Parallel Samples Research Articles

Development of a dual-electrospray ionization source with in-line absorbance-based voltage control.

Emitter tip arrays for electrospray ionization have been used for a variety of MS sample introduction purposes, including detection of multiple sample eluent streams and improved accuracy through parallel infusion of an internal standard. User control is typically required for targeted application of high voltage to specific channels to maximize analyte signal and minimize other background signals. In this communication, an automated approach to applying electrospray voltage only when a detectable analyte is present is described. An in-line absorbance detector is used to identify the presence of an analyte in the fluidic path between the sample introduction valve and the mass spectrometer. A Raspberry Pi-controlled system is then used to apply high voltage to a downstream emitter tip at the MS inlet following a delay volume between the detectors. Demonstration of this technique on two parallel sample channels is reported, including a pulsed voltage application to maximize signal when analytes elute on each channel simultaneously.

Optics-Free, In Situ Swelling Monitoring of Articular Cartilage with Graphene Strain Sensors.

Articular cartilage derives its load-bearing strength from the mechanical and physiochemical coupling between the collagen network and negatively charged proteoglycans, respectively. Current disease modeling approaches and treatment strategies primarily focus on cartilage stiffness, partly because indentation tests are readily accessible. However, stiffness measurements via indentation alone cannot discriminate between proteoglycan degradation versus collagen degradation, and there is a lack of methods to monitor physiochemical contributors in full-stack tissue. To decouple these contributions, here, we developed a platform that measures tissue swelling in full-depth equine cartilage explants using piezoresistive graphene strain sensors. These piezoresistive strain sensors are embedded within an elastomer bulk and have sufficient sensitivity to resolve minute, real-time changes in swelling. By relying on simple DC resistance measurements over optical techniques, our platform can analyze multiple samples in parallel. Using these devices, we found that cartilage explants under enzymatic digestion showed distinctive swelling responses to a hypotonic challenge and established average equilibrium swelling strains in healthy cartilage (4.6%), cartilage with proteoglycan loss (0.5%), and in cartilage with both collagen and proteoglycan loss (-2.6%). Combined with histology, we decoupled the pathologic swelling responses as originating either from reduced fixed charge density or from loss of intrinsic stiffness of the collagen matrix in the superficial zone. By providing scalable and in situ monitoring of cartilage swelling, our platform could facilitate regenerative medicine approaches aimed at restoring osmotic function in osteoarthritic cartilage or could be used to validate physiologically relevant swelling behavior in synthetic hydrogels.

Precise measurement of 226Ra/230Th disequilibria in deep-sea sediments by high-sensitivity ICP-MS

We describe a new method suitable for the precise and accurate determination of 226Ra in porewater and sediment samples using a single-collector sector field ICP-MS (ThermoFisher Element XR) equipped with an Apex-Q desolvation device and a high-sensitivity Jet-X interface. In combination with 230Th measurements in parallel sediment samples, this method allows precise and accurate quantification of the 226Ra/230Th disequilibria in surface sediment cores, thereby enabling the use of this isotope pair as a tracer of solute transfer across the sediment-water interface in the deep ocean. The method integrates a step of isotope dilution with 228Ra as an internal spike, a pre-concentration of Ra and Ba by MnO2 precipitation, and an efficient separation of Ra from other undesirable elements using a cation exchange resin and a Triskem Sr-spec resin. With the inclusion of one or two additional cation resin columns and the use of up to 16 bed-volumes of a lower molarity (1.7 M) HCl eluent, our procedure eliminates the complicated matrix effects persistently encountered in previous studies, and provides a highly purified solution suitable for 226Ra measurement using an Element XR ICP-MS apparatus. Consequently, we are able to determine the activity of 226Ra in ∼20–50 ml of porewater or 100 mg of sediment with an internal precision of ∼1.0% and an accuracy of ∼99.2%. The precise measurements of porewater and solid phase 226Ra in a sediment core from the North Pacific Ocean allowed the distribution coefficient (Kd) of 226Ra to be constrained tightly within a range of 4700–11,600 ml g−1. Moreover, with the aid of a one-dimensional exchange model, the combination of the 226Ra and 230Th measurements allowed us to estimate a 226Ra flux of 1140 ± 20 dpm m−2 y−1 from the sediment core.

Hardware Architecture of a QAM Receiver for Short-Range Optical Communications

Short-reach optical fiber communications systems aim to achieve high throughput, in the order of tens of Gbps. The implementation of these high-speed systems requires parallel processing, which makes low-complexity designs of their subsystems a key to the successful large-scale deployment of this technology. Half-Cycle Nyquist Subcarrier Modulation (HC-SCM) was originally suggested for these systems with the goal of using as much bandwidth as possible and, therefore, achieving high communication rates. Recently, Oversampled Subcarrier Modulation (OVS-SCM) was proposed as an alternative more computational efficient than HC-SCM and also with a better spectral efficiency. This paper proposes a hardware-efficient architecture for an OVS-SCM receiver, which takes into account the inherent parallel processing of these systems. This receiver takes 16 samples in parallel from a 5 GSa/s analog-to-digital converter with a 3.2 GHz 3 dB bandwidth. Design solutions for the frame detection block, the mixer, the resampler, the fractional interpolator, the matched filter and the timing estimator are presented. Our results show that, compared to the HC-SCM receiver, this proposal reduces the computational load of the downconverter stages by 90%. FPGA implementation results are given to demonstrate that our proposal can be implemented in state-of-the-art devices.

Error-guided method combining adaptive learning kriging model and parallel-tempering-based importance sampling for system reliability analysis

This study investigates the rare-event system reliability analysis with numerous failure regions. A novel method, based on the parallel tempering (PT) and importance sampling (IS) technique, is proposed. Surrogate models (kriging model) are built for the true performance function and a probabilistic classification function is derived to predict the failure regions. The PT algorithm is used to obtain points populating all of the predicted failure regions. Representative points are identified using the k-weighted-means clustering method. A Gaussian mixture model is formulated by the representative points and importance samples are simulated accordingly. The optimal training points are selected to update all surrogate models. In the framework of IS, the terminating criterion for assessing the estimation error of the system failure probability is devised. The learning process is terminated at the appropriate stage. The method is termed active learning kriging–parallel tempering–importance sampling (ALK-PT-IS). Four numerical examples illustrate the effectiveness and precision of this method.

Distribution and effect of retained fracturing fluid in pores and fractures of shale: Experimental and field study

Large amounts of fracturing fluid retained in the multi-scale transport channel for shale gas after fracturing, such as the hydraulic fracture networks, natural fractures, matrix pores. Therefore, the distribution and occurrence state of retained fracturing fluids greatly affect shale gas production. The flowback rate of multi-scale pores and fractures of shale and the distribution characteristics of retained fracturing fluid in multi-scale pores and fractures of shale are revealed through the imbibition and flowback experiment of shale with variable fracture width and NMR test, the occurrence state of the retained fracturing fluid during the production process is clarified through flowback experiment with shale sample in parallel and microscopic observation, and suggestions for regulating the retained fracturing fluid under reservoir conditions are put forward through rich field data analysis. The pores with diameter larger than 100 nm are the main contribution space of the flowback fracturing fluid, which provide 74.33% of the flowback fluid. 82.96% retained fracturing fluid was retained in the mesopores (2 nm < pore diameter <50 nm), which are the main existing space for retained fracturing fluid. The main occurrence states of the retained fracturing fluid are liquid bridges, continuous water films or porphyritic water films. The suggestions for regulating the retained fracturing fluid under reservoir conditions are adding drainage aids to reduce the retention of fracturing fluid during fracturing process, reasonably determining the shut-in time to improve the flowback rate in pores and fractures of diameter >0.1 μm during shut-in process, and prolonging the duration of the dominant gas seepage channel and expanding the time of pure liquid seepage by increasing pressure difference during production process.

Investigation of the estrogenic potential of 15 rosé, white and red wines via effect-directed ten-dimensional hyphenation

Wine is consumed for thousands of years all over the world, however, its estrogenic potential is still underexplored. A non-target effect-directed screening was developed to reveal estrogen-like and antiestrogen-like compounds in 15 rosé, white and red wine samples of different origin and grape variety. Normal-phase high-performance thin-layer chromatography multi-imaging detection (NP-HPTLC−UV/Vis/FLD) was combined with the planar yeast estrogen screen (pYES) bioassay or the duplex planar yeast antagonist estrogen screen (pYAES) bioassay on the same adsorbent surface. Up to nine estrogen-like compound zones were detected and further characterized via heart-cut elution from the planar bioautogram to orthogonal reversed phase high-performance liquid chromatography (RP-HPLC) coupled with diode array detection (DAD) and high-resolution tandem mass spectrometry (HRMS/MS). Among the tentatively assigned estrogen-like substances, the HRMS/MS signals pointed to hexylresorcinol and diethyl esters from organic acids for the first time. This highlights the method suitability for non-target complex mixture screening and rapid dereplication. The 10D hyphenation NP-HPTLC−UV/Vis/FLD–pYAES−heart cut–RP-HPLC–DAD–HRMS/MS proved to be an efficient and powerful tool for detecting estrogens as well as antiestrogens in the matrix-rich wine samples. High-throughput capability and substantial reduction in the required resources for analysis were demonstrated by this straightforward hyphenation, if compared to bioassay-guided fractionation. The 10D information (via orthogonal chromatographic, versatile spectrometric and duplex endocrine activity data) obtained during a single chromatographic run for many samples in parallel was advantageous for the tentative molecule assignment.

Programmable Ultrasensitive Molecular Amplifier for Digital and Multiplex MicroRNA Quantification.

Digital bioassays, popularized by digital PCR, provide some of the most robust and accurate methods for nucleic acid quantification. In this chapter, we detail a protocol for digital, isothermal, and multiplex detection of microRNAs, which relies on a recently developed amplification method. Our approach uses programmable ultrasensitive molecular amplifiers (PUMAs) to reveal the presence of target microRNAs randomly isolated in picoliter-size microfluidic droplets. Nonspecific amplification in droplets that do not contain a target is eliminated by an active threshold mechanism. Multiple circuits can be assembled for the multiplex digital detection of up to three targets. We finally present the option of using fluorescent dropcodes to streamline the assay and analyze more than a dozen samples in parallel.

Методика сохранения биологически активных веществ при обработке многокомпонентного побочного рыбного сырья

The conditions of chemical hydrolysis of by-product fish raw materials (skin) of Atlantic herring were determined to obtain a food additive with a maximum yield of biologically active substances. The objects of the study were hydrolysates with a chemical reagent concentration of 0.2% (sample 1), 0.3% (sample 2), 0.4% (sample 3), in which the content of essential amino acids (methionine, lysine and threonine) and fat-soluble vitamins (A, D3, E) were determined by high-performance liquid chromatography. Pretreatment of herring skin with scales was treated with an alkaline reagent with a concentration of 0.05% and a liquid coefficient (LC = 4) for 60 minutes at a temperature of 55 ± 0.5°C, after which the samples were hydrolyzed at the above concentrations for 120 minutes at a temperature of 90 ± 0.5°C. The principal thing in the study was the selection of two parallel samples, excluding the influence of the human factor, which were mathematically processed into two calculations (two chromatograms), and the arithmetic mean of the indicator was derived from the results of two measurements using the LabSolution software. It was found that after pretreatment for 60 minutes, the concentration of 0.3% alkaline reagent is the best for hydrolysis of marine fish by-products (skin) of Atlantic herring at a temperature of 90 ± 0.5°C. Hydrolysis in the presence of such a concentration allowed to obtain the maximum yield of amino acids (2.76; 2.00 and 1.15%) and fat-soluble vitamins (0.16; 0.0039 and 0.29).

Investigating and optimizing a method to determine atmospheric mercury for application in Ho Chi Minh City, Viet Nam

Mercury (Hg) is a toxic of global concern due to its bioaccumulation in the environment and the atmosphere which is an important environment in the biogeochemical cycle of Hg. IO-5 method (US-EPA) is the most common analysis method to measure the concentration of atmospheric Hg. However, few studies have been done in tropical monsoonal areas such as Ho Chi Minh City (HCMC). This paper reported the investigation and optimization of the IO-5 method for WA-5F, NIC with the purpose of measuring the atmospheric Hg in Ho Chi Minh City as well as other tropical monsoon regions. The sampling temperature of gold trap, sampling flow rate and time, use of soda lime, passive Hg infection over time, and concentration repeatability were investigated. The results showed that the NIC gold trap was in good stability with recoveries (H%) always higher than 95% for temperatures of 30‒150oC. Because of the tropical climatology in HCM City, a heater for the gold trap was not necessary. The recoveries were &gt; 95% for sampling rates of 0,3‒2,5 L/min. A sampling rate of 0,5 L/min for 12‒24 hours sampling periods as well as a soda lime trap to protect the gold trap during sampling were recommended. Testing the gold traps blank showed a negligible passive contamination after 30 days of storage, demonstrating the suitability of the procedure for long-term sampling campaigns. In addition, the average difference in the Hg concentration between two parallel samples was less than 10%, consistent with standards from the professional monitoring networks worldwide. In general, applying the IO-5 method in monitoring the atmospheric Hg in HCM City was suitable. Moreover, the obtained result offered a suitable method to collect the atmospheric Hg data in Vietnam as well as in Southeast Asia where the anthropogenic Hg monitoring data were limited.

Defining Selective Neuronal Resilience and Identifying Targets for Neuroprotection and Axon Regeneration Using Single-Cell RNA Sequencing: Experimental Approaches.

A prevalent feature among neurodegenerative conditions, including axonal injury, is that certain neuronal types are disproportionately affected, while others are more resilient. Identifying molecular features that separate resilient from susceptible populations could reveal potential targets for neuroprotection and axon regeneration. A powerful approach to resolve molecular differences across cell types is single-cell RNA-sequencing (scRNA-seq). scRNA-seq is a robustly scalable approach that enables the parallel sampling of gene expression across many individual cells. Here we present a systematic framework to apply scRNA-seq to track neuronal survival and gene expression changes following axonal injury. Our methods utilize the mouse retina because it is an experimentally accessible central nervous system tissue and its cell types have been comprehensively characterized by scRNA-seq. This chapter will focus on preparing retinal ganglion cells (RGCs) for scRNA-seq and pre-processing of sequencing results.

Zmiany w mikrobiomie człowieka oraz w symulatorze ludzkiego ekosystemu drobnoustrojów jelitowych (SHIME®) w odpowiedzi na dietę i suplementację probiotykami

Background. The Simulator of Human Intestinal Microbial Ecosystem (SHIME®) is used to study the behavior of a human microbiome in response to various factors. The aim of this study was to simultaneously demonstrate changes in the microbiome of a human volunteer and in the SHIME® system in response to a change in a diet and probiotic therapy due to a scarcity of published research with similar design. Results and conclusions. The probiotic therapy resulted in a decrease in fasting insulin and glucose (by 18 % and 13 %, respectively), while the increased fiber intake in the experimental diet seemed to reduce triglyceride, total and LDL cholesterol levels (by 27 %, 15 % and 14 %, respectively) in the volunteer's blood. Due to the improvement of the volunteer's metabolic status, changes in her microbiome were observed. Namely, the improvement of glucose homeostasis led to the occurrence of bacteria of the genus Akkermansia in the feces, while the improvement of lipid homeostasis resulted in an increase in the abundance of bacteria of the genus Bacteroides and Bifidobacterium (approx. two and four times, respectively). While changes in the microbiome diversity in the SHIME® luminal fluid (L-SHIME) and in the faeces microbiome were partially similar, the microbiome of SHIME’s intestinal wall (M-SHIME) behaved differently. The characteristic feature of both L- and M-SHIME was a microbiome that differed in its composition compared to the volunteer’s microbiome at parallel sampling points. The results of this study indicate that adjustments to the standard SHIME® experimental protocol should be made to enable the replication of the human microbiome diversity and its changes in the system.

Self-Attention Guidance and Multiscale Feature Fusion-Based UAV Image Object Detection

Object detection on UAV images is a recent research hotspot. Existing object detection methods have achieved good results on general scenes, but there are inherent challenges with UAV images. The detection accuracy of UAV images is limited by complex backgrounds, significant scale differences, and densely arranged small objects. To solve these problems, we propose a UAV image object detection network based on Self-attention Guidance and Multi-scale Feature fusion (SGMFNet). Firstly, we design a Global-Local Feature Guidance module (GLFG). This module can effectively combine local information and global information, which makes the model focus on the object area and reduces the impact of complex background. Secondly, an improved Parallel Sampling Feature Fusion module (PSFF) is designed to efficiently fuse multi-scale features. Thirdly, we design an Inverse-residual Feature Enhancement module (IFE), which is embedded in the front of the newly added detection head to enhance feature extraction on small objects. Finally, we conduct a large number of experiments on the VisDrone2019 dataset. The results show that the proposed SGMFNet outperforms other popular methods, and has achieved good results in many scenarios.

A Novel Architecture for Feature Extraction and Convolution for Image Segmentation of Pathology Detection from Chest X-Ray Images

Neural networks are widely used for the automation of analysis and classification tasks in the field of medical image processing. They have successfully achieved state of the art performance in medical image segmentation and feature extraction techniques. This automatic classification in the medical field is very helpful in developing tools for early detection of dreadful pathologies, like tuberculosis and pneumonia, in areas where access to doctors or radiologists is scarce. In this work, we propose a novel approach for the classification of lung pathologies like tuberculosis and pneumonia by masking them in boundary boxes using convolutional neural networks. Our solution provides a flexible way, by using saved trained models that could be directly employed by the Radiologists. In this paper, we describe the architecture required to achieve such a scalable model which could be used by doctors and radiologists without too much training in the technologies of the times. The proposed convolutional architecture consists of connected components which are parallel residual blocks and sampling layers. The images do not lose their original quality, giving the best error free predictions. We visualize this model to be deployed in labs, providing access to medical imaging expertise to some of the most remote places in the world.

Massively parallel sequencing of endometrial lavage specimens for the detection of cancer-associated mutations in atypical and non-atypical endometrial hyperplasia.

Endometrial hyperplasia (EH), particularly with atypia, is considered an antecedent of endometrial adenocarcinoma. In this study, we aimed to apply massively parallel sequencing of endometrial lavage specimens for the detection of cancer-associated mutations in atypical (AEH) and non-atypical endometrial hyperplasia (NEH). The identified alterations were compared with those detected in tissue samples. Endometrial lavage specimens and parallel biopsy samples (n = 11 for AEH and n = 9 for NEH) were obtained from 18 women (9 with AEH and 9 with NEH) who received an office hysteroscopy for suspected endometrial lesions. All samples were tested for somatic mutations in hotspot regions of 72 cancer-associated genes by massively parallel sequencing. On analyzing sequencing data, the presence of at least one cancer-associated gene mutation was identified in 72.7 and 44.4% of endometrial lavage specimens obtained from women with AEH and NEH, respectively (p = 0.362, 95% confidence interval = 0.72-3.70). The concordance rates between mutations identified in endometrial lavage specimens and endometrial biopsies were 54.5 and 0% from women with AEH and NEH, respectively (p = 0.014). A patient with NEH harbored mutations in endometrial lavage with the same mutations found in the tissue specimen at low allele frequency below detection cutoff, raising the suspicion of missed focal atypia. Endometrial hyperplasia is characterized by a high burden of cancer-associated mutations, particularly in the presence of atypia. Our study, albeit performed with a relatively small number of samples, indicates that their detection by massively parallel sequencing of endometrial lavage is feasible. Our findings may allow tailoring of endometrial biopsies to the individual risk of AEH; additionally, they can pave the way toward less invasive surveillance protocols in patients with known EH.

Comparative Study of the Nutritional Value and Degradation Characteristics of Amaranth Hay in the Rumen of Goats at Different Growth Stages.

The objective of this study was to investigate the rumen degradation characteristics of grain amaranth hay (Amaranthus hypochondriacus) at four different growth stages. The aim of this study was to evaluate the nutritional value of grain amaranth hay at different growth stages by chemical composition, in vivo digestibility, and in situ degradability. Three Boer goats with permanent ruminal fistulas were selected in this study. Amaranthus hay at four different growth stages (squaring stage (SS), initial bloom stage (IS), full-bloom stage (FS) and mature stage (MS)) was crushed and placed into nylon bags. Each sample was set up with three replicates, and two parallel samples were set up in fistulas at each time point. The rumen degradation rates of dry matter (DM), crude protein (CP), neutral detergent fibre (NDF) and acid detergent fibre (ADF) were determined at 0, 6, 12, 24, 36, 48 and 72 h. The results were as follows: (1) The concentration of CP in SS was the highest and was significantly higher than that in other stages (p < 0.05), whereas the contents of NDF and ADF gradually increased with the extension of the growing period and reached a maximum in MS; (2)The degradation of CP in the rumen at 72 h of SS and IS was more than 80%. Compared with other stages, the effective degradability of CP was highest in SS (p < 0.05) and reached 87.05% at 72 h, and the degradation rate was the lowest in MS; and (3) The effective degradability of NDF in IS was the highest (p < 0.05) and reached 69.326% at 72 h. The effective degradability of ADF in MS was the highest (p < 0.05) and reached 65.728% at 72 h. The effective degradability of DM and CP in SS was the highest. In conclusion, among the four stages, IS was superior in chemical composition and rumen degradability characteristics.

Parallel and identical test-retest reliability of the Tower of London test - Freiburg version.

The Tower of London - Freiburg version (TOL-F) was developed in three parallel-test versions (A, B, and C) that only differ in their physical appearance by interchanged ball colors, but not in their cognitive demands. We addressed the question whether the test-retest reliability of an identical problem set differs from the parallel test-retest reliability of a structurally identical problem set with a marginally different physical appearance. Reliabilities were assessed in two samples of young adults over a 1-week interval: In the parallel test-retest sample (n = 93; 49 female), half of the participants accomplished version A at the first session and version B at the second session, while the other half started with version B in the first session and continued with A in the second session. In the identical test-retest sample (n = 86; 48 female), half of the participants performed on version A in both the first and the second session, while the other half went through the same procedure with version B. For overall planning accuracy, intraclass correlation coefficients for absolute agreement were r = .501 for the parallel test-retest and r = .605 for the identical test-retest sample, with Pearson correlations of r = .559 and r = .708 respectively. Greatest lower bound estimates of reliability were adequate to high in the two samples (ranging between .765 and .854) confirming previous studies. Although the TOL-F revealed only moderate intraclass correlations for absolute agreement, it showed some of the highest psychometric indices compared to repeated assessments with other TOL tests.

Non-Conventional Induction Heat Treatment: Effect of Design and Electrical Parameters on Apple Juice Safety and Quality

The proposed non-conventional induction heating, which combines an MSCP and VDC structure, was proved to have excellent thermal effect. Different from other electric field sterilization, this electrotechnology operates with no electrodes, and it is a continuous-flow process with short-duration (about 20 s). In current study, the parameters related to temperature rise were investigated, including applied voltage, frequency, the diameter of the secondary coil and heating tube, as well as their length, etc. It was demonstrated that a smaller diameter of the heating tube, parallel connection sample coils, and higher frequency were beneficial for the inactivation of microorganisms. At 500 Hz, the optimal condition is 800 V, d1 = 2 mm, and L1 = 10 cm. Notably, the system could inactivate all microorganisms and maintained the physicochemical properties of apple juice at 40 kHz. It suggests that this structural design has the potential for industrial applications and the proposed induction heating can realize the rapid sterilization of liquid food without applying electrodes.

Implications of oral streptococcal bacteriophages in autism spectrum disorder

Growing evidence suggests altered oral and gut microbiota in autism spectrum disorder (ASD), but little is known about the alterations and roles of phages, especially within the oral microbiota in ASD subjects. We enrolled ASD (n = 26) and neurotypical subjects (n = 26) with their oral hygiene controlled, and the metagenomes of both oral and fecal samples (n = 104) are shotgun-sequenced and compared. We observe extensive and diverse oral phageome comparable to that of the gut, and clear signals of mouth-to-gut phage strain transfer within individuals. However, the overall phageomes of the two sites are widely different and show even less similarity in the oral communities between ASD and control subjects. The ASD oral phageome exhibits significantly reduced abundance and alpha diversity, but the Streptococcal phages there are atypically enriched, often dominating the community. The over-representation of Streptococcal phages is accompanied by enriched oral Streptococcal virulence factors and Streptococcus bacteria, all exhibiting a positive correlation with the severity of ASD clinical manifestations. These changes are not observed in the parallel sampling of the gut flora, suggesting a previously unknown oral-specific association between the excessive Streptococcal phage enrichment and ASD pathogenesis. The findings provide new evidence for the independent microbiome-mouth-brain connection, deepen our understanding of how the growth dynamics of bacteriophages and oral microbiota contribute to ASD, and point to novel effective therapeutics.

AML MRD By Multiparameter Flow Cytometry Using Laip/Dfn and LSC: Methodological Aspects in a Multicentric Study of the French-Flow MRD AML ALFA Network

Introduction: MRD follow-up is now mandatory for treatment response evaluation in AML clinical trials (ELN 2017 and 2021 guidelines: Heuser et al,Blood doi:10.1182/blood.2021013626.). Beside to molecular biology methods, multiparameter flow cytometry assay represents the most reliable approach. The aim of this study was to implement an harmonized follow up of the patients using a standardized MRD flow approach across 30 French hematology laboratories participating in AML clinical trials. Methods: To obtain superposable results, the network has established recommendations from Wet labs procedure to clinical reports using common panels, flow cytometers settings and analysis strategy. We designed a 2-tubes panel with minimal mandatory 8c markers by tube, according to ELN recommendations to identify the pattern of LAIP/DfN in bulk cells and the LSC (Leukemia Stem Cells) in CD34+CD38- fraction. A « backbone » CD34/CD38/CD45/CD117 was used, completed by CD7,CD56, CD13, CD33, CD19 for the 1st tube and CD90(Thy-1), Mix (CD97+CLL1+TIM3), CD123, CD45RA for the 2nd one. This panel could be used in 8c,10c,12c (HLADR, CD36, CD10, CD200) implemented on Becton Dickinson (CANTO and LYRIC) or Beckman Coulter (NAVIOS and DxFlex) cytometers (Fig 1A). Harmonization of the sensitivity between platforms (18 BD and 12 BC) was performed using 8 Picks Rainbows calibration beads. Immunostaining was performed after "bulk" lysis technique (NH4Cl). A total of minimal 500 000 cells was acquired in each tube and data were analyzed using DIVA/FACSUITE and KALUZA software. Results: To detect any bias between the platforms, firstly we tested 10 EDTA fresh regenerative marrow samples in parallel at 2 platforms (CANTO and NAVIOS). Similarity of the staining index between positive and negative population were compared between the samples and the 2 platforms showing no differences. Secondly, regular QC (Quality Control Sample), of normal bone marrow (from healthy donor) 1/year (a total of 5 since 2017) was shared among the participants centers(Fig1B).The specific events populations (nHSC, MPP, LMPP"like,etc) were evaluated by centers using a common gating strategy. In a third step, standardization of the data analysis strategy obtained in the centers was subsequently evaluated using a virtual quality control(CQA) by sharing MRD FCS data files. About 90% of the locally analyzed data were included in an interval centralized on the average of the series mean +/- 2 SD. During the follow up of the patients, a series of 18 Web educational meeting was implemented to review and validate cases with difficulties regarding analysis interpretation and confrontation with molecular MRD (transcripts, NPM1, WT1, NGS) and also chimerism in allograft follow-up (mainly cases with clonal hematopoiesis or stressed bone marrow). Finally, the harmonized data generated in our group allowed to perform a Computer aided design (CAD) in assessment of AML MRD flow using proprietary developed R script based upon FlowSom. It performs an automated definition of metacluster with abnormal population (by comparison to a set of reference bone marrow) which are quantified in the CD34+ CD117+ space for all samples (diagnosis, follow up and references marrow). It then extracts the metacluster significantly different from that observed for the reference samples (> mean+6sd. It calculates the MRD in % of WBC CD45+. The limits of sensitivity of the CAD method shows that a MRD remains evaluable by CAD up to 0.04% (50 events in 1 000 000). We compared the results between the conventional and the CAD method on 60 MRD samples. The slope of the correlation line was 0.95 with an R2 of 0.97 (Fig 1C). Using the definition threshold of a positive MRD at 0.1%, the agreement of the results between the two methods was evaluated by the Cohen kappa coefficient of 0.87. Conclusions: This methodological validation protocol is a mandatory step to consider the use of MRD flow in AML clinical trials. Choosing a multicentric approach could be challenging, but our first results are promising and showed the feasibility of this concept when: (i) a straight harmonisation of the instruments sensitivity and samples preparation are established, and (ii) training and systematic education among the analytical operators are regularly performed. Finally, our pilot study shows a very strong correlation between conventional method and unsupervised analyses using data generated by the multicentric network. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal