Density Of Specimens Research Articles

Hot-pressed calcium phosphates as a potential bone substitute: structural and mechanical characterization

The compositional similarity of calcium phosphates such as β-TCP and HAp to the inorganic components of human bones makes them excellent candidates for bone substitutes. Regardless of presenting excellent biocompatibility, calcium phosphates present low mechanical strength, which is a major drawback for load-bearing applications. In this sense, achieving Hap or β-TCP with increased density is crucial to enhance their mechanical properties. In the present study, β-TCP and HAp were processed from commercially available powders in order to obtain highly dense specimens aiming to elevate these mechanical properties of calcium phosphates. For this purpose, two sintering strategies were used: in the first, using a single holding time, whereas in the second, two holding times. The obtained phases, their potential degradation, microstructure, porosity, and mechanical strength were investigated. Results revealed that the use of two holding times improved densification, leading to flexural strength improvement, on both materials, but especially on HAp, where a 122% increase was verified.

Pixel-reassigned line-scanning microscopy for fast volumetric super-resolution imaging.

Super-resolution microscopy has revolutionized the field of biophotonics by revealing detailed 3D biological structures. Nonetheless, the technique is still largely limited by the low throughput and hampered by increased background signals for dense or thick biological specimens. In this paper, we present a pixel-reassigned continuous line-scanning microscope for large-scale high-speed 3D super-resolution imaging, which achieves an imaging resolution of 0.41 µm in the lateral direction, i.e., a 2× resolution enhancement from the raw images. Specifically, the recorded line images are first reassigned to the line-excitation center at each scanning position to enhance the resolution. Next, a modified HiLo algorithm is applied to reduce the background signals. Parametric models have been developed to simulate the imaging results of randomly distributed fluorescent beads. Imaging experiments were designed and performed to verify the predicted performance on various biological samples, which demonstrated an imaging speed of 3400 pixels/ms on millimeter-scale specimens. These results confirm the pixel-reassigned line-scanning microscopy is a facile and powerful method to realize large-area super-resolution imaging on thick or dense biological samples.

Living benthic foraminifera from Almirante Câmara and Grussaí canyons and adjacent slope areas (Campos Basin, Southwest Atlantic): Response to trophic and hydrodynamic conditions

Living (rose Bengal stained) benthic foraminiferal faunas, grain size, geochemical parameters (total organic matter, chlorophyll-a, pheophytin-a, molecular markers, and vertical flux of particulate organic matter) were investigated in 14 stations in two submarine canyons Almirante Câmara and Grussaí considered mature and immature, respectively, and on the adjacent continental slope, from 400 to 1300 m water depth, in the southwestern Atlantic Ocean. Samples were retrieved during two campaigns, in the austral winter 2008 and the austral summer 2009. The density, richness, and species composition of the benthic foraminiferal fauna decrease with depth in the canyons and along the adjacent slope transects, reflecting the availability and quality of sedimentary organic matter. Both canyons act as traps of labile organic matter, mainly towards the head of the Almirante Câmara canyon, promoting high specimen density, species richness, and the occurrence of species indicative of pulses of phytodetritus (e.g., Bolivina pacifica, Nonionella opima, and Nonionella stella). Towards the deeper regions of Almirante Câmara canyon and in the adjacent slope area, the lower abundance, diversity, and richness reflect more oligotrophic conditions and an adverse environment in terms of easily metabolizable organic matter. This multiproxy study demonstrates that the canyons in the southeastern Brazilian continental margin play an important role in accumulating organic matter.

Experimental study on the strain energy evolution mechanism of thick and hard sandstone roof in Xinjiang Mining Area

For understanding the mechanical performance and strain energy evolution mechanism of thick hard roof sandstone samples, a sequence of uniaxial compression trials with acoustic emission (AE) monitoring were carried out. The results indicate: (1) The stress-strain curve of the thick hard roof sandstone specimens exhibits distinct stage characteristics. Based on the evolvement of instantaneous axial stiffness, it is separated into Fracture Closure Phase, Elastic Deformation Phase, Steady Fracture Expansion Phase, Unsteady Fracture Expansion Phase, and Post-Peak Phase. (2) The AE energy and cumulative count curves of the thick hard roof sandstone specimens also exhibit significant stage characteristics and can be mutually corroborated with the stage division of the stress-strain curve. (3) Based on the energy conservation principle, the evolution of strain energy density in the thick hard roof sandstone specimens under uniaxial compression loading was analyzed, and plastic strain energy increment was employed to study the stage characteristics of strain energy dissipation. (4) A damage constitutive model for the thick hard roof sandstone specimens was constructed, considering the characteristics of strain energy dissipation. This model effectively describes the stress-strain relationship among the samples, which undergo strain hardening, strain softening, and sudden destruction.

Experimental investigation on the surface and volume homogeneity of additive manufactured fused silica components in the Laser Glass Deposition process

Laser Glass Deposition is an additive manufacturing method to produce individualized structural components out of glass. A CO2 laser is utilized as a heat source to melt fused silica filaments and transform them into a formable viscous state. The fiber filament is fed laterally under a defined angle into the process zone. The viscous filament is deposited layer-by-layer using a 3-axis linear system with an integrated rotational axis. To investigate the surface and volume quality of the additively manufactured fused silica components, fully dense test specimens are analyzed in this paper. Quality characteristics such as surface roughness, formation of boundary layers and optical transparency constitute the focus of the investigations. Consequently, fully dense glass components with homogeneous volume structures without pores and boundary layers and a surface roughness of less than 30 nm were printed successfully.

Influence of initial compaction and confining pressure on the hydraulic conductivity of compacted iron ore tailings

The hydraulic conductivity of iron tailings is an important factor affecting the stability of tailings storage facilities. Stacking compacted filtered ore tailings is a promising alternative for safer tailings disposal. These tailings storage facilities’ internal drainage systems must be designed appropriately to avoid excessive seepage pressure, saturation, and slope failure. In this context, the factors that affect hydraulic conductivity must be adequately evaluated. Therefore, the hydraulic conductivity behavior of compacted iron ore tailings still needs to be investigated. In addition, the effect of high confining pressure on hydraulic conductivity needs to be evaluated. This study investigates the impact of the initial compaction and the confining pressure on hydraulic conductivity. For this, the flexible wall permeameter tests were carried out on filtered ore tailings compacted in three degrees of compaction: 77, 94, and 101% of optimum dry density as determined by the standard Proctor method. These specimens were saturated and then consolidated with different confining pressure. It is possible to affirm that the increased confining pressure decreases the hydraulic conductivity. The hydraulic conductivity decreases by less than one order of magnitude, regardless of the initial compaction. Moreover, the data suggest that hydraulic conductivity’s decay rate depends on initial compaction—the hydraulic conductivity of the loose specimen decays at a greater rate than that of the dense specimens. However, the reductions of the void ratio under the confining pressure of 1600 kPa and 2200 kPa were not enough to decrease the hydraulic conductivity. Thus, under high-stress confining, the hydraulic conductivity becomes practically constant.

Study on Fatigue Performance of Pulsed Tungsten Inert Gas Welding Joint of Duplex Stainless Steel Thin Tube.

To solve the shortage of austenite phase precipitation caused by nitrogen loss in the welding process of UNS S2205 duplex stainless steel (DSS), shielding gas nitriding was investigated by adding different N2 contents in Ar shielding gas during the welding process. A good thin-walled pipe butt joint was formed using the pulsed tungsten inert gas (P-TIG) welding method with Ar-N2 shielding gas. High cycle fatigue tests of the weld joints were conducted to study the effect of shielding gas nitriding on the fatigue properties. Fatigue tests at three stress levels of 225 MPa, 270 MPa, and 360 MPa were carried out on the weld joints with different N2 contents, and the fatigue samples were all fractured in the high temperature heat-affected zone (H-HAZ). Within the current process parameters, the fatigue life of the 4 vol.% N2 welded joints was optimal. Fatigue striations appeared in the fatigue crack propagation zone, and the transient fracture zone was similar to the tensile fracture. Under the low-stress level, the area of the crack propagation zone under 4 vol.% N2 was the highest, the tear ridges all expanded around the crack source area, and the fatigue crack propagation zone presented a radial distribution. The proliferation and expansion of dislocations were mainly carried out in the austenite grains, and the dislocation density of the fatigue specimens under 4 vol.% N2 was smaller than that of the Ar specimens. Shielding gas nitriding effectively improved the balance of the two-phase ratio and the hardness of austenite phase, optimized the internal slip system, inhibited the proliferation of dislocations in the austenite phase, and improved the fatigue life of weld joints.

Energy processes and phase transition in granular assemblies

Granular assemblies are an illustrative example of complex material where unexpected macroscopic properties may emerge when they are subjected to a given loading. The complexity is the consequence of the huge geometrical disorder governed by particle rearrangements, entailing plastic dissipation at contacts. This local dissipation, associated with the global geometric disorder, is probably a key ingredient responsible for various macroscopic features, such as the strain localization in dense granular assemblies leading to the formation of a shear band. Based on a discrete element method (DEM), this manuscript investigates the energy processes at the microscopic scale in granular assemblies along biaxial loading paths for dense and loose assemblies. The localized shear band domain in a dense specimen is inspected. The analysis of elastic processes suggests a maximum capacity for storing elastic energy, giving rise to a phase transition from a homogeneous state to a heterogeneous one. This phase transition is marked by a significant release of elastic energy associated with plastic dissipation. The elastic-to-plastic energy transfer is shown to be a key ingredient to reach the stationary state regime characterized by a unique dynamic equilibrium. It is signaled by the constant ratios of elastic storage and plastic dissipation over the available energy, whatever the initial density of granular assemblies. Finally, the energy processes inside the shear band domain are shown to be largely dominated by intense plastic dissipation. This suggests that the shear band acts as an optimal dissipative structure in dense specimens where elastic mechanisms continue to be active at a much higher level than they are in the outside shear band domain.

Impact of laser scanning strategies on microstructure in laser powder bed fusion (LPBF) of nanoparticle-infused pre-alloyed water-atomized iron powder

This study investigates how two scan strategies impact the microstructural and tensile properties of nanoparticle-infused pre-alloyed water-atomized low-alloy iron powder (Fe-1.50Cu-1.75Ni-0.50Mo) during Laser Powder Bed Fusion (LPBF) under uniform volumetric energy density laser conditions. The two strategies were the rotation method (ROT), in which the laser scan vector was rotated by 67° for each layer (10°, 77°, 144°, 211°, …), and the zig-zag method (ZZ), in which the vector was rotated by 90° for each layer (45°, −45°, …). As a result, the relative density of the ROT specimens was measured to be 95.61 ± 1.01 %, while that of ZZ specimens was 99.76 ± 0.17 %. Furthermore, the surface roughness of ROT specimens was higher than that of ZZ specimens (24.05 μm vs. 10.30 μm). It was found that the scan strategy influences not only the bulk characteristics but also the microstructural changes of the specimens, due to variations in intrinsic heat treatment (IHT). In the ZZ specimens, the regular laser scanning direction resulted in a uniform microstructure across different heights. However, in the ROT specimens, the laser scanning direction varied from layer to layer, with fluctuating heat dissipation influencing the melt pool structure, recrystallized grain distribution, sub-grain cellular structure, and precipitation behavior to obtain lower density and higher surface roughness than those of ZZ samples. Therefore, the samples printed using the ZZ method are found to be advantageous for printing pre-alloyed WA iron powders with uniform microstructure and reliable tensile properties.

Mechanical characterization of visually graded boards from turkish fir and black pine by nondestructive and destructive tests

For the mechanical characterization of Turkish fir and black pine, 400 board specimens with 22 mm× 50 mm × 420 mm were visually graded according to TS 1265 standard. Nondestructive tests were here upon performed using the stress wave method. After specimens were intentionally tested under flatwise bending to research the applicability as an alternative to tension and edgewise bending tests in European strength grading system. According to analyses of variance, the mean values of MOR and MOE differed in four groups at a p<0,05 significance level for visually graded boards. High correlations were found between MOR-MOE (R2=0,837) for fir and MOR-MOE (R2=0,776) for black pine. In addition, correlations of MOR-Knot rate for fir and black pine were respectively R2=0,669 and R2=0,660 showing the effectiveness of flatwise bending tests with the visual grading standard. For nondestructive tests, the mean values of the dynamic modulus of elasticity were very close in between fir and black pine grades while the usage of defect-free density performed better than the density of the whole specimen. Higher strength classes were found for black pine boards (Class 1= C40, Class 2= C27 and Class 3= C22) compared to fir boards (Class 1= C24, Class 2= C22 and Class 3= C18), respectively. Moreover, a simplified nonlinear material model was proposed for numerical modelling, and the results were found in good agreement in terms of the bending stiffness, strength, and deformation capacity of boards especially for class 1 and class 2 in both softwood species.

Crack-free high-strength AA-7075 fabricated by laser powder bed fusion with inoculations of metallic glass powders

High-strength aluminum alloy is widely utilized in aerospace field. The primary thrust of this work was to overcome the issue of hot cracking of AA-7075, which occurs during laser powder bed fusion (LPBF) processing. By employing an innovation inoculation treatment with 5 wt % Zr50.7Cu28Al12.3Ni9 metallic glass (MG) powders, highly dense (relative density >99.2 %) and crack-free composite specimens of AA-7075/MG were successfully fabricated within a broad printing range. The as-printed AA-7075/MG composite specimens exhibited a uniform microstructure with the fine equiaxed grains and the crack mitigation was attributed to the grain refinement, which decreased from 9.9 μm to 0.564 μm after addition of MG powders. The inoculation treatment of MG powders triggered the in-situ formation of Al3Zr particles on the one hand, promoting the formation of fine equiaxed grains with random orientation. On the other hand, in-situ formed nano-sized amorphous particles formed due to the high glass-forming ability of Zr-based MG and rapid cooling rate during LPBF process could strengthen the alloy. The as-printed AA-7075/MG composite specimens exhibited a compressive strength exceeding 1.5 GPa. Meanwhile, it exhibited the highest ultimate tensile stress and yield strength of 513 MPa and 488 MPa, respectively, with an elongation of over 9 %. The excellent mechanical properties are ascribed to a synergistic combination of grain boundary strengthening, precipitation strengthening induced by Al3Zr nanoparticles, solid-solution strengthening, and the effective obstacle of dislocation motion by the nano-sized amorphous particles. The current work presents a novel and cost-effective approach to producing crack-free, high strength aluminum engineering components using LPBF.

Experimental study on the effects of interface dip angle on deformation failure of combined limestone–coal specimens

Uniaxial compression experiments of limestone–coal specimens at different inclination angles (0, 15, 30, 45, and 60°) were conducted using acoustic emission and three-dimensional, extension test digital image correlation, and full-field strain measurement systems to examine how dip angles affect deformation failure. The findings indicate that: (1) specimen groups demonstrate plastic yield characteristics in the pre-peak stage. However, slight variations exist due to inclination angles. (2) The localization zone for deformation evolution closely correlates to primary crack initiation and propagation within coal specimens and to slipping at the rock’s and coal’s interface. Failure in the coal specimen triggers rebound deformation in limestone when the rock coal inclination angle is set at 15°. Both the rebound deformation amount and its rate exhibit upward trends as a function of the inclination angle. (3) The percentage of pre-peak elastic property density in the combined specimen is augmented from 98.56 to 88.08% as the inclination angle augments and reduces to 75.80%. External energy’s conversion into missile performance shows an initial increase followed by a decrease. (4) The energy rate of the acoustic emission (AE) signal exhibits distinct temporal characteristics in the combined specimen that can be associated with quiet, active, and sudden increases.

Quantitative relationship between impact toughness and quasi-static tensile properties of Ti-652 titanium alloy

Opinions regarding the relationship between impact toughness and quasi-static properties of titanium alloys remain divided to this day. In this paper, quasi-static tensile property parameters were introduced into the classical model of elastic-plastic fracture toughness. Utilizing the absorbed energy during the elastic and plastic stages, as conveyed by the integral of the quasi-static tensile stress-strain curve, the description of elastic and plastic work within the fracture toughness model was provided. Additionally, for the first time, a quantitative model relating impact toughness to quasi-static tensile property parameters in high-strength titanium alloys was proposed and applied to the high-strength Ti-652 titanium alloy. Decoupling the elastic and plastic segments of stress-strain curve, it was found that plasticity contributes more to impact toughness than strength, especially for high-strength metallic materials. Comparing the mechanical properties of Ti-652 titanium alloy with 9 kinds of microstructure, M9 was found to have the highest impact toughness (53.713 J/cm2) which features a duplex microstructure and underwent more severe plastic deformation during the test of samples, leaving shear bands and “ripple-like” patterns on the quasi-static tensile and impact fractures. Besides, statistics of dislocation density assisted by TEM images demonstrate that the dislocation density (ρ̅GND=4.82 ×1014 m−2) of high-impact toughness specimens are apparently higher than that (ρ̅GND=3.86 ×1014 m−2) of low-impact toughness specimens. Severe plastic deformation increased the energy consumption during the fracture process, resulting in high impact toughness of specimen.

Utilization of Steel Slag Waste as Construction Materials of Rigid Pavements

Roads play an important role in realizing the development and distribution of development results in certain areas. Pavement performance shows a decreased durability over time-related to how long the pavement construction can carry out its functions without experiencing fatal damage. This study aims to identify the effect of using steel slag as a substitute for filler with steel slag content of 0%, 1%, 2%, and 3% on concrete compressive and flexural strength values. This study used an experimental method by conducting experiments for testing the compressive and flexural strength of concrete specimens. The results showed that the use of steel slag as a substitute for filler obtained the average compressive strength value of MPa, 21.87 MPa, 22.17 MPa, and 28.47 MPa in concrete aged 28 days with steel slag content of 0%, 1%, 2% and 3% 25.07 respectively. The average flexural strength of concrete aged 28 days with steel slag content of 0%, 1%, 2% and 3% were 3.84 MPa, 2.83 MPa, 3.41 MPa, and 4.09 MPa respectively. The composition of 3% steel slag was the optimal composition as a filler replacement material. The pozzolanic properties of steel slag could increase the durability and density of the concrete specimen, but it required a long curing time and the increase in the initial strength of concrete slows down.

A numerical study on the multi-cycle self-burrowing of a dual-anchor probe in shallow coarse-grained soils of varying density

Development of self-burrowing probes that can penetrate soils without the aid of external reaction force from drill rigs and trucks would facilitate site characterization activities and deployment of sensors underneath existing structures and in locations with limited access (e.g., toe of dams, extraterrestrial bodies). Successful deployment of self-burrowing probes in the field will require several cycles of expansion, penetration, and contraction motions due to the geometric constraints and the increase in soil strength with depth. This study explores the multi-cycle performance of a dual-anchor self-burrowing probe in granular assemblies of varying density using discrete element modeling simulations. The simulated probe consists of an expandable top shaft, expandable bottom shaft, and a conical tip. The expansion of the shafts are force-controlled, the shaft contraction and tip advancement are displacement-controlled, and the horizontal tip oscillation is employed to reduce the penetration resistance. The performance of the self-burrowing probe in terms of self-burrowing distance is greater in the medium dense specimen than in the dense and loose specimens due to the high magnitude of anchorage force in comparison with penetration resistance. For all three soil densities, most of the mechanical work is done by tip oscillation; however, this accounts for a greater percentage of the total work in the denser specimen. Additionally, while tip oscillation aids in enabling self-burrowing to greater depths, it also produces a greater work demand. The results presented here can help evaluate the effects of soil density on probe prototypes and estimate the work requited for self-burrowing.

Longitudinal wave propagation analysis in Entangled metallic wire materials using an improved wave and finite element method

This paper proposes a novel concept and methodology to study longitudinal wave propagation in entangled metallic wire material (EMWM), a porous structural network consisting of spatial helix wires. The numerical model of EMWM is constructed using representative volume elements (RVE) obtained through X-ray tomography and skeletonization of a standard specimen. By increasing the approximate periodic boundary, the improved wave and finite ele-ment method (WFE) is employed to model EMWM as approximately periodic structures, allowing for numerical simulations of wave propagation characteristics. Frequency bands and resonance modes of EMWM are calculated to analyze the characteristics of one-dimensional wave propagation. The numerical results show that EMWM exhibits unique frequency band characteristics compared to typical periodic porous materials. In the frequency range of 15 to n n 35 kHz, the first pass band corresponds to the tensile-compressive motion of the entire specimen, while the higher pass bands correspond to localized vibrations within the spatial wires. Additionally, the frequency bands can be adjusted across a wide range based on the relative density of EMWM specimens. The improved WFE method and obtained numerical data can facilitate the application of EMWM on high frequency vibration isolation.

STUDY OF PHYSICAL AND PHOTOLUMINESCENCE PROPERTIES OF ERBIUM IONS DOPED BARIUM-TELLURITE GLASSES

Tellurite base glass doped with Erbium ions have been synthesized by melting-quenching method. X-ray diffractogam is used to verification of the glassynature of samples. Differential scanning calorimetry determine the glass transition temperature. Density (ρ) of specimens was calculated by Archimedess principle. Other physical parameters as oxygen packing density (OPD), Erbium ion concentration (CL) in the specimen, molar volume (Vm), intermolecular distance Ri, Polaron radius Rp, erbium ion concentration and Molar index of refraction Rm, were computed. In order to study the optical characteristics of the specimens, for instance, coefficient of absorption, forbidden energy gap (Eg), refractive index, and Urbach Energy (E), UV-visible absorption characterizations of Ba-Te glasses was performed in the range of 340-1000 nm. PL characterization has performed for photoluminescence study.

Enamel remineralization potential and antimicrobial effect of a fluoride varnish containing calcium strontium silicate.

ObjectivesTo investigate enamel remineralization and antimicrobial effect of sodium fluoride (NaF) varnish containing calcium strontium silicate (CSR). MethodsCSR was synthesized by sol-gel process and incorporated in 5 % NaF varnish at three different concentrations (1 %, 2 %, and 4 % w/v). The treatment/control groups were: 1 % CSR+NaF, 2 % CSR+NaF, 4 % CSR+NaF, NaF, and no treatment. Strontium and fluoride release from the varnishes was evaluated. Sound enamel specimens (n = 6) were demineralized, varnish-treated, and subjected to remineralization cycle. Mineral density of enamel specimens was evaluated using micro-CT. Antimicrobial effect of the varnishes on Streptococcus mutans and Lactobacillus acidophilus biofilms was assessed using confocal laser scanning microscopy. The HGF-1 cytotoxicity of the varnishes was examined using CCK-8 assay. ResultsBoth 2 % and 4 % CSR+NaF varnishes showed significantly higher F release and remineralization potential than NaF varnish (p < 0.05). Dead bacterial proportion of 4 % CSR+NaF varnish was significantly higher than NaF varnish (p < 0.05). The CFUs values of both S. mutans and L. acidophilus were significantly lower in 4 % CSR+NaF group than NaF group (p < 0.05). No significant difference in cell viability was observed among the groups (p > 0.05). ConclusionsIncorporation of 4 % CSR in a NaF varnish significantly enhanced its enamel remineralization and antimicrobial potential with no cytotoxic effect. Clinical significanceDental caries is a major public health problem globally. The study highlights the great potential of CSR-doped NaF varnish as a novel anti-caries agent with synergistic remineralizing and antimicrobial properties to combat early enamel caries lesions in the general population.

Laboratory investigation of foamed concrete prepared by recycled waste concrete powder and ground granulated blast furnace slag

Foamed concrete is a primary choice for building envelopes due to its good thermal inertia, controllable density and excellent fire resistance. Because of the high carbon emissions and energy consumption, it has become an increasingly urgent need to find a material that can replace cement. In particular, how to effectively use solid waste is an important trend and a significant challenge. In this study, foamed concrete was prepared by recycled waste concrete powder (RP) and ground granulated blast furnace slag (GGBS) using wet foam mixing method. By measuring the stability time of preformed foam and setting process of paste, as well as the compressive strength, dry density, porosity and thermal conductivity of specimen, the effects of alkali activator dosage, CaCl2·6H2O dosage and foam content on the properties of foamed concrete were discussed. The pore structure and reaction mechanism were analyzed by means of image analysis and X-ray diffraction. The results indicate that the compressive strength of specimen increased and then decreased with the increase of alkali content, and reached the maximum value at 5% of alkali activator. But the porosity and dry density were less affected by the amount of alkali activator. The addition of CaCl2·6H2O significantly shortened the setting process of paste, which made the paste and preformed foam achieve a better matching state. Thus the compressive strength and thermal inertia were markedly improved, and the pores were effectively refined. Foamed concrete with different density grades can be prepared by changing preformed foam content. With the increase of foam content, compressive strength and thermal inertia were obviously decreased, and the pore distribution was gradually coarsening. Furthermore, the thermal conductivity and porosity conformed to ME2 model. The activity of both RP and GGBS can be effectively activated, the two played a coordinating and complementary role to achieve the effect of synergistic utilization of solid waste.

B-069 Performance Evaluation of a Ma2 Autoantibodies ELISA

Abstract Background Ma-2 is a neuronal nucleolar protein with purported involvement in RNA transcription and apoptosis regulation. Patients with Ma2 antibodies present with autoimmune encephalitis (limbic or brainstem). Ma-2 antibodies are a high-risk marker for paraneoplastic neurologic cause for autoimmune encephalitis (&amp;gt;75% of patients having either testicular cancer or non-small cell lung cancer). In older patients, there is a higher frequency of small cell lung cancer or other cancers especially when Ma 1 antibodies are also detected. Identification of Ma2 antibodies is important to help diagnose autoimmune encephalitis, guide the cancer search and aid treatment selection. The objective of this study was to develop and document the performance of a qualitative enzymatically amplified sandwich-type Ma2 Antibody ELISA. Methods The Ma2 ELISA was produced in-house utilizing commercially available protein and was performed on a Hamilton Star liquid handling instrument equipped with a plate washer and plate reader. Each assay plate included patient samples along with 3 independent negative serum calibrators and 4 quality controls (Both positive and negative controls per matrix [CSF or serum]) each tested in duplicate. Mean optical densities (OD) for patient specimens were compared to the average OD of the negative serum calibrators to generate a ratio. A ratio cut-off of 8 was selected for reporting positivity based on preliminary studies and was validated during the method performance assessment. To assess assay performance we measured precision (3 positive pools at or above the cut-off ratio for each specimen type were tested 20 times in one assay and in duplicate across 10 independent assays), accuracy comparing to the current reference method at Athena® Diagnostics (N = 10 positives / 40 negatives), analytical specificity (20 samples with high titer autoantibodies directed against other proteins), and the impact of common interferences (lipids, bilirubin, hemolysate were spiked into 2 positive and 1 negative sample per interference). Clinical performance of the assay for differentiating healthy donors without disease (N = 140) and those with non-Ma2 neurological autoimmunity (N = 23) from those with clinical phenotypes consistent with Ma-2 associated encephalitis (N = 8) was measured. Results Serum intra-assay imprecision (coefficient of variation; %CV) ranged from 3.0–6.5%. Inter-assay imprecision ranged from 21–29%. CSF intra-assay imprecision ranged from 3.5–10%. Inter-assay imprecision ranged from 13–15.5%. Accuracy was assessed via qualitative comparison to a reference method [western blot or nanoliter scale immunoassay]. There was perfect agreement between methods. To assess analytical specificity, samples with high concentrations of immunoglobulins (hypergammaglobulinemia [N = 10] or Systemic Lupus Erythematosus [N = 10]) were tested in serum (and spiked into CSF); all were negative. Interference studies were conducted on 2 positive Ma2 serum and CSF samples, spiked for highly hemolytic (1000 mg/dL), lipemic (2000 mg/dL) and icteric (60 mg/dL) conditions. All samples remained positive under given interferents. A negative sample was tested for each matrix, both remained negative. Clinical specificity was 100% with all samples from patients without Ma-2 encephalitis testing negative (N = 183). All ELISA positives identified in this study had phenotypes consistent with Ma-2 autoimmune encephalitis (N = 8). Conclusion The performance of the Ma2 ELISA was acceptable for clinical laboratory implementation.