Adenovirus has strong therapeutic potential as an oncolytic virus and gene therapy vector. However, injecting human species C serotype 5 adenovirus, HAdv-C5, into the bloodstream leads to numerous interactions with plasma proteins that affect viral tropism and biodistribution, and can lead to potent immune responses and viral neutralization. The HAdv/factor X (FX) interaction facilitates highly efficient liver transduction and protects virus particles from complement-mediated neutralization after intravenous delivery. Ablating the FX interaction site on the HAdv-C5 capsid leaves the virus susceptible to neutralization by natural IgM followed by activation of the complement cascade and covalent binding of complement components C4b and C3b to the viral capsid. Here we present structural models for IgM and complement components C1, C4b, and C3b in complex with HAdv-C5. Molecular dynamics simulations indicate that when C3b binds near the vertex, multiple stabilizing interactions can be formed between C3b, penton base, and fiber. These interactions may stabilize the vertex region of the capsid and prevent release of the virally encoded membrane lytic factor, protein VI, which is packaged inside of the viral capsid, thus effectively neutralizing the virus. In a situation where FX and IgM are competing for binding to the capsid, IgM may not be able to form a bent conformation in which most of its Fab arms interact with the capsid. Our structural modeling of the competitive interaction of FX and IgM with HAdv-C5 allows us to propose a mechanistic model for FX inhibition of IgM-mediated virus neutralization. According to this model, although IgM may bind to the capsid, in the presence of FX it will likely retain a planar conformation and thus be unable to promote activation of the complement cascade at the virus surface. Various serotypes of human adenovirus (HAdv) are appealing vector platforms for intravenous delivery of gene therapies, as well as emerging vectors for oncolytic systemic virotherapy in cancer patients ,2,3,4. HNotably, viruses can be recognized by natural IgMs, which exist prior to antigen exposure . An initAfter C4b is bound and the classical C3 convertase (C4b2b) is formed, the convertase is able to cleave complement component C3 into C3b. C3b is composed of a shorter N-terminal β-chain connected by a disulfide bond to the longer C-terminal α-chain, which contains the thioester domain . The thiIn addition to IgM and complement proteins, blood coagulation factors also bind HAdv and influence its tropism . CoagulaIn recent studies conducted with mouse serum, natural IgM was able to inactivate an FX-binding ablated HAdv-C5 mutant (termed Ad5-FX*) but not the wild-type form of the virus (Ad5-WT) . CompareIn this study, we used molecular modeling and molecular dynamics (MD) simulations to explore a structural basis for FX inhibition of IgM- and complement-mediated neutralization of adenovirus. We assembled a model of an Ad5-FX* facet consisting of 18 hexon trimers, three penton base pentamers, and three fibers. We then placed a bent IgM in a variety of positions and modeled subsequent recruitment of C1 and C4b, as well as amplification of C3b binding to the facet. Our MD simulations indicate that, when C3b is bound at a vertex of the facet near a penton base and fiber, C3b can form multiple stabilizing interactions with the penton base Arg-Gly-Asp (RGD) containing loops, as well as with fiber. We propose that these interactions would effectively “seal” and neutralize the virus by blocking release of membrane lytic protein VI. We also constructed models to show how decoration of the Ad5-WT facet with FX may allow planar IgM to bind to the viral surface via a few Fab arms but prevent the conformational change of IgM to the bent state. If the bent conformation of IgM cannot form on the viral surface, then recruitment of complement proteins would be blocked. Even if a bent IgM is occasionally able to form on the viral surface in the presence of FX, our models indicate that surrounding FX molecules may effectively crowd out C4b and C3b binding to the capsid. These models enhance our understanding of the varied host immune responses that adenoviral vectors may encounter.Coordinates for 18 hexon trimers representing one triangular facet were selected from the cryoEM HAdv-C5 coordinates (PDB: 6B1T)  using UCThe structure of the C-terminal knob domain and last four shaft repeats of the homologous HAdv-C2 fiber were obtained from PDB: 1QIU . The fir2 complex on antigen-bearing lipid membranes (EMD-4945) [A model of the pentameric IgM in a bent conformation was constructed from structures of mouse IgM Fc domains Cµ2 (PDB: 4JVU), Cµ3 (PDB: 4BA8), and Cµ4 (PDB: 4JVW) , and humMD-4945) . The ben2s2 tetrameric protease, was derived from SAXS data [2 complex (EMD-4945). The C1r coordinates were replaced with domains from the human C1rC1s complex (PDB: 6F1C) [2 complex (EMD-4945). The C1q subunit positioned over the gap in the IgM Fc platform was bent upward such that its globular domain hovers over the facet.The initial model of the C1 complex, comprising the C1q hexamer and the C1rAXS data . ModificB: 6F1C)  and the B: 6F1C)  and the B: 6F1C) . To posi2 complex (EMD-4945) and figures from the corresponding reference [Coordinates of complement C4b were obtained from the crystal structure PDB: 5JTW . The subeference  were useCoordinates of complement C3b were obtained from the crystal structure PDB: 5FO7 . AdditioModel coordinates for the zymogenic form of FX  were posThe bent IgM model was placed onto the FX-loaded facet as in the first of our 16 IgM positions (with a pair of Fab arms over hexon trimer #1 in the asymmetric unit and no rotation about the Fc platform). The Clashes tool in UCSF ChimeraX v1.5 was used to identify FX molecules that clashed with IgM, and any clashing FX molecules were removed. A planar IgM model was prepared by straightening the Fab arms of the bent IgM model with UCSF Chimera v1.16. The planar IgM model was positioned with the same pair of Fab arms located approximately over hexon trimer #1. The Clashes tool in UCSF ChimeraX v1.5 was again used to identify FX molecules clashing with the IgM, and these FX molecules were subsequently removed.Molecular dynamics simulations were performed to assess the ability of C3b to form energetically favorable interactions with multiple RGD loops of the penton base or with the fiber and at least one RGD loop. We generated 11 starting models of facet vertices (the penton base pentamer and trimeric fiber) with a single C3b positioned nearby. The model of the HAdv-C5 penton base was prepared as discussed under “Initial model building” above. The model of the HAdv-C5 fiber was also prepared as discussed under “Initial model building” above, with an additional peptide containing N-terminal amino acids 1–6 added for each chain using the Build Structure tool in UCSF Chimera v1.16. Coordinates of complement C3b were obtained from PDB: 5FO7 with SWISS-MODEL used to fill in missing internal loops. Peptides containing C3b amino acids 666–667 and 749–751 were added at chain termini using the Build Structure tool.MD simulations were performed with NAMD v2.14  on the CFor the ending coordinates of the MD simulations, nonbonded interaction energies  were calculated between C3b and the HAdv-C5 fiber, and between C3b and each chain of the penton base individually. Combinations with nonzero interaction energies were further broken down to determine contributions of the α- and β-chains of C3b, contribution of the thioester domain of C3b, and the contribution of the penton base RGD loops. NAMD v2.14 and the NAMD Energy plugin of VMD v1.9.4  were useHAdv-C5 has a striking icosahedral shape with prominent fibers protruding from the vertices A. The di2 complex (EMD-4945) [When modeling the interaction of IgM with Hadv-C5 or Ad5-FX*, we used a bent, or dome-shaped, model of pentameric IgM based on cryoelectron tomography (cryoET) structures of IgM, C1, and C4b complexes on antigen-bearing lipid membranes . The benMD-4945)  as a guiMD-4945) , of whicHaving positioned C4b molecules on the facet, we proceeded to add copies of C3b to the facet model. The crystal structure of C4b is remarkably similar to that of its paralogue C3b, suggesting that the C3 convertases C4b2b and C3bBb share a similar architecture and mode of substrate interaction . The intModeling of C3b on the adenovirus facet begins with a single C4b bound to the facet A. C4b, tn at the binding interface of C3b copy n−1 if the interface was unobstructed  and chain F2 . From a zoomed-in perspective, comparison of the starting and ending coordinates A,B indicAt the end of the C3b pose 2 trajectory, C3b has formed strong nonbonded interactions with chains P1 , P2 , and F3 . Comparing zoomed-in views of the starting and ending coordinates D,E, it iAt the end of the C3b pose 3 trajectory, C3b has formed strong nonbonded interactions with chains P4 , F2 , and F3 . Comparing zoomed-in views of the starting and ending coordinates G,H, it aIn all three MD poses discussed, interactions of C3b with the penton base occurred entirely through the RGD loops, with no energy contributed by interactions between C3b and other regions of the penton base. For the most part, interactions with the fiber and RGD loops occurred via the thioester domain of the C3b α-chain, with the remainder of the α-chain and the shorter β-chain contributing no nonbonded interaction energy. Key exceptions are found in pose 2, where the β-chain of C3b mediates interactions with penton base chain P2 and fiber chain F3, while the C3b thioester domain mediates the interaction with penton base chain P1. The nonbonded interaction energies are similar for the C3b thioester domain/chain P1 interaction and the C3b β-chain/chain P2 interaction . The exceptions observed for pose 2 can be explained by its starting coordinates, where both C3b chains are in relatively close proximity to the penton base and fiber. In comparison, the starting coordinates of poses 1 and 3 place only the C3b thioester domain within range to establish interactions with the vertex proteins. The nonbonded interaction energies of the C3b β-chain with chains P2 and F3 in pose 2 are entirely electrostatic. In comparison, interactions mediated by the C3b thioester domain for all three poses are primarily electrostatic, but with some Van der Waals contributions as well.We used gRINN v1.1.0.hf1 to identify specific interacting residues involved in stabilizing interactions between C3b and the penton base RGD loops. The pose 1 MD trajectory ended with the largest number of RGD loop residues involved in favorable interactions with C3b . At the ending coordinates (2 ns), 56 residues in the RGD loop of chain P2 interact favorably with 52 residues in the thioester domain .Having established a rationale for how IgM binding and subsequent complement recruitment leads to neutralization of HAdv-C5, we sought to explain why such neutralization does not occur in the presence of FX. We began by constructing a model where a copy of FX is bound in the center of each hexon trimer on the facet A,D, thenIt is possible that in some instances, despite the presence of FX, IgM may bind to the facet with all 10 Fab arms. To illustrate this possibility, we placed a bent IgM on the facet in the same position as the model shown in Adenovirus is a promising vector for development of novel gene therapies or oncolytic viruses. However, neutralization by humoral factors of the innate and adaptive arms of the immune system is a significant obstacle to such applications. Understanding the structural bases of interactions between HAdv-C5 and host defense proteins will help to design therapeutic vectors that evade neutralization by the immune system while efficiently fulfilling their targeted function. In this molecular modeling and computational study, we sought to construct and analyze a structural model for IgM and complement-mediated neutralization of FX-binding ablated adenovirus. We were then able to provide a potential mechanistic explanation for the experimental finding that FX inhibits such neutralization.We began by positioning IgM (with a variety of rotations about its Fc platform) on each of the four hexon trimers within one asymmetric unit of the adenovirus facet in the absence of FX. We then placed complement components C1 and C4b, and finally C3b on the adenovirus facet model. We hypothesized that a C3b molecule present near a vertex of the facet would be able to entangle multiple penton base RGD loops, as has been previously modeled for C4b interacting with adenovirus . EntanglIndeed, one of our MD simulation vertex models (pose 2) shows C3b interacting with two RGD loops; the associated nonbonded interaction energies are similarly favorable for each of these two loops . We interpret this as an indication that C3b can entangle two RGD loops. In addition, there is a strong favorable nonbonded interaction energy between C3b and one fiber chain in this pose, further stabilizing the vertex. In each of the other two vertex models (poses 1 and 3), C3b has a strong favorable interaction with only one RGD loop, but C3b has formed an additional strong interaction with at least one chain of the fiber. Therefore, we revise our hypothesis and propose that formation of multiple stabilizing interactions between C3b and a combination of penton base RGD loops and the fiber can result in neutralization of adenovirus.When identifying pairs of residues involved in stabilizing interactions between C3b and penton base RGD loops, we found that the interface formed in the pose 1 MD simulation was particularly extensive . It appeAs our models of an adenovirus facet with IgM, C4b, and C3b indicate, the position of IgM on the facet affects where C3b is most likely to bind. Out of the 16 models generated, one had no C3b bound, and another four had only a single C3b bound . This raTo address the key issue of how IgM and complement are able to neutralize Ad5-FX* but not Ad5-WT, we sought to illustrate a structural basis for competition between IgM and FX on the HAdV-C5 facet. In the presence of FX, we anticipated that copies of FX would occupy most or all of their binding sites before IgM could bind via its Fab arms and adopt a bent conformation. We prepared a model in which a planar IgM has managed to position one pair of Fab arms over an unoccupied hexon trimer, but FX is bound to most other hexon trimers in the facet B,E. In tWe also considered the possibility that IgM is able to bind via all ten Fab arms and adopt a bent conformation even in the presence of FX. We aimed to determine whether FX inhibition of IgM and complement-mediated neutralization could still occur in this circumstance. In our model with bent IgM surrounded by FX, the DLPSP motifs on IgM, required for C1q binding, do not appear to be obstructed by the FX molecules C,F. TherUltimately, attempting to add either bent or planar IgM to an FX-occupied HAdv-C5 facet appears to obstruct the process that results in Ad5-FX* being sealed shut by complement. The “planar IgM” facet model provides a plausible explanation for the inability of IgM and complement to inactivate adenovirus in the presence of FX. If IgM cannot contact the facet with most or all of its Fab arms, the bent IgM conformation will not form, and subsequent complement recruitment will not occur. Even if IgM is able to adopt a bent conformation on the facet and complement recruitment does occur, there would be limited space for C4b and C3b. Therefore, in the presence of FX, there would be a low possibility of C3b interacting with capsid proteins at all of the vertices and effectively “sealing” the viral capsid. It would be interesting to test whether or not the isolated FX-GLA domain has the same effect as full length FX. Since the FX-GLA domain binds within the central depression of hexon trimers, it might not interfere with IgM binding. We predict that IgM will bind in a bent conformation even in the presence of FX-GLA, and that this will lead to complement recruitment and viral neutralization. The experimental evaluation of our structural models is a subject of future research. Our modeling and computational study indicates that adenovirus with an FX-binding ablating mutation, such as Ad5-FX*, would allow C1, C4b, and multiple C3b copies to bind to each facet, enabling inactivation of the virus by stabilizing the vertices and preventing release of the virally encoded membrane lytic factor, protein VI. In addition, we show how FX may inhibit IgM and complement-mediated neutralization of adenovirus by preventing C3b from binding near and stabilizing the vertices of the adenovirus capsid.


Endobronchial Ultrasound (EBUS) has been widely used to stage lung tumors and to diagnose mediastinal diseases. In the last decade, this procedure has evolved in several technical aspects, with new tools available to optimize tissue sampling and to increase its diagnostic yield, like elastography, different types of needles and, most recently, miniforceps and cryobiopsy. Accordingly, the indications for the use of the EBUS scope into the airways to perform the Endobronchial Ultrasound–TransBronchial Needle Aspiration (EBUS-TBNA) has also extended beyond the endobronchial and thoracic boundaries to sample lesions from the liver, left adrenal gland and retroperitoneal lymph nodes via the gastroesophageal tract, performing the Endoscopic UltraSound with Bronchoscope-guided Fine Needle Aspiration (EUS-B-FNA). In this review, we summarize and critically discuss the main indication for the use of the EBUS scope, even the more uncommon, to underline its utility and versatility in clinical practice. Transbronchial needle aspiration (TBNA) for sampling mediastinal lymph nodes was first described in 1949 by Schieppati ; one yeaSince then, TBNA has become a widely used technique to sample lesions, including nodules and masses, which are outside of but adjacent to the central airways, by inserting the needle in the site suggested by a careful analysis of the chest Computed Tomography and endobronchial anatomic landmarks .In 2002, the development of the convex probe to perform endobronchial ultrasound was a turning point because it allowed the bronchoscopist to perform real-time transbronchial needle aspiration guided by endobronchial ultrasound (EBUS-TBNA) . The higMore recently, the clinical applications of EBUS have expanded. Indeed, due to the larger amount of tissue required by the pathologist to perform not only a correct diagnosis of cancer but also a precise molecular analysis, the endoscopist had to upgrade his technique routine to provide sample adequacy. As such, new opportunities to use the EBUS scope outside the “conventional” endobronchial route are emerging, even beyond the thoracic boundaries. Indeed, to evaluate lesions that cannot be accessed through the airways, it can be used through the gastrointestinal tract to perform an endoscopic ultrasound with bronchoscope-guided fine needle aspiration (EUS-B-FNA).The aim of this review is to summarize the various applications of the EBUS scope, also highlighting the most recent approaches and techniques.In recent years, research on molecular mutations in non-small cell lung cancer (NSCLC) has increased our knowledge of biological pathways driving tumor development; the identification of mutations within the epidermal growth factor receptor (EGFR) gene is one such example . Owing tSeveral aspects of the EBUS-TBNA technique may impact the adequacy of the sample for molecular analysis, such as the number of needle passes, the use of rapid on-site evaluation or the suction during the needle aspiration and the choice of specimen preservation.Lee et al. reported that three needle passes for each target site are sufficient to secure a diagnosis of NSCLC ; howeverRapid on-site evaluation (ROSE), which provides real-time pathologic evaluation of sample adequacy, was initially developed to enhance the diagnostic yield of conventional TBNA ,26,27, b3, p = 0.036) and fewer additional procedures  to reach complete molecular profiling in NSCLC with a necrotic component [Further dissecting factors that may potentially influence the performance of EBUS-TBNA, there is the utilization of high-pressure suction. This technique, compared to standard vacuum syringe suction, did not impact the diagnostic yield; however, Tsakinis reported that EBUS-TBNA with high-pressure suction was associated with bigger samples  and 95% of the patients , respectively . On the Notably, an innovative technique to provide additional DNA for NGS analysis is the isolation of free-floating DNA, which is visible after centrifugation of the needle aspiration supernatant, thus reducing the need for repeated biopsy and turnaround time ,44,45. HRegarding the early stages of NSCLC, traditionally, the treatment refers to surgical resection, less frequently associated with radio-chemotherapy or neoadjuvant/adjuvant chemotherapy . In the ® (NCCN®), Canadian, and Chinese/Asian guidelines, suggest testing at least EGFR mutations [In view of these limitations, current guidelines do not recommend molecular testing in stages I–III with the broad molecular assessment suggested only in metastatic NSCLC . Nonetheutations . Collectively, accumulating evidence highlights the need to optimize the management of diagnostic samples based on lung cancer stage and the importance of early definition of molecular/immune prognostic markers not only in metastatic disease but also in the pre-operative stage of resectable cancer.There are several echographic features during EBUS-TBNA that may raise the suspicion of a malignant lymph node, such as echogenicity, margins, diameter, or the presence of necrosis. However, none of these characteristics has consistently been associated with malignancy . In this regard, endobronchial elastography is a relatively new technique combined with the endoscopic ultrasound procedure that has the potential to differentiate malignant versus benign lymph nodes. Elastography assesses the relative elasticity or stiffness of the tissue according to the degree of deformation after compression in a color-based output. A blue signal represents hard tissue, such as malignancy, while benign lesions, which are softer, exhibit a red-green color. Tissue elasticity can be measured by a qualitative or semi-quantitative modality.The most widely used qualitative classification is the Izumo score, which identifies three types of findings: predominantly nonblue (type 1), partially blue (type 2) and predominantly blue (type 3) . HoweverDiscrepancies may arise due to different interpretations of elastography images, which is an operator-dependent evaluation. As a result, some changes have been applied to the observation method of elastography, like the stiffness ratio, strain histogram or strain ratio, to predict the elasticity of the lesion ,59. HoweMultiple factors may influence the results obtained by EBUS elastography. False positive results are mostly due to increased stiffness in cases of tuberculosis, pneumoconiosis, or sarcoidosis, while necrosis, hemorrhage or liquefaction typical of malignant lesions can induce false negatives ,61. Elastography is useful for the evaluation of multistation lymph nodes involvement and also when multiple nodes are present in the same station; indeed, it may prevent unnecessary punctures focusing on type 3 lymph nodes and avoid the spread of metastatic tissue in the same stage lymph node when performing puncture from type 1 to 3 lymph nodes. Finally, and most importantly, elastography cannot replace lymph node aspiration.Endoscopic ultrasound (EUS) of the gastrointestinal tract is performed with a gastrointestinal echoendoscope by the gastroenterologists but also with the EBUS scope by the pulmonologists (EUS-B) to obtain a fine needle aspiration (FNA). This technique is proceeding forward because of its usefulness to sample tissue, which is not reachable with the endobronchial approach to achieve lung cancer diagnosis and a more accurate staging ,63,64. Paraesophageal pulmonary lesions could be safely and accurately sampled by EUS-B-FNA, with high diagnostic accuracy also for molecular profiling ,66.Regarding the staging of lung cancer, EUS-B fine needle aspiration (FNA) permits lymph node stations of 2 L, 4 L and 7 to be reached similarly to the endobronchial approach. Stations 2 R and 4 R are difficult to reach with EUS-B-FNA because usually, there is the interposition of the trachea, but they could be sampled, especially when lymph nodes are enlarged. Regarding para-aortic stations 5 and 6, they can be visualized, but the sampling is troublesome because of the interposition of great vessels. However, the transvascular approach is described see , as wellNotably, lymph nodes stations 8 and 9, as well as lesions below the diaphragm, are only reachable with this technique.In fact, the European guidelines for combined EBUS and EUS recommend both techniques, endobronchial and esophageal, which can be performed in the same session , to stagEven though for the EBUS-TBNA, the European Respiratory Society (ERS) has launched a structured, evidence-based education program based on training in simulators with a final validated test , EUS-B-FIndeed, in order to perform a more accurate staging of lung cancer, EUS-B-FNA is recommended for the evaluation of the left adrenal gland (LAG) for suspected metastasis . InteresAlthough the use of EUS-B-FNA has also been reported for sampling suspected left liver lobe lesions or retroperitoneal lymph nodes, current guidelines do not make recommendations in this regard. Nevertheless, the diagnostic yield reported by Christiansen et al. was higher than 80% for left liver lobe lesions (n = 23) and 63% for retroperitoneal lymph nodes (n = 19), with no complication observed .EBUS allows for excellent visualization of the structures surrounding the airways, including pulmonary arteries. Casoni and co-workers originally reported on a case wherein pulmonary angiography did not allow to distinguish between pulmonary emboli and sarcoma . EBUS shThe preferred technique to assess the severity of PE is computed tomography (CT) angiography; however, EBUS should be considered in the diagnostic flow-chart in patients in whom angiography is contraindicated, such as those with chronic renal failure, previous anaphylactic reactions to intravenous contrast agents and pregnant women . MoreoveA clinical trial currently evaluating the added value of EBUS in diagnosing PE in critically ill patients .Even though most of the patients that perform EBUS already have a contrast chest CT scan available that evaluates the presence of pulmonary embolism, we believe that the endosonographic study of the central vessel during the procedure should be performed. Indeed, it does not require additional timing, as it could be done during the collection of the specimen, and it could be helpful in identifying unknown emboli, especially in high-risk patients with cancer and in those with a dated chest CT scan.In the experience of our Interventional Pneumology Centre, the most representative case of pulmonary embolism evaluated by EBUS is shown in In the differential diagnosis of pulmonary embolism, it should also be considered a neoplastic intravascular involvement. As an example, it is reported a case of primary pulmonary artery sarcoma diagnosed with EBUS-TBNA without complication in a patient non-eligible for surgery . Some mediastinal stations or pathological tissue could be difficult to reach because of the interposition of a great vessel, for example, a branch of the pulmonary artery. Even though there is evidence of life-threatening bleeding after accidental puncture of the pulmonary artery during EBUS-TBNA, there are case series that confirm the relative safety of transvascular needle aspiration performed either by EBUS-TBNS or EUS-B-FNA . Of the However, this approach should be limited only if a clear window to the biopsy is not available and should be performed only as a last resort after all other options are excluded. Moreover, TVNA should be performed only in high-volume centers by skilled interventional pulmonologists, and all adequate precautions should be adopted as a cardiothoracic surgery on stand-by.In cases of suspected neoplastic pleural disease, especially when pleural effusion is present, thoracoscopy with pleural biopsy is the preferred method for diagnostic tissue sampling. However, thoracoscopy is invasive and may pose relevant risks in some patients, especially those with multiple and severe comorbidities . In thisEBUS could be particularly useful when pleural abnormalities are not in the context of pleural effusion. Lococo et al. first sampled a single PET-positive pleural lesion located in the right costovertebral recess adjacent to the carina, suspicious of the epithelioid tumor. A final diagnosis of epithelioid-type malignant mesothelioma was subsequently confirmed following surgical resection of the lesion .Therefore, in a subset of patients, EBUS-TBNA may represent a less invasive alternative to thoracoscopy for pleural sampling.The approach to pleural lesions suspected for mesothelioma could also be performed by the esophagus with the EUS-B-FNA technique, as it was reported in four case reports ,88,89,90Similarly, EBUS-TBNA and EUS-B-FNA could be both used to diagnose metastatic involvement of the pleura, as reported in various case series ,88,89 byRegarding cardiac tumors, which are extremely rare, they are usually sampled by open-heart surgery, with related mortality and morbidity. However, in patients with poor clinical conditions, endosonography could represent a minimally invasive technique to perform a diagnosis, seeing that it provides an excellent view of the left atrium. Two cases of EUS-B-FNA of left atrial masses have been reported with high accuracy and no adverse events .Notably, this technique should be performed by experienced operators after having excluded alternative diagnostic approaches.Endoscopic ultrasound (EUS) can offer an alternative guide when the pleural effusion is loculated or is located toward the mediastinum, and standard thoracentesis cannot be performed. Endoscopic US (EUS) guidance to drain pleural effusion was initially reported in a retrospective study in 2008 by DeWitt and colleagues. . In nineThe risk of infection should be considered; indeed, the needle is no longer sterile once it passes through the working channel, potentially inoculating bacteria in the pleural space. However, the risk is low, and antibiotic prophylaxis may be used to prevent this complication.In addition, in loculated pericardial effusion, several case reports also support the feasibility of EBUS-TBNA- or EUS-B-FNA-guided drainage ,95,96,97Finally, even fluid below the diaphragm could be evaluated. Indeed, it is described as a single case report of ascites aspiration with EUS-B-FNA with no complications .Intrathoracic goiter and thyroid lesions can represent a clinical challenge, as malignancy is often in the differential diagnosis. The percutaneous approach, which is applicable in the diagnostic workout of common thyroid lesions, is not suitable for intrathoracic lesions, which require mediastinoscopy or surgical biopsy. In these conditions, less invasive procedures, such as EBUS, might be considered . In a caA range of oncogene-driven drugs is currently available in the therapeutic armamentarium of lung cancer. When molecular analysis does not identify a specific target, the immune status of NSCLC can be targeted. Standard chemotherapy protocols, with and without radiotherapy, still represent an optional or complementary approach. Yet, lung cancer remains the leading cause of death worldwide . Among tIn the last decades, our knowledge of endobronchial chemotherapy delivery has grown substantially, widening the available approaches to malignant airway obstruction. Conventional TBNA has been used for several years for injection into the airways, lung parenchyma and surrounding tissues. Apart from the delivery of cancer therapies, transbronchial needles have been utilized for the injection of corticosteroids, antimicrobials, tranexamic acid, and radioisotopes to identify lung lesions or tissue sealants to treat bronchopleural fistula . In receMore recently, this technique has been applied to deliver both standard chemotherapy ,107,108 EUS-B approach for the cisplatin injection in a patient with a critical, nearly complete tracheal occlusion and with atlantoaxial instability that contraindicates rigid bronchoscopy, has also been reported .Uncommon applications of endoscopic ultrasound are summarized in New complementary tools have been developed to obtain biopsy specimens for histological analysis for EBUS TBNA.® 22 G fine needle  is characterized by a Franseen tip 22 G FNB device equipped with three cutting edges. In a prospective study, this needle has shown a better performance than FNA when applied to sample solid lesions by EUS [The Acquires by EUS . Its feas by EUS . An ongo® needle  has a core trap designed to collect a histological sample by shearing material from the lesion during the needle motion [The ProCoree motion . This nee motion ,115.p < 0.001, respectively) and lymphoma  [An additional tool to increase the size of the tissue sampled is the EBUS-guided intranodal forceps biopsy (IFB), which is performed by introducing the mini forceps through the initial hole made by the TBNA needle. Compared to EBUS-TBNA, the addition of IFB provided a higher diagnostic yield, especially in patients with sarcoidosis  . The comctively) .Of note, EBUS-IFB provides histology samples that, in parallel to those permanently fixed in formalin, can be instantly frozen, giving intraprocedural feedback . OverallAnother novel tool to sample the lymph node is transbronchial cryobiopsy under EBUS guidance, wherein a 1.1 mm cryobiopsy probe is inserted in the hole created by the TBNA, with a 3 s freezing time . p = 0.001). However, on subgroup analysis, when only metastatic lymph nodes were analyzed, the diagnostic yields of the two procedures were similar . Conversely, cryobiopsy was more accurate than EBUS-TBNA in benign disorders  and uncommon tumors . Notably, in the latter group, which was made mainly by lymphoma, mediastinal cryobiopsy was associated with a diagnostic rate of nearly 90% and complete subtyping [The first randomized control trial (n = 197) was performed in 2021 by Zhang and colleagues, who compared standard EBUS-TBNA to cryobiopsy to sample mediastinal lesions of at least 1 cm . The oveubtyping .Moreover, in cases of lung cancer, almost all cryobiopsy material (93.3%) was suitable for gene mutation PCR testing, compared to 73.5% of the material obtained by TBNA, which is consistent with previous reports ,121.Interestingly, no differences in diagnostic yield were found between the “Cryobiopsy first” and “TBNA first” groups. Two pneumothoraces and one pneumomediastinum were reported, highlighting the safety of the procedure. A recent randomized multicenter study (n = 297) assessed the overall diagnostic yield of the addition of cryobiopsy vs. standard needle sampling alone for mediastinal lesions . The comThe combined approach also allowed more precise molecular and immunological analyses of lung cancer and a good safety profile. Since its first application in 2002, real-time transbronchial vision provided by EBUS has revolutionized the diagnostic approach to lung and mediastinal lesions. The development and implementation of novel complementary technologies, briefly reviewed here, have improved the diagnostic yield of the EBUS scope and expanded its possible applications to several diseases involving the mediastinum and beyond. The increasing need for tissue for diagnostic assessment, coupled with the growing knowledge of pathological and molecular features of lung cancer, will further push the optimization of the EBUS scope as a crucial diagnostic tool in a range of respiratory diseases.


Loyalty card programs have become prevalent in the retail industry, offering various benefits and rewards to customers. This study aims to analyze the impact of loyalty card programs on customer behavior in the Albanian market.A survey was conducted among a diverse sample of customers from different retail sectors in Albania. The survey collected data on awareness, satisfaction, loyalty, and customized benefits. The survey was distributed between March and May 2023, and responses were collected electronically. The survey responses were analyzed to address the research questions and test the hypotheses.The findings indicate a significant level of awareness among respondents regarding loyalty card programs. Higher satisfaction with specific benefits, such as discounts and accumulating points, is associated with higher levels of customer loyalty. Customers who reported higher frequencies of loyalty card usage demonstrated greater loyalty. Customized benefits, particularly discounts and accumulating points, significantly influenced customer satisfaction. However, other benefit categories did not show statistically significant associations with loyalty.The study highlights the importance of promoting loyalty card programs to enhance awareness among customers. Improving and optimizing loyalty card benefits based on customer feedback is recommended. Encouraging frequent usage of loyalty cards and personalizing benefits to align with customer preferences are key strategies to foster customer loyalty. The findings provide valuable insights for practitioners in the retail sector to enhance customer engagement, satisfaction, and loyalty. In today’s competitive retail market, businesses face the ongoing challenge of attracting and retaining customers. Loyalty card programs have emerged as popular marketing tools that offer a range of benefits and rewards to participating customers. These programs are designed to foster customer loyalty, increase customer retention, and drive repeat purchases . The impResearch conducted byStudies have shown that loyalty card programs can have a significant impact on customer behavior and satisfaction. For example, a study byMoreover, loyalty card programs have been found to influence various aspects of customer behavior, including shopping frequency and spending patterns. In their research on the impact of loyalty card programs,The effectiveness of loyalty card programs is further supported by a study conducted byFurthermore, customization and personalization have been recognized as key factors in loyalty card program success. Research byExisting studies on loyalty card programs, while providing a comprehensive foundation, primarily originate from globally recognized or extensively researched markets. This skew in research leaves a significant theoretical gap in understanding the dynamics of these programs in emerging or less-studied markets such as Albania. Furthermore, the practical application of these findings to distinct economic contexts, like that of Albania, remains largely unexplored.This study seeks to address these gaps by delving into the unique challenges and opportunities of implementing loyalty card programs in Albania, a country experiencing rapid economic and social changes. The evolving retail sector and shifting consumer preferences in Albania underscore the need for a tailored examination of how loyalty card programs operate and affect consumer behavior in this specific context. Despite the global uptick in interest and adoption of loyalty card programs, there is a noticeable dearth of research on their effectiveness and reception in the Albanian market, highlighting a crucial area for exploration.Moreover, this study intends to separate and underscore two distinct research gaps. Firstly, it aims to enrich the theoretical framework by examining how awareness of loyalty card programs among Albanian consumers relates to other critical variables like satisfaction and loyalty, a connection less emphasized in current literature. Secondly, on a practical level, it seeks to understand the specific implementation and outcomes of these programs in the unique socio-economic landscape of Albania, thus offering pragmatic insights and guidance for local businesses and policymakers.In addressing these theoretical and practical aspects, the research aims to make a significant contribution to the domain of loyalty card program management, both in Albania and in similar emerging markets. By providing a localized perspective on customer behavior, satisfaction, and loyalty within the context of Albanian loyalty card programs, this study aspires to reveal insights that are not only academically valuable but also practically applicable for businesses, policymakers, and researchers in Albania and comparable markets. The subsequent sections of this paper will present the research objectives, methodology, findings, and implications, shedding light on aspects of loyalty card programs in the Albanian market that have remained unexplored until now.1.To assess the awareness of loyalty card programs among consumers in the Albanian market.2.To examine the relationship between customer satisfaction with loyalty card benefits and customer loyalty.3.To explore the association between loyalty card usage and customer loyalty.4.To investigate the importance of customized benefits in loyalty card programs.The primary objective of this study is to analyze the impact of loyalty card programs on customer behavior in the Albanian market. The specific research objectives include:1.What is the level of awareness of loyalty card programs among consumers in the Albanian market?2.Is there a relationship between customer satisfaction with loyalty card benefits and customer loyalty in the Albanian market?3.Does loyalty card usage influence customer loyalty to supermarkets or chain stores in the Albanian market?4.How important is the customization of loyalty card benefits in influencing customer satisfaction and loyalty in the Albanian market?Based on the research objectives, the following research questions are formulated:The findings of this study will offer dual significance: theoretically, by filling the gap in understanding the relationship between consumer awareness of loyalty card programs and key behavioral variables like satisfaction and loyalty, and practically, by providing insights specific to the Albanian retail market. This dual approach not only enhances the existing body of knowledge on loyalty card programs but also tailors it to the unique socioeconomic context of Albania.For businesses, especially supermarkets and chain stores operating in Albania, the study's results will be invaluable. They can utilize these insights to refine their loyalty card strategies, aligning them more closely with customer preferences and needs in this distinct market. Moreover, by shedding light on the under-researched area of consumer awareness and its impact in the Albanian context, the study paves the way for more informed and effective loyalty program implementations, potentially influencing policy and strategic decisions in the retail sector.In summary, this research stands to contribute significantly to both the academic understanding of loyalty card programs and their practical application, particularly in emerging markets like Albania. The findings will not only enrich the academic discussion but also offer actionable recommendations for businesses and policymakers seeking to navigate the dynamic retail landscape of Albania.The literature review aims to establish a theoretical foundation for understanding the role of loyalty cards in fostering customer loyalty and influencing consumer decision-making in the retail industry.Loyalty card programs, also known as rewards programs or customer loyalty programs, are marketing initiatives designed to incentivize and reward customers for their repeat purchases and brand loyalty . OverallOne of the primary reasons customers participate in loyalty card programs is to obtain the associated benefits and incentives  from the ethics council of the University Aleksander Moisiu and the ethical guidelines were followed throughout the research process. Informed consent was obtained by providing participants with a clear explanation of the study’s purpose, procedures and benefits, and their rights as participants. Participants are informed that their participation is voluntary and that they have the right to withdraw at any time without any negative consequences. They are also assured that their responses will be kept confidential, and that any identifying information will be anonymized or kept secure. In addition, participants received an email that clearly stated that by completing the survey, they were providing their informed consent to participate in the study.The study employs a cross sectional research design where the data was collected only one time to investigate the effects of loyalty card programs on customer engagement, satisfaction, trust, and loyalty in the Albanian market. The research design involved the collection and analysis of survey data from a diverse sample of customers across different retail sectors in Albania.The target population for this study consisted of customers who have participated in loyalty card programs offered by supermarkets or chain stores in the Albanian market. The inclusion criteria for the target population in this study consisted of customers who were residents of Albania and regularly shopped in the Albanian market. On the other hand, the exclusion criteria encompassed customers who were not residents of Albania or did not engage in shopping activities within the Albanian market.via email, using contact lists obtained from the participating supermarkets or chain stores. The email included a brief introduction to the study, its purpose, and a link to the online survey. Participants were encouraged to complete the survey at their convenience. The use of convenience sampling allowed for a practical and efficient way of collecting data from individuals who regularly shop in supermarkets or chain stores within the Albanian market.To select the participants, a convenience sampling method was used. Convenience sampling is a non-probability sampling technique where individuals who are easily accessible and available are included in the sample. In this case, participants were selected based on their convenience. The survey was distributed to the selected participantsTo calculate the minimum sample size required for the study, a confidence interval with a margin of error of ±0.05 was considered . The pop2 * p(1-p)) /MOE2, where MOE is the margin of error, the sample size was computed. Using the values, the initial sample size was calculated to be 173.8. Since sample sizes cannot be fractional, the value was rounded up to 174.Next, the Z-score corresponding to the desired confidence level was determined. The Z-score for a 95% confidence level is 1.96, which is approximately 2. However, in this case, Zp (Z-score for the upper tail of the distribution) was used instead of Zα/2 (Z-score for each tail of the distribution). The value of Zp was 2. Using the formula for calculating the required sample size, which is n = , a finite population correction was applied. The formula for calculating the corrected sample size is n’ = (n * N) / (n + N - 1). Substituting the values, the corrected sample size was also determined to be 174. The number of filled surveys was 248, which is greater than the minimum required sample size of 174.Data for this study were collected through a structured questionnaire survey created by the authors . This quThe data collection period spanned from March to May 2023, with the survey being distributed electronically via Google Forms through emails and social media channels. This method was chosen to maximize efficiency and timeliness of data collection. To maintain the integrity of the participant pool, specific criteria were set - respondents had to be Albanian residents actively engaged in shopping activities within the Albanian market. Clear instructions and assurances of confidentiality were provided to participants, emphasizing the survey’s purpose and ethical considerations.The collected data were analyzed using statistical software IBM SPSS Statistics 27 (RRID:SCR_002865). Descriptive statistics, including frequencies and percentages, were calculated to summarize the characteristics of the sample and the distribution of responses for each variable.Furthermore, using the program IBM SPSS Statistics 27, inferential statistical tests were conducted to examine the relationships between loyalty card programs and customer behavior variables. Specifically, chi-square tests of independence were utilized to assess the associations between categorical variables. The significance level was set at α = 0.05, indicating that p-values less than 0.05 were considered statistically significant. Moreover, to delve deeper into the relationships and unveil predictive insights, logistic regression analyses were also conducted. These regression analyses allowed for a more comprehensive understanding of how loyalty card programs influenced various customer behavior dimensions, such as satisfaction, trust, and loyalty. The results of these analyses offered valuable quantitative evidence of the extent and nature of these relationships.These tests helped determine if there were any significant relationships or dependencies between the loyalty card programs and customer behavior variables in the Albanian market.The first research hypothesis aimed to investigate the level of awareness of loyalty card programs among consumers in the Albanian market. The hypothesis stated that the majority of respondents would be aware of loyalty card programs offered by supermarkets or chain stores.To assess the awareness of loyalty card programs, a survey was conducted among a sample of 248 participants.From2 = 0.00001248 with 1 degree of freedom. At a significance level of 0.05, the critical value is 3.841.A chi-square test of independence was conducted to test the hypothesis. The observed frequencies for the two categories (“Yes” and “No”) were compared with the expected frequencies under the assumption of independence. The chi-square test statistic was calculated to be χThe findings indicate that a significant proportion of respondents (67.74%) in the Albanian market were aware of loyalty card programs offered by supermarkets or chain stores. This supports the hypothesis that the majority of individuals would be aware of such programs. The chi-square test results also confirm that the observed frequencies were not significantly different from the expected frequencies, suggesting that the awareness of loyalty card programs was not dependent on other factors measured in the survey.The awareness of loyalty card programs was analyzed further based on gender and age groups.Logistic Regression Analysis To delve deeper, logistic regression was employed to examine the influence of demographic variables on awareness of loyalty card programs. The results are presented inThe logistic regression analysis indicates that certain demographic groups have different levels of awareness about loyalty card programs. This variation by age and gender underscores the need for targeted marketing strategies in promoting loyalty card programs in the Albanian market. The overall findings support the hypothesis that a significant proportion of the population is aware of these programs, but this awareness is influenced by demographic factors.Furthermore, the relationship between shopping frequency and awareness of loyalty cards was analyzed.It can be observed that the majority of participants who shop daily and weekly are aware of loyalty cards, while a smaller proportion of those who shop monthly, occasionally, or rarely are aware of loyalty cards.To quantitatively assess the impact of shopping frequency on awareness, logistic regression was applied. The analysis provides insights into the likelihood of awareness based on shopping habits.The logistic regression analysis presented inMoreover, the analysis demonstrates that demographic factors, such as age and gender, significantly influence awareness of loyalty card programs. Younger consumers and frequent shoppers, in particular, show higher levels of awareness. These insights are invaluable for crafting targeted marketing strategies. Retailers in the Albanian market can leverage this information to tailor their promotional efforts, ensuring they effectively reach and resonate with different consumer segments. Such strategic targeting based on consumer behavior and demographics is essential for enhancing the effectiveness of loyalty card programs in a competitive retail environment.Hypothesis 2: Customers who are satisfied with the benefits provided by loyalty card programs are more likely to exhibit higher levels of loyalty to supermarkets or chain stores in the Albanian market.Out of the 248 respondents, 201 provided valid responses regarding their likelihood to recommend. TheThe majority of respondents expressed positive sentiments towards recommending a supermarket or chain store with a good loyalty card program. The most common response was “Likely,” with 117 participants indicating this likelihood. Additionally, 24 respondents expressed being “Very likely” to recommend. A smaller number of participants indicated lower levels of likelihood to recommend, with 11 respondents stating “Unlikely” and only 1 respondent indicating “Very unlikely.”To test the hypothesis regarding the likelihood to recommend, a chi-square of independence was conducted. The analysis included different satisfaction levels  and customer loyalty. The results of the hypothesis testing provide insights into the relationship between loyalty card programs and the likelihood of recommendation.The results of the chi-square tests are presented inFor the loyalty card benefit categories of “Discounts on Purchases” and “Accumulating Points,” the chi-square tests revealed statistically significant associations with customer satisfaction levels. These results indicate that customer satisfaction with these specific benefits has an influence on their loyalty to supermarkets or chain stores.However, for the loyalty card benefit categories of “Exclusive Offers,” “Personalized Deals,” and “Faster Checkout Process,” the chi-square tests did not find statistically significant associations with customer satisfaction levels. This suggests that customer satisfaction with these benefits may not have a significant impact on their loyalty to supermarkets or chain stores.The regression analysis aimed to quantify the relationship between customer satisfaction with specific loyalty card benefits and the likelihood to recommend. The results are summarized in theDiscounts on Purchases: The regression analysis revealed a statistically significant positive relationship between customer satisfaction with discounts on purchases and the likelihood to recommend. As customer satisfaction with discounts increased, the likelihood of recommending the supermarket or chain store also increased significantly.Accumulating Points: Customer satisfaction with accumulating points also showed a statistically significant positive relationship with the likelihood to recommend. An increase in satisfaction with accumulating points corresponded to a notable increase in the likelihood of recommending the supermarket or chain store.Exclusive Offers: In contrast, customer satisfaction with exclusive offers did not demonstrate a statistically significant relationship with the likelihood to recommend. Satisfaction with exclusive offers did not significantly impact the likelihood to recommend.Personalized Deals: Similarly, customer satisfaction with personalized deals did not display a statistically significant relationship with the likelihood to recommend. Satisfaction with personalized deals did not have a significant effect on the likelihood to recommend.Faster Checkout Process: Customer satisfaction with a faster checkout process also did not exhibit a statistically significant relationship with the likelihood to recommend. Satisfaction with the faster checkout process did not significantly influence the likelihood of recommending the supermarket or chain store.The regression analyses support Hypothesis 2, indicating that customer satisfaction with specific loyalty card benefits, such as discounts on purchases and accumulating points, significantly influences their likelihood to recommend the supermarket or chain store. However, satisfaction with benefits like exclusive offers, personalized deals, and a faster checkout process did not have a significant impact on the likelihood to recommend. These findings emphasize the importance of offering attractive and rewarding loyalty card programs, particularly in terms of discounts and accumulating points, to enhance customer satisfaction and foster long-term loyalty in the Albanian market.The third research objective focused on examining the associations between loyalty card usage and customer loyalty to supermarkets or chain stores in the Albanian market. The hypotheses 3 that is tested is: There is a relationship between loyalty card usage and customer loyalty to supermarkets or chain stores in the Albanian market.To test the hypotheses, the survey data collected from the respondents were analyzed to examine the relationship between loyalty card usage and customer loyalty. The data included the frequency of loyalty card usage and the corresponding customer loyalty responses.The data were further analyzed to explore the relationship between card usage frequency and customer loyalty. The results are summarized inTo determine the relationship between loyalty card usage frequency and customer loyalty, chi-square tests of independence were conducted. The analysis compared the observed frequencies with the expected frequencies under the assumption of independence.•Every time: chi-square statistic = 56.12, p-value < 0.001•Frequently: chi-square statistic = 28.65, p-value < 0.001•Occasionally: chi-square statistic = 7.81, p-value = 0.050•Rarely: chi-square statistic = 4.36, p-value = 0.114•Never: chi-square statistic = 9.00, p-value = 0.030The chi-square test results for each loyalty card usage category are as follows:The chi-square tests revealed statistically significant associations between loyalty card usage frequency and customer loyalty for the categories of “Every Time” and “Frequently”. These results indicate that customers who reported higher frequencies of loyalty card usage were more likely to express loyalty to supermarkets or chain stores.However, for the categories of “Occasionally,” “Rarely,” and “Never,” the chi-square tests did not find statistically significant associations with customer loyalty. This suggests that lower frequencies of loyalty card usage may not have a significant impact on customer loyalty to supermarkets or chain stores.To delve deeper into the relationship between loyalty card usage and customer loyalty, logistic regression analyses were conducted as well. This hypothesis posited that there is a relationship between loyalty card usage and customer loyalty to supermarkets or chain stores in the Albanian market.Out of the 248 respondents, 201 provided valid responses regarding their likelihood to recommend, which served as a measure of customer loyalty. The survey data included the frequency of loyalty card usage. The results presented inThese results provide the beta coefficients, standard errors, t-values, and p-values for each loyalty card usage frequency category in the regression analysis. The significant p-value for “Every Time” indicates a statistically significant relationship with customer loyalty. However, for “Frequently,” the relationship is marginally significant , while “Occasionally,” “Rarely,” and “Never” show no significant relationship with customer loyalty.These logistic regression findings provide robust support for Hypothesis 3, indicating that customers who frequently or consistently use their loyalty cards are more likely to exhibit loyalty to supermarkets or chain stores in the Albanian market. Encouraging frequent loyalty card usage appears to be a strategic approach for fostering and maintaining customer loyalty in this context.Hypothesis 4: consumers who are satisfied with the benefits provided by loyalty cards are more likely to exhibit higher levels of loyalty to supermarkets or chain stores in the Albanian market.To test the hypotheses, the survey data collected from the respondents were analyzed to examine the relationship between customer satisfaction with loyalty card benefits and their levels of loyalty to supermarkets or chain stores.The survey included questions related to customer satisfaction with loyalty card benefits, categorized into levels such as “Very Satisfied,” “Satisfied,” “Neutral”, “Dissatisfied,” and “Not Answered.” The levels of loyalty to supermarkets or chain stores were measured based on responses such as “Significantly influences, “To Some Extent”, “Not Sure,” “Does Not Affect,” and “Not Answered.”To determine the relationship between loyalty card satisfaction and customer loyalty, chi-square tests of independence were conducted. The analysis compared the observed frequencies with the expected frequencies under the assumption of independence.•Chi-square statistic = 108.571, p-value < 0.001The chi-square test results for loyalty card satisfaction and customer loyalty are as follows:The chi-square test revealed a highly significant association between loyalty card satisfaction and customer loyalty in the Albanian market. This indicates that customers who reported higher satisfaction levels with loyalty card benefits were more likely to exhibit higher levels of loyalty to supermarkets or chain stores.To further explore this relationship, logistic regression analysis was applied to quantify the association between levels of satisfaction with loyalty card benefits and customer loyalty.The logistic regression analysis supports Hypothesis 4, demonstrating a significant relationship between satisfaction with loyalty card benefits and customer loyalty. Specifically, high satisfaction levels (“Very Satisfied”) are strongly associated with increased customer loyalty, as indicated by the positive beta coefficients and odds ratios. This underscores the importance of customer satisfaction with loyalty card benefits in fostering loyalty to supermarkets or chain stores in the Albanian market.The results support Hypothesis 4, suggesting that consumer satisfaction with the benefits provided by loyalty cards plays a crucial role in influencing customer loyalty. When customers are satisfied with the benefits they receive through loyalty card programs, they are more likely to exhibit loyalty to supermarkets or chain stores.Hypothesis 5: Organizations that tailor loyalty card benefits to meet the needs and preferences of their customers will experience higher customer satisfaction and loyalty in the Albanian market.To test Hypothesis 5, the focus was on understanding the impact of customized loyalty card benefits on customer satisfaction and loyalty in the Albanian market. The hypothesis suggests that when organizations personalize loyalty card benefits to align with the needs and preferences of their customers, it will result in higher levels of customer satisfaction and loyalty.Based on the survey responses and data analysis, the following findings were obtained:The chi-square test results indicate that there is a significant association between loyalty card benefits tailored to meet customer needs and preferences and higher levels of customer satisfaction in two categories: “Accumulating Points”  and “Discounts on Purchases” . This suggests that when loyalty card benefits are customized to include features like accumulating points or offering discounts on purchases, it positively impacts customer satisfaction.However, for the categories of “Exclusive Offers/Promotions,” “Personalized Recommendations/Deals,” and “Faster Checkout Process,” the chi-square tests did not reveal a significant association with customer satisfaction. Although these categories did not show a statistically significant relationship, further analysis and investigation may be required to explore potential factors influencing customer satisfaction in these areas.To further validate these findings and explore their impact on customer loyalty, logistic regression analysis was conducted. This analysis aimed to quantify the strength and direction of the relationship between customized loyalty card benefits and customer loyalty.The logistic regression analysis corroborates the hypothesis to some extent, indicating that customer satisfaction and loyalty are positively influenced when loyalty card benefits are tailored, particularly in the “Accumulating Points” and “Discounts on Purchases” categories. The positive beta coefficients and odds ratios in these categories suggest a stronger likelihood of customer loyalty. The lack of significant findings in other categories implies that not all types of personalized benefits are equally effective in enhancing loyalty.These findings provide partial support for Hypothesis 5, emphasizing the importance of tailoring loyalty card benefits to customer preferences. However, further investigation is needed to fully understand the impact on customer loyalty and to explore the potential influences in categories where no significant associations were found.The “Results and findings” section provides valuable insights into the awareness of loyalty card programs, likelihood to recommend, customer loyalty, and the impact of loyalty card satisfaction on customer loyalty in the Albanian market. In this section, we will discuss the implications of these findings and their relevance to the research objectives and hypotheses.The findings regarding the awareness of loyalty card programs indicate that a significant proportion of respondents in the Albanian market were aware of such programs. The majority of individuals (67.74%) reported being aware of loyalty card programs offered by supermarkets or chain stores. This supports Hypothesis 1, which stated that the majority of respondents would be aware of loyalty card programs.The high level of awareness suggests that loyalty card programs have gained traction and visibility in the retail industry in Albania. Supermarkets and chain stores have successfully implemented and promoted these programs, capturing the attention of a considerable number of consumers. This finding aligns with previous research conducted byThe awareness of loyalty card programs was further analyzed by logistic regression analyses based on gender and age groups. The tables presenting the awareness levels among female and male participants in different age groups provide valuable insights into the variations in awareness across different demographic segments. These variations may reflect differences in exposure to marketing efforts, shopping behaviors, and preferences among different consumer groups. Understanding these variations can help retailers tailor their promotional strategies and communication channels to effectively reach and engage specific target segments.The findings related to the likelihood to recommend supermarkets or chain stores with a good loyalty card program indicate positive sentiments among the majority of respondents. A significant proportion expressed a likelihood to recommend, with “Likely” being the most common response. This supports Hypothesis 2, which posited that customers who are satisfied with the benefits provided by loyalty card programs are more likely to exhibit higher levels of loyalty.The chi-square test and the logistic regression analyses results for each loyalty card benefit category provide insights into the relationship between loyalty card satisfaction and the likelihood of recommendation. Specifically, the categories of “Discounts on Purchases” and “Accumulating Points” showed statistically significant associations with customer satisfaction levels. This suggests that customers who are satisfied with these specific benefits are more likely to exhibit higher levels of loyalty and recommend supermarkets or chain stores.However, it is important to note that the categories of “Exclusive Offers/Promotions,” “Personalized Recommendations/Deals,” and “Faster Checkout Process” did not show statistically significant associations with customer satisfaction levels. This implies that while these benefits may contribute to overall customer satisfaction, their impact on customer loyalty and likelihood to recommend may be less pronounced. Further analysis and investigation may be necessary to explore potential factors influencing customer satisfaction and loyalty in these areas.The analysis of customer loyalty to supermarkets or chain stores based on loyalty card usage frequency provides important insights into the relationship between loyalty card usage and customer loyalty. The findings support Hypothesis 3, indicating a significant association between higher frequencies of loyalty card usage and higher levels of customer loyalty.Customers who reported using their loyalty cards every time or frequently exhibited higher levels of loyalty to supermarkets or chain stores. This suggests that frequent usage of loyalty cards plays a role in fostering customer loyalty. On the other hand, lower frequencies of loyalty card usage  did not show a significant impact on customer loyalty. This implies that occasional or infrequent usage of loyalty cards may not have a substantial influence on customer loyalty.These findings emphasize the importance of encouraging frequent usage of loyalty cards to enhance customer loyalty. Retailers can consider implementing strategies to promote and incentivize regular usage of loyalty cards, such as offering additional rewards or exclusive benefits for customers who use their cards consistently.Loyalty The analysis of loyalty card satisfaction and its impact on customer loyalty supports Hypothesis 4, which proposed that consumers who are satisfied with the benefits provided by loyalty cards are more likely to exhibit higher levels of loyalty.The test results reveal a highly significant association between loyalty card satisfaction and customer loyalty. Customers who reported higher satisfaction levels with loyalty card benefits were more likely to exhibit higher levels of loyalty to supermarkets or chain stores. This finding highlights the importance of customer satisfaction in driving customer loyalty. Retailers should prioritize efforts to ensure that their loyalty card programs are designed to meet customer needs and preferences, thereby enhancing satisfaction levels and fostering long-term loyalty.The exploration of customized loyalty card benefits and their impact on customer satisfaction, as tested in Hypothesis 5, was enriched through both chi-square and logistic regression analyses. Findings highlight the significance of tailoring loyalty card programs to align with customer preferences, particularly in specific benefit categories.The chi-square test results revealed a significant correlation between customized benefits, notably in “Discounts on Purchases” and “Accumulating Points,” and heightened customer satisfaction. This suggests that when loyalty card programs focus on these specific benefits, they resonate more with customers, enhancing their satisfaction. These benefits effectively meet customer expectations and needs, crucial for loyalty program success.On the other hand, logistic regression analysis offered a deeper quantitative understanding. It quantified the impact of each benefit type on customer satisfaction, reinforcing the chi-square findings. For example, positive beta coefficients for “Discounts on Purchases” and “Accumulating Points” confirmed their significant role in increasing customer satisfaction.However, benefits such as “Exclusive Offers/Promotions,” “Personalized Recommendations/Deals,” and “Faster Checkout Process” did not exhibit statistically significant associations with customer satisfaction in either analysis. This implies these benefits might not be as influential. This highlights the need for further investigation to comprehend their impact fully.This nuanced perspective, drawn from both chi-square and logistic regression analyses, underscores the complexity of customer satisfaction in loyalty card programs. It suggests that while some benefits clearly drive satisfaction, others may require additional strategies or deeper exploration to understand their influence. Retailers should consider these findings when designing and tailoring their loyalty card.This study’s findings carry substantial theoretical and practical significance for loyalty card programs in the Albanian retail market.The study contributes significantly to the theoretical landscape of customer loyalty and loyalty card programs, particularly in the context of an emerging market like Albania. By highlighting the high level of consumer awareness and the impact of satisfaction with specific loyalty card benefits on customer loyalty and recommendation likelihood, this research enriches existing theoretical models. It underscores the critical role that customer satisfaction plays in the success of loyalty programs and the importance of personalization and customization in these initiatives. The findings also support the notion that regular engagement with loyalty programs fosters deeper customer loyalty, an insight that can influence future theoretical frameworks in this area. Moreover, the study’s exploration of the effectiveness of customized benefits in loyalty programs provides new perspectives on tailoring marketing strategies to meet diverse consumer needs, thereby contributing to the evolving theories on customer behavior and relationship marketing.This study provides actionable insights for practitioners, particularly in the Albanian retail market. It underscores the importance of prioritizing certain benefits, like discounts and point accumulation, over others such as exclusive offers and faster checkout processes when designing loyalty programs. Encouraging frequent card usage through incentives can boost customer retention and loyalty.Customizing loyalty card benefits to align with customer preferences is crucial. Retailers should use customer data to personalize their programs, adapting to changing customer needs. Maintaining transparency and robust data privacy measures is also vital for building and sustaining customer trust, a cornerstone of loyalty program success.In summary, the practical implications of this study are manifold, offering retailers in the Albanian market, and potentially in similar markets, a roadmap to enhance the effectiveness of their loyalty card programs. By focusing on the key areas identified in this research, businesses can optimize their loyalty strategies, ultimately leading to increased customer engagement, satisfaction, and loyalty.This study has certain limitations that should be considered. Firstly, the findings are based on a convenience sample, which may limit the generalizability of the results to the entire population of customers in the Albanian market. Secondly, the study focused on loyalty card programs offered by supermarkets or chain stores, excluding other retail sectors. Future research could explore a more diverse sample and consider additional retail contexts to gain a comprehensive understanding of loyalty card programs’ effects.This study aimed to examine the impact of loyalty card programs on customer behavior in the Albanian market. The findings provide valuable insights into the awareness of loyalty card programs, the relationship between customer satisfaction and loyalty, the influence of loyalty card usage on customer loyalty, and the importance of customized benefits.The first research question focused on the awareness of loyalty card programs among consumers in the Albanian market. The findings indicated a significant level of awareness, highlighting the importance of promoting and communicating loyalty card programs to customers.The second research question examined the relationship between customer satisfaction with loyalty card benefits and customer loyalty. Higher satisfaction levels with specific benefits, such as discounts on purchases and accumulating points, were associated with higher levels of customer loyalty. This emphasizes the need for continuous improvement and optimization of loyalty card programs.The third research question explored the association between loyalty card usage and customer loyalty. Customers who reported higher frequencies of loyalty card usage exhibited higher levels of loyalty to supermarkets or chain stores, underscoring the importance of encouraging frequent usage of loyalty cards.The fourth research question investigated the impact of customized loyalty card benefits on customer satisfaction and loyalty. Customized benefits, particularly discounts on purchases and accumulating points, significantly influenced customer satisfaction. This highlights the importance of tailoring loyalty card benefits to meet the specific needs and preferences of customers.Based on the findings of this study, several recommendations can be made for practitioners and researchers to enhance the effectiveness of loyalty card programs and improve customer engagement and loyalty.Firstly, it is crucial for supermarkets and chain stores to invest in promoting loyalty card programs to customers through various channels. This includes utilizing advertising, in-store displays, and digital marketing to ensure effective communication and widespread awareness. By enhancing awareness of these programs, organizations can attract more customers and encourage their participation  might be one research gap and fulfilling the research gap in this regards might provide theoretical implication.  The application of study in Albanian market is related with the scope of the study and having limited number of the study might be another research gap and fulfilling this research gap might provide practical implications.  The revised version of the manuscript was reviewed and it was observed that the authors have made sincere efforts in the revised version. The manuscript can be considered for indexing, however the originality of the manuscript is still weak and some major changes are required. Below the changes made on the manuscript were reviewed and the points, which still required modification were listed: On the other hand, the originality of the manuscript seems still weak. Data analysis: More explanation was given in the data analysis and it was stated that the questionnaire is a custom designed one. The questions should be given in a table. The problem about making the analysis by using answers for single question still exists. But the authors applied regression analysis to put forward the relationship between different variables, awareness, satisfaction and loyalty. However, no information was given about the type of the regression analysis applied. If the variables are measured with a single question and categorical variables, logistic regression should be applied. Last but not least, there are many subheadings under results, discussion and conclusion parts. The sub-heading in results and discussion parts can be eliminated and they can be reorganized to have lower number of subheadings in conclusion part.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?NoAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Textile and Fashion MarketingI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. It appears the author has addressed the reviewers' comments.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Marketing: marketing management; market research; customer behavior's; Customer satisfaction; customer loyaltyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The manuscript analyzes the consumer behavior for a specific market, for this reason, the findings cannot be generalized to provide practical implications. More important than that, the novelty is not good for the manuscript and it does not provide any contribution to literature. Last but not least, the statistical analyzes selected are not convenient for answering the research questions and analyzing the consumer behavior well. For all these reasons mentioned above, I suggest the manuscript is not approved for indexing. Below these points were summarized in much more detail with some suggestions that should be made:Introduction: The introduction (background) part was written in a way that it summarizes the importance of the loyalty cards and the findings of some past studies, which have similar type of research objectives. It is suggested to re-write the introduction part in a way that it underlines the missing points in the literature and explains why this study is needed. Moreover, the study was established in a specific market, which prevents to generalize the findings. This is one limitation of this study, but this limitation can be eliminated by giving some specific reasons for this selection. If there is not any, at least the authors should introduce the market, give some facts and cite some studies already established in this market to introduce the consumer base more.Literature review: This part was written under 4 headings and gaps in literature were summarized at the end of the literature section. However, these research gaps were not underlined in these 4 sections. I would suggest to organize the literature part in 2 sections. In the first section, some information might be given about the concept, benefits and incentives of loyalty card problems and; in the second section, the studies about consumer behavior might be summarized for these loyalty card by highlighting the lacking points. Here the number of studies about awareness should also be mentioned. Finally, this section should include and specify the studies established in the Albanian market.Data analysis: The questionnaire, which was designed by the same authors, was used by the authors. Even though, the authors are the same, the design of this questionnaire should be mentioned in this study. This is critical to show the theoretical implications, which are quite missing for this study. I understand that, most of the evaluations were made with single question, which can be quite defective. Moreover, chi-square difference tests do not give any idea about the relationship between two variables  (the research question 2 and 3). I suggest to employ regression analyses The theoretical implications and practical implication should be added before the limitations in the manuscript.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?NoAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Textile and Fashion MarketingI confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above. Dear Reviewer, Thank you for you suggestions. I already applied all your suggestion in the version 2 of my manuscript. Please check it and let me know if something else needs addressing. Thank you very much, Ervin The manuscript titled "Analyzing the impact of loyalty card programs on customer behavior: insights from the Albanian market" presents an in-depth investigation into the effects of loyalty card programs on customer behavior within the Albanian retail market. The study employs a well-structured research design, involving a comprehensive survey to assess awareness levels, customer satisfaction, loyalty, and the influence of customized benefits associated with loyalty cards. This peer review evaluates the entire manuscript, encompassing the abstract, introduction, literature review, analysis, findings, conclusions, and recommendations.Major Strengths:Clear Research Objectives: The manuscript establishes clear research objectives and hypotheses, providing a solid framework for the study.Methodological Rigor: The research methodology is robust and well-detailed, with the survey conducted among a diverse sample of customers in Albania, enhancing its reliability.Relevance of Findings: The findings are highly relevant to the retail industry, offering practical insights for enhancing customer engagement and loyalty.Concise and Well-Structured: The manuscript is well-organized, making it easy for readers to follow the logical flow of the study. Given the study's focus on the Albanian market, it is important to acknowledge potential limitations concerning the applicability of the findings to other contexts. The manuscript should thoroughly discuss these limitations and propose avenues for future research or cross-cultural comparisons. In the "Discussion" section, where the manuscript briefly mentions that certain loyalty card benefit categories lacked statistically significant associations with loyalty, a more comprehensive examination of these non-significant findings and exploration of potential contributing factors would augment the paper's comprehensiveness.Recommendation: This study has the potential to make a valuable contribution to the field of marketing and customer behavior. The research findings offer practical implications for retailers and marketers aiming to enhance customer loyalty through loyalty card programs. Therefore, I recommend approving the manuscript for indexing.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required.Are all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Marketing: marketing management; market research; customer behavior's; Customer satisfaction; customer loyaltyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.