Steps Of Protocol Research Articles

Polyferrocenylsilane Block Copolymer Spherulites in Dilute Solution

Self-assembly of block copolymers (BCP) into uniform 3D structures in solution is an extremely rare phenomenon. Furthermore, the investigation of general prerequisites for fabricating a specific uniform 3D structure remains unknown and challenging. Here, through a simple one-pot direct self-assembly (heating and cooling) protocol, we show that uniform spherulite-like structures and their precursors can be prepared with various poly(ferrocenyldimethylsilane) (PFS) BCPs in a variety of polar and non-polar solvents. These structures all evolve from elongated lamellae into hedrites, sheaf-like micelles, and finally spherulites as the annealing temperature and supersaturation degree are increased. The key feature leading to this growth trajectory is the formation of secondary crystals by self-nucleation on the surface of early-elongated lamellae. We identified general prerequisites for fabricating PFS BCP spherulites in solution. These include corona/PFS core block ratios in the range of 1-5.5 that favor the formation of 2D structures as well as the development of secondary crystals on the basal faces of platelets at early stages of the self-assembly. The one-pot direct self-assembly provides a general protocol to form uniform spherulites and their precursors consisting of PFS BCPs that match these prerequisites. In addition, we show that manipulation of various steps in the direct self-assembly protocol can regulate the size and shape of the structures formed. These general concepts show promise for the fabrication and optimization of spherulites and their precursors from semicrystalline BCPs with interesting optical, electronic, or biomedical properties using the one-pot direct self-assembly protocol.

Improved Genome Editing via Oviductal Nucleic Acids Delivery (i-GONAD): Protocol Steps and Additional Notes.

The clustered regularly interspaced short palindromic repeats (CRISPR) technology has made it possible to produce genome-edited (GE) animals more easily and rapidly than before. In most cases, GE mice are produced by microinjection (MI) or by in vitro electroporation (EP) of CRISPR reagents into fertilized eggs (zygotes). Both of these approaches require ex vivo handling of isolated embryos and their subsequent transfer into another set of mice (called recipient or pseudopregnant mice). Such experiments are performed by highly skilled technicians (especially for MI). We recently developed a novelgenome editing method, called "GONAD (Genome-editing via Oviductal Nucleic Acids Delivery)," which can completely eliminate the ex vivo handling of embryos. We also made improvements to the GONAD method, termed "improved-GONAD (i-GONAD)." The i-GONAD method involves injection of CRISPR reagents into the oviduct of an anesthetized pregnant female using a mouthpiece-controlled glass micropipette under a dissecting microscope, followed by EP of the entire oviduct allowing the CRISPR reagents to enter into the zygotes present inside the oviduct, in situ. After the i-GONAD procedure, the mouse recovered from anesthesia is allowed to continue the pregnancy to full term to deliver its pups. The i-GONAD method does not require pseudopregnant female animals for embryo transfer, unlike the methods relying on ex vivo handling of zygotes. Therefore, the i-GONAD method can reduce the number of animals used, compared to the traditional methods. In this chapter, we describe some newer technical tips about the i-GONAD method. Additionally, even though the detailed protocols of GONAD and i-GONAD have been published elsewhere (Gurumurthy et al., Curr Protoc Hum Genet 88:15.8.1-15.8.12, 2016 Nat Protoc 14:2452-2482, 2019), we provide all the protocol steps of i-GONAD in this chapter so that the reader can find most of the information, needed for performing i-GONAD experiments, in one place.

3D-printed-tooth for socket and soft tissue preservation in the aesthetic zone: Clinical protocol and case report

Background: Since 1965, Dental Implantology and rehabilitation have evolved significantly, changing the paradigms in oral rehabilitation, from single-tooth replacement to complex maxillofacial reconstructions secondary to trauma or pathologic entities.Methods: The main goal of this article is throughout the preoperative, surgical and restorative phase, trace the path to copy the technique and get similar results in coincident patients. A brand new protocol step by step is written and detailed from previous techniques with the aid of CAD-CAM Technologies. Clinical Results: Soft tissue management on provisional with critical and subcritical emergence maintenance and select the most accurate healing abutment. The technique must be used precisely to achieve similar outcomes.Conclusions: Dental Implants play an essential role in oral rehabilitation. We must update and analyze treatments, co-working hand-by-hand with digital technologies. Further prospective randomized clinical studies are required to objectively measure this technique's clinical outcomes, which we are expecting to publish in the future.

"Dispatcher, Can You Help Me? A Woman Is Giving Birth". A Pilot Study of Remote Video Assistance with Smart Glasses.

Smart glasses (SG) could be a breakthrough in emergency situations, so the aim of this work was to assess the potential benefits of teleassistance with smart glasses (SG) from a midwife to a lifeguard in a simulated, unplanned, out-of-hospital birth (OHB). Thirty-eight lifeguards were randomized into SG and control (CG) groups. All participants were required to act in a simulated imminent childbirth with a maternal-fetal simulator (PROMPT Flex, Laerdal, Norway). The CG acted autonomously, while the SG group was video-assisted by a midwife through SG (Vuzix Blade, New York, NY, USA). The video assistance was based on the OHB protocol, speaking and receiving images on the SG. The performance time, compliance with the protocol steps, and perceived performance with the SG were evaluated. The midwife's video assistance with SG allowed 35% of the SG participants to perform the complete OHB protocol. No CG participant was able to perform it (p = 0.005). All OHB protocol variables were significantly better in the SG group than in the CG (p < 0.05). Telemedicine through video assistance with SG is feasible so that a lifeguard with no knowledge of childbirth care can act according to the recommendations in a simulated, unplanned, uncomplicated OHB. Communication with the midwife by speaking and sending images to the SG is perceived as an important benefit to the performance.

Tissue RNA Integrity in Visium Spatial Protocol (Fresh Frozen Samples).

The transcriptome of a tissue can be acquired both by single-cell RNAseq (scRNA-seq) and by spatial transcriptomics (ST). The dissociation step, which is mandatory in scRNA-seq methods, might lead to the loss of fragile cells and of spatial information, thus limiting the acquisition of the tissue cellular organization. Spatial transcriptomics methods moderate the above-mentioned issues and provide single-cell transcripts detection over an intact fresh frozen tissue section. Visium platform, commercialized from 10× Genomics, provides a whole transcriptome spatial transcriptomics platform, which does not require dedicated instruments, other than those available in any pathology laboratory. In spatial transcriptomics, proper tissue handling is mandatory to preserve the morphological quality of the tissue sections and the integrity of mRNA transcripts. Proper tissue handling is critical for downstream library preparation and sequencing performance. In this chapter, we describe the most critical steps of Visium protocol on fresh frozen tissues and we provide indications on how to interpret the data obtained from the quality control analysis recommended during the workflow.

Efficacy of a typical clean-in-place protocol against in vitro membrane biofilms.

This study evaluates the effectiveness of a typical clean-in-place (CIP) protocol against in vitro biofilms on whey reverse osmosis (RO) membranes developed under static condition. Bacterial isolates obtained from RO membrane biofilms were used to develop single and multispecies biofilms under laboratory conditions. A typical commercial CIP protocol was tested against the 24-h-old biofilms, and included 6 sequential treatment steps based on alkali, surfactant, acid, enzyme, a second surfactant, and a sanitizer treatment step. Experiments were conducted in 4 replicates and the data were statistically analyzed. The results revealed a variation in the resistance of mixed-species biofilms against the individual steps in the sequential CIP protocol. The overall 6 steps protocol, although resulted in a greater reduction, also resulted in the detection of survivors even after the final sanitizer step, reflect the ineffectiveness of the CIP protocol for complete removal of biofilms. Posttreatment counts of 0.71 log after the sequential CIP of mixed-species biofilm revealed the resistance of biofilm constitutive microbiota. Mixed-species biofilms, constituting different genera including Bacillus, Staphylococcus, and Streptococcus, were observed to be more resistant than most of the single-species biofilms. However, among the single-species biofilms, significantly different resistance pattern was observed for Bacillus isolates compared with the other bacterial isolates. All 5 isolates of Bacillus were found resistant with survivor counts of more than 1.0 log against the sequential CIP protocol tested. Thus, it can be concluded that the tested CIP protocol had a limited effectiveness to clean membrane biofilms formed on the whey RO membranes.

Vigorous Growing of Donor Plantlets by Liquid Overlay in Subcultures Is the Key to Cryopreservation of Endangered Species Pogostemon yatabeanus.

Cryopreservation is a unique option for the long-term conservation of threatened plant species with non-orthodox or limitedly available seeds. However, the wide application of cryopreservation for the protection of wild flora is hampered by some reasons: limits of source material available, difficulties in in vitro propagation, needs to re-optimize protocol steps for new species, etc. In this study, using an endemic and endangered Korean species, Pogostemon yatabeanus, we investigated subculture medium and supplements on in vitro growth of donor plants: medium strength, gelling agents, liquid overlay, plant hormones, and activated charcoal. Subculture conditions of each cycle tested significantly impacted on height and dry weight of subcultured donor plantlets. Among the treatments tested, the overlay of the liquid medium on top of gellan gum-gelled medium significantly increased the growth of shoots and roots. In the droplet-vitrification procedure, the survival and regeneration of cryopreserved shoot tips were critically impacted by the dry weight of donor plantlets (CORELL = 0.85~0.95) which was affected by the following subculture conditions. Moreover, every subsequent subculture cycle before cryopreservation positively or negatively impacted post-cryopreservation regeneration. This study highlights the vigor of donor plantlets for post-cryopreservation regeneration and provides practices for the revitalization of donor plants during subcultures.

Fixation free laparoscopic obliteration of inguinal hernia defects with the 3D dynamic responsive scaffold ProFlor

Laparoscopic TAPP/TEP approaches are well-established options for the cure of inguinal hernias. As in the open approach, mesh fixation and poor-quality biologic response represent controversial questions and are a source of concerns. Furthermore, hernia defect patency represents another problem which seems not well acknowledged among surgeons. These problems are considered the cause of frequent intra and postoperative complications. To overcome these concerns, recently a different concept of cure has emerged. Based on a newly developed dynamic responsive 3D scaffold named ProFlor, a permanent hernia defect obliteration has been finalized. Following its inherent centrifugal expansion due to its dynamic responsivity, this hernia device is positioned fixation free within the defect and induces a probiotic biological response allowing for the re-establishment of the degenerated inguinal barrier. A laparoscopic approach with the 3D scaffold has been tested on 71 patients to demonstrate its effectiveness in reducing intra and postoperative complications. The operated patients presented with bilateral and/or recurrent inguinal hernia. Overall, 122 hernia defects were obliterated with 119 dynamic responsive scaffolds. The procedures were carried out from January 2018 to January 2022 with a defined protocol and detailed procedural steps. The laparoscopic technique with the 3D hernia scaffold allowed for fixation free placement, permanent defect obliteration and dynamically induced regenerative effects. The technique proved effective in reducing intra and postoperative complications. In particular, early postoperative pain and discomfort significantly decreased. No chronic pain and no recurrences were reported during follow up. The results achieved with the described laparoscopic technique seem to embody an innovative concept for inguinal hernia repair. Fixation free, dynamic responsive, permanent defect obliteration, histologically proven regenerative effects are the distinctive features of this 3D scaffold. It seems to embody a more physiological and pathogenetically coherent concept of cure, thus improving treatment results of this widespread disease.

SIMULATION MODELING OF A HIGH-THROUGHPUT OYSTER CRYOPRESERVATION PATHWAY.

The genetic resources of oysters in Louisiana and the Gulf of Mexico are threatened due to high unexplained oyster mortality. Germplasm repositories are collections of cryopreserved genetic material stored alongside associated information that are used to protect genetics resources and facilitate breeding programs in agricultural industries. Therefore, there is great need for oyster repositories. Development of repositories has been slow despite research on high-throughput cryopreservation protocols because of logistical complexities. The goal of this study was to begin to address the gap between cryopreservation research and repository development in oyster aquaculture by modeling a cryopreservation protocol to understand and improve the process. The steps of a high-throughput cryopreservation protocol were defined and mapped in a process flow diagram. A simulation model was created using time study data, and key bottlenecks in the process were identified. Finally, model variations using alternate types of devices (tools or equipment) were created to address the identified bottlenecks. The model was found to accurately simulate the cryopreservation process. Parameters such as number of straws frozen per oyster, batch size, and number of operators significantly affected how the model performed and device choices produced substantial improvements. Simulation modeling has the potential to inform how cryopreservation pathways and repository systems in aquatic species should be structured and operated. There is ample opportunity for future work such as analyzing the impacts of production scale on cryopreservation processes.

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells.

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

Drama Therapy Family Mask Protocol in a forensic setting: Participant and therapist experience

In this clinical commentary, steps of a Drama Therapy Family Mask Protocol, implemented in a carceral setting, are discussed, using a case example. Steps include: mask making, ‘intermingling’, reparative family drama and participant reflection. Masks as a projective technique may provide a greater sense of safety in a forensic setting to support the participant’s experience while exploring their family drama. An autoethnographic response to the implementation of this protocol is processed through art and written response.

Bond strength performance according to the adhesive technique used in universal adhesive systems, literature review

Introduction: Adhesive dentistry faces complex clinical situations, which require the dentist to be able to choose the best adhesive protocol offered by universal adhesives. Objective: To present current information about the performance in bond strength according to the etching technique used in universal adhesives, evaluating the total etching technique, selective enamel etching, self-etch technique and bonding used in special clinical situations. Methodology: A search was carried out in the PubMed electronic database using as keywords “universal adhesive”, “total etch technique”, “selective enamel etching”, “self-etch”. Results: In the total acid etching technique, the acid gel to be used must be considered, as well as the type of adhesive. Selective enamel etching seeks to obtain that strong anchorage to the enamel that can only be obtained using a phosphoric acid gel, allowing the self-etching quality of the adhesive to act on dentin. The use of the self-etch technique will simplify the clinical steps in the adhesive protocol, however adhesion may decrease consequently. If there is any type of alteration in the substrate to be adhered, the resulting adhesive force could be modified, regardless of the adhesive technique used. Conclusion: Universal adhesives will allow the clinician to choose the adhesive protocol, depending on the clinical situation they are facing, as long as the clinician pays attention to aspects such as the acid gel to be used, the adhesive application time and the conditions of the substrate to adhere.

The PortaLyzer, a DIY tool that allows environmental DNA extraction in the field

The PortaLyzer is a portable homemade device that allows researchers to perform bead-beating steps commonly found in environmental DNA (eDNA) extraction protocols in the field without access to power. This allows researchers to preserve in situ organism abundance by beginning eDNA extraction quickly. The PortaLyzer is composed of a variable speed, battery-powered multi-tool and a vortexer adapter plate. We used the PortaLyzer, in conjunction with the Qiagen DNEasy PowerSoil Pro kit, to successfully field process samples taken from the forelands of the Sólheimajökull and Kvíárjökull glaciers in Iceland, as well as soil samples acquired from a prairie in Indiana. Additionally, we provide evidence that samples held in Buffer CD2 of the DNEasy PowerSoil Pro Kit may be transported to traditional lab spaces and processed up to one month after the initial protocol steps, and still provide an equivalent DNA quality and abundance yield as those processed the same day. These improvements to DNA extraction protocols give researchers more flexibility while sampling, shipping and processing eDNA samples.

Effect of Aerobic Exercise on Rehabilitation of Autistic Child

Objective: To determine the effect of aerobic exercises on cardiopulmonary endurance among Autistic child of different age groups. Methodology: The study was RCT The sampling technique used was Non-probability convenient technique for selection then randomization was done through lottery method. The sample size of 30 was divided into two groups Group A and Group B. Group A was asked to perform stationary cycling for 5 minutes or as per their schedule of daily activities and Harvard step test. Group B was asked to perform Stationary cycling for 10 minutes with warm-up and cool-down for 5 minutes each before and after exercise. Non parametric test was applied for results. Results : comparison of both groups showed Significant improvement in sBP, dBP, HR post training and stationary cycling speed shown significant improvement on week 5 with P value <0.05. Harvard step test post training showed significant difference with P value <0.05. CARS showed significant improvement post training in Experimental group. Conclusion: In this study it was concluded that stationary cycling and Harvard step test are beneficial in order to manage maladaptive behavior of autism spectrum disorder. Stationary cycling with warm up 5 minutes and cool down 5 minutes protocol and Harvard step test are less demanding exercise protocol to increase cardio-respiratory fitness. There was increase in Lung volume capacities after exercise protocol. Keywords: Autism, CARS, Harvard step test, sBP, dBP

Improved Dye Survival in Expansion Microscopy through Stabilizer-Conjugated Linkers.

Expansion microscopy (ExM) has been widely used to detect biomolecules in cultured cells and tissue samples due to its enablement of super resolution imaging with conventional microscopes, via physical expansion of samples. However, reaction conditions inherent to the process bring about strong fluorescent signal loss during polymerization and digestion and thus limit the brightness of the signal obtained post expansion. Here, we explore the impact of stabilizer-containing organic fluorophores in ExM, as a mitigation strategy for this radical-induced dye degradation. Through direct conjugation of 4-nitrophenylalanine (NPA) to our previously developed trifunctional reagents, we validate and demonstrate that these multifunctional linkers enable visualization of different organelles with improved fluorescent intensity, owning to protection of the dyes to radical induced degradation as well as to photoprotection upon imaging. At this point, we cannot disentangle the relative contribution of both mechanisms. Furthermore, we report anchoring linkers that allow straightforward application of NPA or Trolox to commercially available fluorophore-conjugated antibodies. We show that these anchoring linkers enable complete retention of biological targets while increasing fluorophore photostability. Our results provide guidance in exploring these stabilizer-modified agents in ExM and methods for increased signal survival through the polymerization steps of the ExM protocols.

Revisiting the protocol for determining submicromolar concentrations of ozone in the water treated by advanced oxidation processes.

Ozone is formed at submicromolar concentrations from photolysis of many oxyanions and oxidants in water and contributes importantly to the degradation of emerging contaminants and inactivation of pathogenic microorganisms in the natural and engineered aquatic systems. In this study, we identified and discussed the critical limitations of the commonly-used protocols using cinnamic acid (CNA) as a probe compound to determine the submicromolar-level ozone and proposed a modified protocol that overcomes those limitations. Our experimental investigation demonstrated that the radicals (e.g., HO•) formed from photolysis of oxyanions and oxidants, other than ozone, could also oxidize CNA and form benzaldehyde, resulting in the overestimation of ozone concentrations by using the commonly-used protocols. Moreover, the benzaldehyde formed from ozone-CNA reactions could be degraded by the radicals, leading to the underestimation of ozone concentrations by using the commonly-used protocols. A new protocol with high accuracy and precision was proposed and the rationales for each operational step of the new protocol were explained in detail and supported with justifications. The new protocol was compared with two commonly-used protocols in determining the concentration of ozone in the same water sample treated by the UV/chlorine process at three different UV wavelengths. The wavelength-dependent overestimation/underestimation of the ozone concentrations by using the two commonly-used protocols was well demonstrated and explained by the overlooked interferences of radicals in the protocols.

Classical Shadows With Noise

The classical shadows protocol, recently introduced by Huang, Kueng, and Preskill [Nat. Phys. 16, 1050 (2020)], is a quantum-classical protocol to estimate properties of an unknown quantum state. Unlike full quantum state tomography, the protocol can be implemented on near-term quantum hardware and requires few quantum measurements to make many predictions with a high success probability. In this paper, we study the effects of noise on the classical shadows protocol. In particular, we consider the scenario in which the quantum circuits involved in the protocol are subject to various known noise channels and derive an analytical upper bound for the sample complexity in terms of a shadow seminorm for both local and global noise. Additionally, by modifying the classical post-processing step of the noiseless protocol, we define a new estimator that remains unbiased in the presence of noise. As applications, we show that our results can be used to prove rigorous sample complexity upper bounds in the cases of depolarizing noise and amplitude damping.

A microfluidic electrochemical aptasensor for highly sensitive and selective detection of A549 cells as integrin α6β4-containing cell model via IDA aptamers

There is a growing interest in developing microfluidic biosensors for the accurate and reproducible analysis of various biomarkers obtained from liquid biopsy. This paper reports a novel microfluidic electrochemical aptasensor for determination of A549 cells as integrin α6β4-containing cell model and circulating tumor cell (CTC) model of NSCLC, based on target-induced structure switching mode. The conformational change of IDA aptamer structure with an affinity towards A549 cells, in the absence and presence of A549 cells allowed selective and sensitive detection of A549 cells. The microfluidic biosensor consisted of a microchip integrated with a screen-printed gold electrode functionalized with SH-IDA aptamers via covalent chemistry. Target solution containing A549 cells in Phosphate-buffered saline could be introduced to the microchip for analysis. Upon exposing to redox probe, a reduction in peak current occurred. Required flow sequencing for various steps of the detection protocol was performed using on-chip gas-actuated microvalves with programmable operation. The microfluidic biosensor provided a wide linear dynamic range of 50–5 × 105 cells/mL and allowed determination of A549 cells with a detection limit of 14 cells/mL. Furthermore, the microfluidic biosensor was efficiently used for detection of A549 cells in complex matrices such as human serum. Our novel microfluidic aptasensor presents an enabling analytical platform to perform various detections of low volume biomarkers with high accuracy and reproducibility.

Optimization of a sample preparation workflow based on UHPLC-MS/MS method for multi-allergen detection in chocolate: An outcome of the ThRAll project

Developing reliable methodologies for detecting and quantifying allergens in processed food commodities is crucial to support food business operators in allergen risk assessment and properly implementing precautionary allergen labels whenever required to safeguard the health of allergic consumers. Multiple Mass Spectrometry (MS) methods have been developed so far and applied for single and multi-allergen detection in foods, generating a heterogeneous literature on this topic, with little attention paid to the extraction and the digestion steps, crucial in delivering accurate allergen measurements.This investigation carried out within an international consortium specifically built up to convey a prototype MS based reference method, reports on the first part of the method development, namely the optimization of the sample preparation protocol for six allergens detection (cow's milk, hen's egg, soy, peanut, hazelnut, and almond) in chocolate. The latter was chosen as model complex food matrix, having a high lipid and polyphenol content.Different steps of the sample preparation protocol have been taken into consideration: (i) sampling, (ii) composition of the extraction buffer, (iii) protein purification, (iv) protein enzymatic digestion, (v) peptide purification and pre-concentration, and some experiments were carried out by two independent laboratories and two different MS platforms to provide a first assessment of the robustness of the method under development. Fifty target peptides were monitored in multiple reaction monitoring mode and validated in different laboratories to trace the six allergenic ingredients in the incurred chocolate and the best performing protocol for sample preparation was identified. This work paves the way of the forthcoming full analytical validation of a prototype reference method for MS-based allergen quantification.

Impact of Pancreatitis-Associated Protein on Newborn Screening Outcomes and Detection of CFTR-Related Metabolic Syndrome (CRMS)/Cystic Fibrosis Screen Positive, Inconclusive Diagnosis (CFSPID): A Monocentric Prospective Pilot Experience.

Pancreatitis-Associated Protein (PAP)-based Cystic Fibrosis (CF) newborn bloodspot screening (NBS) protocols detect less CFTR-Related Metabolic Syndrome (CRMS)/CF Screen Positive, Inconclusive Diagnosis (CFSPID). We prospectively evaluated the impact of PAP as the second step of the CF NBS protocol, before the CFTR genetic analysis, on NBS outcomes and CRMS/CFSPID detection in the Tuscany region, Italy. In parallel to the usual protocol (IRT/DNA, protocol 1), PAP was analyzed in IRT-positive infants (IRT/PAP/DNA, protocol 2) from 1 June 2020 until 31 May 2022. We defined an infant as NBS positive if PAP was >1.8 μg/L for IRT value 99th percentile-100 μg/L or >0.6 μg/L for IRT value >100 μg/L. To increase the positive predictive value (PPV) of protocol 2, we retrospectively lowered the upper IRT range value from 100 to 90 μg/L (modified protocol 2). We identified 8 CF and 13 CRMS/CFSPID with protocol 1, 5 CF and 5 CRMS/CFSPID with protocol 2 and 8 CF and 5 CRMS/CFSPID with modified protocol 2. With the PAP-based protocols, we observed a reduction of sweat tests, healthy carrier detection and a significant increase in PPV to 15.38%. Further data are needed in order to evaluate the outcomes of CRMS/CFSPID after a long follow-up.