation are essential components of all flow cytometers. Flow cytometers now typically rely on multiple laser wavelengths allowing excitation of a constantly increasing variety of fluorescent probes. The expanding use of spectral flow cytometry to increase the magnitude of multiparametric analysis is also changing the significance of laser choice in cytometry. In this chapter, we review the lasers available for flow cytometry and provide guidance in choosing laser wavelengths and characteristics to best match the needs of modern cell analysis by both conventional and spectral cytometry. We also discuss the recent advances in laser technology as the push to expand the palette of laser wavelength for cytometry continues.

Novel automated antifungal susceptibility testing system for yeasts based on dual-detection algorithm of turbidimetry and colorimetry.

Introduction. The increasing prevalence and growing resistance of fungi present a significant peril to public health. There are only four classes of antifungal medicines available today, and few candidates are in clinical trials.Hypothesis/Gap Statement. Rapid and sensitive diagnostic techniques are lacking for most fungal pathogens, and those that do exist are expensive or hard to obtain.Aim. This study aimed to evaluate the feasibility of a novel automated antifungal susceptibility testing system, Fungus AST, in comparison to the broth microdilution method (BMD) recommended by the Clinical and Laboratory Standards Institute (CLSI).Methodology. A total of 101 clinical Candida spp. isolates were collected from the Zengcheng Branch of Nanfang Hospital and subjected to antifungal susceptibility testing. Antifungal susceptibility was assessed using the Fungus AST method and the BMD.Results. In this study, we introduce a novel automated antifungal susceptibility testing system, Fungus AST, which detects the turbidity and/or colour intensity of microdilution wells using a four-wavelength detection technology in real time and is designed to match the growth characteristics of strains over time. Based on our analysis, all reportable ranges of Fungus AST were suitable for clinical fungal isolates in PR China. Within ±twofold dilutions, reproducibility was 100 %. Considering the BMD as a referenced method, ten antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole, voriconazole, posaconazole, itraconazole, amphotericin B, 5-flucytosine and nystatin) showed an essential agreement of >95 %. The category agreement of five antifungal agents (anidulafungin, caspofungin, micafungin, fluconazole and voriconazole) was excellent at >90 %. One Candida albicans isolate and voriconazole showed a major error (ME) (1.7 %), and no other ME or very ME agents were found.Conclusion. Given the above, it can be argued that the utilization of Fungus AST is a discretionary automated approach. More improvements are needed in Fungus AST compared to the BMD system for a wider range of clinical isolates, including different types of fungi.

In Situ Oral Metabolism Analysis of Astringent Compounds in Tea by Paper Spray Mass Spectrometry, Electrospray Mass Spectrometry, Turbidimetry, and Sensory Evaluation.

The phenolic compounds (PCs) are the primary components responsible for the astringency of tea infusions, and this astringency is intricately linked to the in situ oral metabolism of PCs in saliva. Initially, a total of 54 PCs were identified in tea infusions by electrospray mass spectrometry (ESI-MS). Subsequently, an in vivo metabolism analysis of PCs during varying drinking times and oral locations was conducted by both paper spray mass spectrometry (PS-MS) and sensory evaluation. The metabolism of PCs within oral saliva was a prolonged process, the residual PCs were distributed across diverse oral regions after drinking tea infusion, and the higher residual PC content reflected the stronger astringency intensity. Furthermore, an in vitro metabolism analysis of PCs under varied reaction temperatures and durations was performed by ESI-MS and turbidimetry. As the reaction time extended, more PCs in tea was interacting with saliva. Moreover, the higher temperatures facilitated this interaction between PCs and saliva. Therefore, this investigation establishes a foundation for further elucidating the mechanisms underlying astringency formation.

Measuring of Alpha-1 Antitrypsin Concentration by Nephelometry or Turbidimetry.

Most clinical laboratories quantify alpha-1 antitrypsin using either nephelometry or turbidimetry techniques because they are commercially available, amenable to automation, and precise. Both methods are based on light scatter. The foundation of both techniques is based on incubation of the specimen with anti-AAT polyclonal antibody solution, a polymer matrix between endogenous AAT and the reagent antibodies forms, leading to production of light-scattering large particles. Although these two terms are sometimes used synonymously, technically speaking they are not.Nephelometry measures the amount of turbidity or cloudiness of a solution by directly quantifying the intensity of the light scattered by insoluble particles in the sample. Therefore, this technique measures the light that passes through the sample, with the detector being placed at an angle from the sample. Turbidimetry is the process of measuring the loss of intensity of the light transmitted linearly through a sample caused by the scattering effect of insoluble particles. The decrease in light transmission is measured compared to a reference, and the absorbed light is quantified.Beyond specific technical differences between both techniques, there are two major differences between the two procedures that may influence the results. First, the concentration of the sample and the resulting intensity of scattered light relative to the intensity of the light source is one major factor. Second, the size of the scattering particles is also a key differentiating factor. This chapter describes the technical requirements, the different protocols, and the clinical applicability of these two techniques in the diagnosis of alpha-1 antitrypsin deficiency.

Individual polysaccharide quantification in polyvalent pneumococcal conjugate vaccine using rate nephelometry.

The multivalent pneumococcal conjugate vaccine (PCV) contains purified polysaccharides of different serotypes conjugated to a carrier protein. Testing the final formulated product for individual serotype polysaccharide content is critical in vaccine quality control which requires an assay specific to each serotype polysaccharide present in the formulated product. Antibodies specific to the serotypes specific polysaccharide were used in rate nephelometry assay for quantifying individual serotype polysaccharides in the formulated vaccine. Generally, native polysaccharide (NP) have been used as reference standard. However, the polysaccharide antigen in the vaccine product is in the conjugate form (CRM197 linked) and hence using NP as a reference standard may not be suitable. Activated quenched polysaccharide (AQP) as a reference standard in rate nephelometry would be more appropriate. The epitope structure of AQP closely represents the polysaccharide-protein conjugate drug product (DP) after trypsin digestion. Hence, AQP was evaluated as a novel reference standard for the accurate and precise determination of individual polysaccharides in the multivalent DP. Rate nephelometry assay using AQP could be used for DP release and stability for monitoring time-dependent changes in the product and establishing the shelf life. A similar strategy could be applied to test and release monovalent or multivalent polysaccharide-protein conjugate vaccines (Meningococcal, Haemophilus influenza Type B, Typhoidal, and non-typhoidal salmonella).

Shape Discrimination of Individual Aerosol Particles Using Light Scattering.

We established an experimental apparatus by combining polarized light scattering and angle-resolved light scattering measurement technology to rapidly identify the shape of an individual aerosol particle. The experimental data of scattered light of Oleic acid, rod-shaped Silicon dioxide, and other particles with typical shape characteristics were analyzed statistically. To better study the relationship between the shape of particles and the properties of scattered light, the partial least squares discriminant analysis (PLS-DA) method was used to analyze the scattered light of aerosol samples based on the size screening of particles, and the shape recognition and classification method of the individual aerosol particle was established based on the analysis of the spectral data after nonlinear processing and grouping by particle size with the area under the receiver operating characteristic curve (AUC) as reference. The experimental results show that the proposed classification method has a good discrimination ability for spherical, rod-shaped, and other non-spherical particles, which can provide more information for atmospheric aerosol measurement, and has application value for traceability and exposure hazard assessment of aerosol particles.

Nonlinear multi-magnon scattering in artificial spin ice.

Magnons, the quantum-mechanical fundamental excitations of magnetic solids, are bosons whose number does not need to be conserved in scattering processes. Microwave-induced parametric magnon processes, often called Suhl instabilities, have been believed to occur in magnetic thin films only, where quasi-continuous magnon bands exist. Here, we reveal the existence of such nonlinear magnon-magnon scattering processes and their coherence in ensembles of magnetic nanostructures known as artificial spin ice. We find that these systems exhibit effective scattering processes akin to those observed in continuous magnetic thin films. We utilize a combined microwave and microfocused Brillouin light scattering measurement approach to investigate the evolution of their modes. Scattering events occur between resonance frequencies that are determined by each nanomagnet's mode volume and profile. Comparison with numerical simulations reveals that frequency doubling is enabled by exciting a subset of nanomagnets that, in turn, act as nanosized antennas, an effect that is akin to scattering in continuous films. Moreover, our results suggest that tunable directional scattering is possible in these structures.

Clinical relevance of leukocyte-associated endotoxins measured by semi-automatic synthetic luminescent substrate method.

A newly developed semi-automatic synthetic luminescence substrate (SALS) method for measuring endotoxin was compared with the existing turbidimetric kinetic assay (TKA) using leukocyte-rich plasma to verify its usefulness. As a result, the endotoxin levels by this method were higher than that by the existing assay in most specimens, and the time required for measurement was much shorter. In addition, the leukocyte-rich plasma endotoxin level minus the plasma endotoxin levels were named leukocyte-associated endotoxin, and these levels per one leukocyte were compared. As a result, those levels were highly correlated with the endotoxin measurement levels of leukocyte-rich plasma. The correlation coefficient of SALS method was superior to the existing TKA method, the endotoxin level by this method may be close to true endotoxin levels.

Underwater polarization imaging for visibility enhancement of moving targets in turbid environments.

Polarization imaging techniques have more prominent advantages for imaging in strongly scattered media. Previous de-scattering methods of polarization imaging usually require the priori information of the background region, and rarely consider the effect of non-uniformity of the optical field on image recovery, which not only reduces the processing speed of imaging but also introduces errors in image recovery, especially for moving targets in complex scattering environments. In this paper, we propose a turbid underwater moving image recovery method based on the global estimation of the intensity and the degree of polarization (DOP) of the backscattered light, combined with polarization-relation histogram processing techniques. The full spatial distribution of the intensity and the DOP of the backscattered light are obtained by using frequency domain analysis and filtering. Besides, a threshold factor is set in the frequency domain low-pass filter, which is used to adjust the execution region of the filter, which effectively reduces the error in image recovery caused by estimating the DOP of the backscattered light as a constant in traditional methods with non-uniform illumination. Meanwhile, our method requires no human-computer interaction, which effectively solves the drawbacks that the moving target is difficult to be recovered by traditional methods. Experimental studies were conducted on static and moving targets under turbid water, and satisfactory image recovery quality is achieved.

Comparison of the ELISA method with the nephelometric method for determination of serum amyloid A in patients with COVID 19.

This study aimed to examine the agreement of the serum amyloid A (SAA) values determined using the ELISA test and the nephelometric automated method. This study included 80 serum samples obtained from patients with COVID-19. Samples were determined using ELISA and the nephelometric method. Wilcoxon signed ranks test showed a statistically significant difference in the calculated median values (Z = -2.432, p = 0.015). The correlation between methods was statistically significant (r = 0.603, p < 0.0001). Bland Altman analysis showed a bias of 56.6 mg/L and a relative bias of 7.4% between the methods. The results of this study indicate that further studies are needed that will examine the compliance between the ELISA and the nephelometric method for determining SAA, and the results must be carefully interpreted based on the method used.

Study on the Mechanism of False Low Measurement of IgG-Binding (Affinity) IgM Type M Protein by Turbidimetric Immunoassay.

BACKGROUND: The purpose of this study was to investigate the immunological and physical characteristics of IgM-λ type M-protein from patients who were measured low in the turbidimetric immunoassay (TIA) IgM assay without error codes for high concentration to determine the cause of the false low levels and to clarify the mechanism of their occurrence. METHODS: Materials were IgM patient samples and 8 serum samples from other IgM M-protein patients as controls. Patient samples were assayed by the TIA method, in which five manufacturers and six models (two reagent manufacturers) share the principle, and the BN ProSpec method (nephelometric method), which has a different principle. Dilution linearity tests, IgG addition experiments, isoelectric point electrophoresis, and hydrophobic chromatography were performed on patients and subjects. In addition, the binding capacity of γ-globulin by BIACORE was also examined. RESULTS: The reaction curve of the patient IgM curved downward when the concentration of IgM exceeded 20 g/L, and no error code was obtained. In the measurement by the TIA method of five manufacturers and six models, patient IgM was measured at a false low level with no error code obtained in undiluted dilution by any of the instruments and reagents, but could be measured without any problem by the nephelometric method. In addition, in the patient IgG addition experiment, only patient IgM showed a false low level under high IgG concentration. Furthermore, the binding capacity of patient IgM to γ-globulin (IgG) by BIACORE was significantly higher than that of the control IgM-type M protein. CONCLUSIONS: Patient IgM has an affinity (binding capacity) for IgG and forms an IgM-IgG complex under conditions of high IgG concentration. It was speculated that this complex inhibited the reaction with the anti-IgM antibody and the absorbance of the second reaction did not increase, suggesting a false low.

Establishing the lower bacterial concentration threshold for different optical counting techniques.

This work compares several physical and optical techniques used in fundamental research and industrial applications to detect bacteria in water. Optical techniques such as, UV-absorbance spectroscopy, laser particle counting, turbidimetry and Z-Sizer light scattering, and a direct observational physical technique, the plate count method, were compared when measuring the concentration of E.coli in tenfold dilution from a stock solution. Estimates of the detection threshold limit of E.coli for the different optical counting techniques and the relationship between colony-forming units (CFU) and tenfold dilutions was established. Optical methods have generated interest due to the rapid response of just minutes, non-destructive approach and minimal sample preparation but their use is still limited to concentrations of up to 4 Log E.coli/mL. In contrast, the plate count method is still a reliable technique for water quality analysis despite its long response time of 24 h.

Chicken antibodies are highly suitable for particle enhanced turbidimetric assays.

Antibody-based assays are commonly used in clinical laboratories for analyzing plasma, serum and other samples for particular protein markers. Although such assays have been traditionally based on antibodies raised in mammals (e.g., mice, rabbits, goats), there are several advantages of using avian antibodies (IgY) raised in chickens, including production volumes, costs, and ethical/animal welfare considerations. A further disadvantage of using mammalian IgG in such assays is the potential for agglutination when exposed to rheumatoid factor (RF) in serum. However, when used in the free form the immune complexes formed with avian antibodies have been reported to have less ability than those formed with mammalian antibodies to cause the light scatter which are used for instrument measurement. In addition, when the amount of antigen exceeds the maximum precipitating point in relation to the amount of antibody, there is a rapid decline in the absorbance values of the immune complexes (antigen excess) when IgY is used. However, when avian antibodies are conjugated to a substrate and used in particle enhanced turbidimetric assays (PETIA), these problems are avoided. Here we investigated three clinical assays using chicken antibodies, one using free (unbound) IgY and two with IgY-based PETIA. The IgY PETIA demonstrated a strong scatter response, even at high antigen concentrations in contrast to the steep decline seen with free IgY antibodies. IgY PETIA reagents can provide test results with low coefficient of variation (<1% for duplicate samples). We also investigated the effect of RF on agglutination of mammalian antibodies (IgG from mouse, rabbit, sheep, and human) and chicken antibodies. Whereas agglutination was observed with all the mammalian antibodies in the presence of RF, this was not observed at all with chicken IgY. Our results support the growing body of evidence that chicken egg yolks can thus be a valuable source of antibodies for use in PETIA in clinical laboratories.

Spatially resolved reflectance from turbid media having a rough surface. Part II: experiments.

Spatially resolved reflectance measurements are a standard tool for determining the absorption and scattering properties of turbid media such as biological tissue. However, in literature, it was shown that these measurements are subject to errors when a possible rough surface between the turbid medium and the surrounding is not accounted for. We evaluated these errors by comparing the spatially resolved reflectance measured on rough epoxy-based samples with Monte Carlo simulations using Lambertian surface scattering, the Cook-Torrance model, and the generalized Harvey-Shack model as surface scattering models. To this aim, goniometric measurements on the epoxy-based samples were compared to the angularly resolved reflectance of the three surface models to estimate the corresponding model parameters. Finally, the optical properties of the phantoms were determined using a Monte Carlo model with a smooth surface.

A method to obtain purified free light chain monomers and dimers from urine samples of patients with multiple myeloma.

Antibody light chains are synthesized in excess by plasma cells, and this excess can be secreted into biological fluids as dimers or monomers in various proportions. Structural differences between monomers or dimers of free light chains (FLC) can affect their biological functions and possibly their pathogenicity. They also may exhibit differential immune reactivity, perhaps explaining discrepant quantifications when measured by different immunoreagents. Having purified FLC monomers and dimers available can be useful for studying their properties. Here we propose a simple preparatory procedure to purify FLC monomers and dimers from urine samples of patients with plasma cell disorders. Two representative urine samples containing lambda or kappa FLC were loaded into a nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel strips containing separate monomers and dimers were excised, electroeluted, and the FLC recovered. The FLC were recovered from SDS-PAGE gel in sufficient amounts to be quantified by UV and two automated nephelometric assays immunochemical. The procedure was found to be simple, reproducible, and with a high yield, thus offering the opportunity to compare different assays. Not all urine samples are suitable for this procedure, but this approach allows for the purification of FLC monomers and dimers from many selected urine samples which maintain their oligomeric organization.

Quantitation of IgG Subclasses in Serum Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Serum IgG subclasses (IgGSC) are measured for a number of indications, but the most common are the identification of selective immunodeficiency disease and the diagnosis of IgG4-related disease (IgG4RD). Traditional nephelometric (IN) assays can suffer from two issues impacting the accuracy of the results: (1) hook effect and (2) antibody cross-reactivity between the subclasses. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is not vulnerable to these modes of interference and therefore serves as an excellent and relatively inexpensive means of diagnosing and/or monitoring the relevant clinical conditions.We describe a semiautomated and simple method for the accurate and precise measurement of IgGSC from 20 µL of serum using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method following digestion of serum proteins in 96-well plate format. Due to the high abundance of the target proteins, no specialized sample preparation (such as solid phase extraction) is required. Twenty microliters are injected to the LC-MS/MS system. Quantitation is performed against a five-point duplicate linear calibration curve prepared in blank matrix. The assay calibration range is 0.38-7.74 g/L for IgG1, 0.24-4.46 g/L for IgG2, 0.038-0.752 g/L for IgG3, 0.025-0.435 g/L for IgG4, and 0.62-15.5 g/L for total IgG. Total IgG is used as an internal quality control marker and is compared to the sum of the four subclass results. Total imprecision in clinical production has been observed to be 5.1-10.6% for in-house prepared control materials having IgGSC mean values in the range of 0.38-8.43 g/L for IgG1, 0.22-3.76 g/L for IgG2, 0.0387-0.721 g/L for Ig3, and 0.0279-1.46 g/L for IgG4. Limit of quantitation (LoQ) was determined to be 0.29 g/L for IgG1, 0.22 g/L for IgG2, 0.019 g/L for IgG3, and 0.0067 g/L for IgG4.

The Applicability of Current Turbidimetric Approaches for Analyzing Fibrin Fibers and Other Filamentous Networks.

Turbidimetry is an experimental technique often used to study the structure of filamentous networks. To extract structural properties such as filament diameter from turbidimetric data, simplifications to light scattering theory must be employed. In this work, we evaluate the applicability of three commonly utilized turbidimetric analysis approaches, each using slightly different simplifications. We make a specific application towards analyzing fibrin fibers, which form the structural scaffold of blood clots, but the results are generalizable. Numerical simulations were utilized to assess the applicability of each approach across a range of fiber lengths and diameters. Simulation results indicated that all three turbidimetric approaches commonly underestimate fiber diameter, and that the "Carr-Hermans" approach, utilizing wavelengths in the range of 500−800 nm, provided <10% error for the largest number of diameter/length combinations. These theoretical results were confirmed, under select conditions, via the comparison of fiber diameters extracted from experimental turbidimetric data, with diameters obtained using super-resolution microscopy.

Influence of Lambertian surface scattering on the spatially resolved reflectance from turbid media: a computational study.

The determination of the optical properties in turbid media plays an essential role in medical diagnostics and process control. The method of spatially resolved reflectance measurements is a frequently used tool to evaluate the reduced scattering coefficient as well as the absorption coefficient. In most cases a smooth interface is assumed between the medium under investigation and the surrounding medium. However, in reality, a rough surface is present at the interface, which alters the light interaction with the surface and volume of the turbid medium. Hence, the idea behind this paper was to investigate the influence of rough surfaces on the spatially resolved reflectance and thus on the determination of the optical properties of turbid media. Particularly, the influence of a Lambertian scattering surface on the result of Monte Carlo simulations of a spatially resolved reflectance setup is shown. In addition, we distinguish between the different interaction modes of surface scattering on the spatially resolved reflectance. There is a strong influence of roughness when the light enters and leaves the turbid medium. Furthermore, the simulations show that, especially for small reduced scattering coefficients and absorption coefficients, large errors in the determination of the optical properties are obtained.

Efficacy of antifungal agents against fungal spores: An in vitro study using microplate laser nephelometry and an artificially infected 3D skin model.

Dermal fungal infections seem to have increased over recent years. There is further a shift from anthropophilic dermatophytes to a growing prevalence of zoophilic species and the emergence of resistant strains. New antifungals are needed to combat these fungi and their resting spores. This study aimed to investigate the sporicidal effects of sertaconazole nitrate using microplate laser nephelometry against the microconidia of Trichophyton, chlamydospores of Epidermophyton, blastospores of Candida, and conidia of the mold Scopulariopsis brevicaulis. The results obtained were compared with those from ciclopirox olamine and terbinafine. The sporicidal activity was further determined using infected three-dimensional full skin models to determine the antifungal effects in the presence of human cells. Sertaconazole nitrate inhibited the growth of dermatophytes, molds, and yeasts. Ciclopirox olamine also had good antifungal activity, although higher concentrations were needed compared to sertaconazole nitrate. Terbinafine was highly effective against most dermatophytes, but higher concentrations were required to kill the resistant strain Trichophyton indotineae. Sertaconazole nitrate, ciclopirox olamine, and terbinafine had no negative effects on full skin models. Sertaconazole nitrate reduced the growth of fungal and yeast spores over 72 h. Ciclopirox olamine and terbinafine also inhibited the growth of dermatophytes and molds but had significantly lower effects on the yeast. Sertaconazole nitrate might have advantages over the commonly used antifungals ciclopirox olamine and terbinafine in combating resting spores, which persist in the tissues, and thus in the therapy of recurring dermatomycoses.

Associations between the von Willebrand Factor-ADAMTS13 Axis, Complement Activation, and COVID-19 Severity and Mortality.

BACKGROUND: Endothelial and complement activation were both associated with immunothrombosis, a key determinant of COVID-19 severity, but their interrelation has not yet been investigated. OBJECTIVES: We aimed to determine von Willebrand factor (VWF) antigen (VWF:Ag) concentration, VWF collagen binding activity (VWF:CBA), a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity (ADAMTS13:Ac), and their ratios in hospitalized COVID-19 patients, and to investigate how these parameters and their constellation with complement activation relate to disease severity and in-hospital mortality in COVID-19. METHODS: Samples of 102 hospitalized patients with polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 positivity were included in our observational cohort study. Patients were stratified according to the peak severity of COVID-19 disease in agreement with the World Health Organization ordinal scale. Twenty-six convalescent plasma donors with previous COVID-19 disease formed the control group. VWF:Ag concentration and VWF:CBA were determined by enzyme-linked immunosorbent assay (ELISA); ADAMTS13:Ac was determined by fluorescence resonance energy transfer. Complement C3 and C3a were measured by turbidimetry and ELISA, respectively. Clinical covariates and markers of inflammation were extracted from hospital records. RESULTS: VWF:Ag and VWF:CBA were elevated in all groups of hospitalized COVID-19 patients and increased in parallel with disease severity. ADAMTS13:Ac was decreased in patients with severe COVID-19, with the lowest values in nonsurvivors. High (> 300%) VWF:Ag concentrations or decreased (< 67%) ADAMTS13:Ac were associated with higher risk of severe COVID-19 disease or in-hospital mortality. The concomitant presence of decreased ADAMTS13:Ac and increased C3a/C3 ratio-indicating complement overactivation and consumption-was a strong independent predictor of in-hospital mortality. CONCLUSION: Our results suggest that an interaction between the VWF-ADAMTS13 axis and complement overactivation and consumption plays an important role in the pathogenesis of COVID-19.