Adhesion Defects Research Articles

On the sensitivity of bondline control specimens with respect to the detection of adhesion defects

ABSTRACT Adhesive bonding is a highly efficient joining technology for the repair of fibre-reinforced plastics. However, in civil aviation, its application is currently limited to non-critical joints. In safety critical cases, certification authorities demand proof of sufficient bond strength for each individual joint. Today, there is no practicable inspection method that can provide this kind of evidence. Recently, a novel method has been proposed that may resolve this issue. This so-called Bondline Control Technology (BCT) is a new type of adhesion test that provides proof of bond quality and simultaneously serves as a robust surface pretreatment for adhesive bonding. The present study investigates on the capability of the BCT to detect adhesion defects. This is done by means of in-lab tests on bonded specimens with adhesion defects of defined severity. The detection sensitivity of BCT coupons (Bondline Control Specimens, BCS) is compared with double-cantilever beam specimens (DCB specimens), which are known to be particularly capable of detecting adhesion defects.

Pectin methyltransferase QUASIMODO2 functions in the formation of seed coat mucilage in Arabidopsis

Pectin, cellulose, and hemicelluloses are major components of primary cell walls in plants. In addition to cell adhesion and expansion, pectin plays a central role in seed mucilage. Seed mucilage contains abundant pectic rhamnogalacturonan-I (RG-I) and lower amounts of homogalacturonan (HG), cellulose, and hemicelluloses. Previously, accumulated evidence has addressed the role of pectin RG-I in mucilage production and adherence. However, less is known about the function of pectin HG in seed coat mucilage formation. In this study, we analyzed a novel mutant, designated things fall apart2 (tfa2), which contains a mutation in HG methyltransferase QUASIMODO2 (QUA2). Etiolated tfa2 seedlings display short hypocotyls and adhesion defects similar to qua2 and tumorous shoot development2 (tsd2) alleles, and show seed mucilage defects. The diminished uronic acid content and methylesterification degree of HG in mutant seed mucilage indicate the role of HG in the formation of seed mucilage. Cellulosic rays in mutant mucilage are collapsed. The epidermal cells of seed coat in tfa2 and tsd2 display deformed columellae and reduced radial wall thickness. Under polyethylene glycol treatment, seeds from these three mutant alleles exhibit reduced germination rates. Together, these data emphasize the requirement of pectic HG biosynthesis for the synthesis of seed mucilage, and the functions of different pectin domains together with cellulose in regulating its formation, expansion, and release.

The scaffold protein NEDD9 is necessary for leukemia-cell migration and disease progression in a mouse model of chronic lymphocytic leukemia

The scaffold protein NEDD9 is frequently upregulated and hyperphosphorylated in cancers, and is associated with poor clinical outcome. NEDD9 promotes B-cell adhesion, migration and chemotaxis, pivotal processes for malignant development. We show that global or B-cell-specific deletion of Nedd9 in chronic lymphocytic leukemia (CLL) mouse models delayed CLL development, markedly reduced disease burden and resulted in significant survival benefit. NEDD9 was required for efficient CLL cell homing, chemotaxis, migration and adhesion. In CLL patients, peripheral NEDD9 expression was associated with adhesion and migration signatures as well as leukocyte count. Additionally, CLL lymph nodes frequently expressed high NEDD9 levels, with a subset of patients showing NEDD9 expression enriched in the CLL proliferation centers. Blocking activity of prominent NEDD9 effectors, including AURKA and HDAC6, effectively reduced CLL cell migration and chemotaxis. Collectively, our study provides evidence for a functional role of NEDD9 in CLL pathogenesis that involves intrinsic defects in adhesion, migration and homing.

ARVCF catenin controls force production during vertebrate convergent extension

The design of an animal's body plan is encoded in the genome, and the execution of this program is a mechanical progression involving coordinated movement of proteins, cells, and whole tissues. Thus, a challenge to understanding morphogenesis is connecting events that occur across various length scales. Here, we describe how a poorly characterized adhesion effector, Arvcf catenin, controls Xenopus head-to-tail axis extension. We find that Arvcf is required for axis extension within the intact organism but not within isolated tissues. We show that the organism-scale phenotype results from a defect in tissue-scale force production. Finally, we determine that the force defect results from the dampening of the pulsatile recruitment of cell adhesion and cytoskeletal proteins to membranes. These results provide a comprehensive understanding of Arvcf function during axis extension and produce an insight into how a cellular-scale defect in adhesion results in an organism-scale failure of development.

VEGFR2 signals with VE‐cadherin and PECAM to facilitate leukocyte transendothelial migration

The migration of leukocytes out of the blood and into tissue is a critical component of the inflammatory process. To gain access to the target tissue and exert their beneficial or deleterious effects, leukocytes undergo a complex series of adhesive interactions with endothelial cells, which line the vessel wall. In the putative committed step in this process, the leukocyte must find the junction between adjacent endothelial cells and crawl through. This process, commonly referred to as transendothelial migration (TEM), has long been known to require homophilic interaction between PECAM on leukocyte and the endothelial cells. Recently our group showed that the process also involves calcium signaling originating through the cation channel TRPC6.We hypothesized that PECAM may be functioning in TEM using some of the interactions recently shown to be involved in its role in the mechanotransduction of fluid sheer stress. Perhaps engagement of leukocyte PECAM with endothelial cell PECAM at the cell border provides the sheer stress in this case. Here we report the unexpected role of a macromolecular PECAM complex that facilitates signaling to TRPC6 in TEM. Specifically, we show that endothelial cell PECAM binds to VEGFR2 and VE‐cadherin via the transmembrane domain of VE‐cadherin. Swapping the VE‐cadherin transmembrane domain with that of N‐cadherin abrogated TEM. Likewise, deletion of the cytoplasmic c‐terminal tail of PECAM also abolished this signaling cascade. Stimulation of this complex with antibodies against PECAM induces the phosphorylation of VEGFR2 on tyrosine Y1175. Mutation of that critical residue to phenylalanine (Y1175F) prevented VEGFR2 phosphorylation and blocked TEM. Furthermore we show that this complex signals specifically to PLC1γ (and not PLCβ3) leading to its phosphorylation. Blocking its function with the inhibitor U73122 also blocked TEM. Diacylglycerol liberated by PLCγ1 activates TRPC6.We also confirmed the role of VEGFR2 in vivo using to separate models of inflammation. First, we examined the recruitment of neutrophils to inflamed skin using the croton oil dermatitis model. In control animals, neutrophils are robustly recruited to and migrate across post‐capillary venules. In VEGFR2 knockout (KO) mice, most neutrophils are found inside the vessel with a corresponding decrease in those found out in tissue. Similarly, live animal imaging (intravital microscopy) of the inflamed cremaster muscle showed no defect in neutrophil rolling or adhesion in the VEGFR2 KO animals, but a profound disruption in their ability to undergo TEM compared to control wild type mice.Taken together, these data confirm the role of a VEGFR2/PECAM/VE‐cadherin mechanotransduction complex that signals through PLC1γ to facilitate leukocyte TEM upstream of the action of TRPC6. Uncovering this signaling pathway opens up several new targets for potential therapeutic intervention.

ARHGAP29 Regulates Keratinocyte Migration through the RhoA/ROCK Pathway

Genetic variants in Rho GTPase Activating Protein 29 (ARHGAP29) have been associated with non‐syndromic cleft lip and/or palate, one of the most common human birth defects. Orofacial clefting results from the failure of facial processes to fuse during development which can be caused by defects in proliferation, adhesion, and/or migration. ARHGAP29 is widely expressed and preferentially interacts with the small GTPase RhoA, a critical modulator of the actin cytoskeleton required for cell morphology and migration. Therefore, we hypothesized that ARHGAP29 would play a critical role in keratinocyte migration. To test this hypothesis, we knocked down ARHGAP29 in a human keratinocyte cell line using both shRNA and CRISPR technologies. To assess migration, we used an in vitro scratch assay on a confluent keratinocyte monolayer and followed scratch closure over 24 hours. ARHGAP29 knockdown keratinocytes displayed significantly increased cell area and significant delays in scratch closure. By tracing single cells throughout the duration of scratch closure, we found that ARHGAP29 knockdown cells were significantly slower and showed defects in directionality that were rescued with a ROCK inhibitor, Y‐27632, suggesting these migration defects are downstream of the RhoA pathway. Collectively, our results demonstrate that ARHGAP29 regulates keratinocyte morphology and promotes migration speed and directionality through the RhoA/ROCK pathway, providing potential insight into its importance in craniofacial development.

Epidermolysis Bullosa in children: the central role of the pediatrician

Epidermolysis bullosa (EB) is a severe hereditary disease characterized by defective epithelial adhesion causing mucocutaneous fragility. The major types are EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB) and more than 35 EB subtypes. Another very rare type of EB is Kindler EB (KEB). Clinically, it is a very heterogeneous disease which ranges from localized to extensive skin lesions with frequent multisystem extra cutaneous involvement. The role of a pediatrician-dermatologist cooperation within a multidisciplinary team is fundamental for both the diagnosis and management contributing to these patients’ better life expectancy. Aim of this study is to describe clinical and laboratory characteristics of the main EB subtypes focusing on nutritional and gastrointestinal aspects, providing information to aid the paediatric management of children with EB. This retrospective study reviewed the cases of 160 pediatric EB patients (76 male and 84 female): 31 patients affected by EBS (mean age ± SD: 4.37 ± 7.14), 21 patients affected by JEB (mean age ± SD: 9.26± 17.30) and 108 with DEB (mean age ± SD: 11.61 ± 13.48). All patients were admitted at the Bambino Gesù Children’s Hospital in Rome, between June 2005 to June 2020. The reduced gastrointestinal absorption, chronic losses, esophageal stenosis and chronic inflammatory state, represent the basis of nutritional problems of EB patients. In particular, anemia represents one of the most important complications of DEB patients which could require transfusion-dependent patterns. Malnutrition, vitamin deficiencies and anemia have been related to growth delay in EB patients. A specific diet with a balance of all macronutrients is required and improving caloric intake with sugar limitations is fundamental to prevent dental caries and tooth decay typical of EB patients. While sepsis proved to be the major cause of morbidity and mortality in younger patients, squamous cell carcinoma was mostly observed in older patients, especially those affected by DEB. Patients with EB require regular monitoring for complications and sequelae with a frequency of evaluations which varies based on age and EB subtypes. Cooperation among medical teams involving paediatricians, dermatologists, specialist clinicians including nutritionists such as families and patient’s association is fundamental to approach the disease and improve the quality of life of these patients.

Molecular Characterization of Three Tandemly Located Flagellin Genes of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a motile, opportunistic pathogen. The flagellum, which is involved in swimming, swarming, adhesion, and biofilm formation, is considered a virulence factor for motile pathogens. Three flagellin genes, fliC1, fliC2, and fliC3, were identified from the sequenced S. maltophilia genome. FliC1, fliC2, and fliC3 formed an operon, and their encoding proteins shared 67–82% identity. Members of the fliC1C2C3 operon were deleted individually or in combination to generate single mutants, double mutants, and a triple mutant. The contributions of the three flagellins to swimming, swarming, flagellum morphology, adhesion, and biofilm formation were assessed. The single mutants generally had a compromise in swimming and no significant defects in swarming, adhesion on biotic surfaces, and biofilm formation on abiotic surfaces. The double mutants displayed obvious defects in swimming and adhesion on abiotic and biotic surfaces. The flagellin-null mutant lost swimming ability and was compromised in adhesion and biofilm formation. All tested mutants demonstrated substantial but different flagellar morphologies, supporting that flagellin composition affects filament morphology. Bacterial swimming motility was significantly compromised under an oxidative stress condition, irrespective of flagellin composition. Collectively, the utilization of these three flagellins for filament assembly equips S. maltophilia with flagella adapted to provide better ability in swimming, adhesion, and biofilm formation for its pathogenesis.

Immunomodulation of Telmisartan-Loaded PCL/PVP Scaffolds on Macrophages Promotes Endogenous Bone Regeneration.

Biomaterial-immune system interactions play an important role in postimplantation osseointegration to retain the functionality of healthy and intact bones. Therefore, appropriate osteoimmunomodulation of implants has been considered and validated as an efficient strategy to alleviate inflammation and enhance new bone formation. Here, we fabricated a nanostructured PCL/PVP (polycaprolactone/polyvinylpyrrolidone) electrospinning scaffold for cell adhesion, tissue ingrowth, and bone defect padding. In addition, telmisartan, an angiotensin 2 receptor blocker with distinct immune bioactivity, was doped into PCL-/PVP-electrospun scaffolds at different proportions [1% (TPP-1), 5% (TPP-5), and 10% (TPP-10)] to investigate its immunomodulatory effects and osteoinductivity/conductivity. Telmisartan-loaded scaffolds displayed in vitro anti-inflammatory bioactivity on lipopolysaccharide-induced M1 macrophages by polarizing them to an M2-like phenotype and exhibited pro-osteogenic properties on bone marrow-derived mesenchymal stem cells (BMSCs). Histological analysis and micro-CT results of a rat skull defect model also showed that the telmisartan-loaded scaffolds induced a higher M2/M1 ratio, less inflammatory infiltration, and better bone regenerative patterns. Furthermore, activation of the BMP2 (bone morphogenetic protein-2)-Smad signaling pathway was found to be dominant in telmisartan-loaded scaffold-mediated macrophage-BMSC interactions. These findings indicate that telmisartan incorporation with PCL/PVP nanofibrous scaffolds is a potential therapeutic strategy for promoting bone healing by modulating M1 macrophages to a more M2 phenotype at early stages of postimplantation.

Thirty Years of Thermal Barrier Coatings (TBC), Photothermal and Thermographic Techniques: Best Practices and Lessons Learned

With the adoption of thermal barrier coatings (TBCs) to protect the hot parts of gas turbines at the end of the last century, the characterization of their thermophysical properties and the evaluation of their state of integrity has become necessary. That leads to the development and/or the adaptation of photothermal and thermographic techniques to meet the newly requirements that have emerged. Whereas this is not an all-encompassing review of all the literature on the subjects, this work summarizes the research and the development of techniques specifically aimed at measuring the thermophysical properties, the characterization of the microstructure and the identification of adhesion defects to the metal component of the TBCs. In the following, the best practices, the lessons learned, the successes and the defeats experienced along almost thirty years will be summarized.

RIPK4 regulates cell-cell adhesion in epidermal development and homeostasis.

Epidermal development and maintenance are finely regulated events requiring a strict balance between proliferation and differentiation. Alterations in these processes give rise to human disorders such as cancer or syndromes with skin and annexes defects, known as ectodermal dysplasias (EDs). Here, we studied the functional effects of two novel receptor-interacting protein kinase 4 (RIPK4) missense mutations identified in siblings with an autosomal recessive ED with cutaneous syndactyly, palmoplantar hyperkeratosis and orofacial synechiae. Clinical overlap with distinct EDs caused by mutations in transcription factors (i.e. p63 and interferon regulatory factor 6, IRF6) or nectin adhesion molecules was noticed. Impaired activity of the RIPK4 kinase resulted both in altered epithelial differentiation and defective cell adhesion. We showed that mutant RIPK4 resulted in loss of PVRL4/nectin-4 expression in patient epidermis and primary keratinocytes, and demonstrated that PVRL4 is transcriptionally regulated by IRF6, a RIPK4 phosphorylation target. In addition, defective RIPK4 altered desmosome morphology through modulation of plakophilin-1 and desmoplakin. In conclusion, this work implicates RIPK4 kinase function in the p63-IRF6 regulatory loop that controls the proliferation/differentiation switch and cell adhesion, with implications in ectodermal development and cancer.

Proline-rich tyrosine kinase Pyk2 regulates deep vein thrombosis.

Deep vein thrombosis results from the cooperative action of leukocytes, platelets, and endothelial cells. The proline-rich tyrosine kinase Pyk2 regulates platelet activation and supports arterial thrombosis. In this study, we combined pharmacological and genetic approaches to unravel the role of Pyk2 in venous thrombosis. We found that mice lacking Pyk2 almost completely failed to develop deep venous thrombi upon partial ligation of the inferior vena cava. Pyk2-deficient platelets displayed impaired exposure of phosphatidylserine and tissue factor expression by endothelial cells and monocytes was completely prevented by inhibition of Pyk2. In human umbilical vein endothelial cells (HUVEC), inhibition of Pyk2 hampered IL-1β-induced expression of VCAM and P-selectin, and von Willebrand factor release. Pyk2-deficient platelets showed defective adhesion on von Willebrand factor and reduced ability to bind activated HUVEC under flow. Moreover, inhibition of Pyk2 in HUVEC strongly reduced platelet adhesion. Similarly, Pyk2-deficient neutrophils were unable to efficiently roll and adhere to immobilized endothelial cells under venous flow conditions. Moreover, platelets and neutrophils from Pyk2-knockout mice showed defective ability to form heterogeneous aggregates upon stimulation, while platelet monocyte interaction occurred normally. Consequently, platelet neutrophil aggregates, abundant in blood of wild-type mice upon inferior vena cava ligation, were virtually undetectable in Pyk2-knockout mice. Finally, we found that expression of Pyk2 was required for NETosis induced by activated platelets. Altogether our results demonstrate a critical role of Pyk2 in the regulation of the coordinated thromboinflammatory responses of endothelial cells, leukocytes and platelets leading to venous thrombosis. Pyk2 may represent a novel promising target in the treatment of deep vein thrombosis.

RBFOX2 is required for establishing RNA regulatory networks essential for heart development.

Human genetic studies identified a strong association between loss of function mutations in RBFOX2 and hypoplastic left heart syndrome (HLHS). There are currently no Rbfox2 mouse models that recapitulate HLHS. Therefore, it is still unknown how RBFOX2 as an RNA binding protein contributes to heart development. To address this, we conditionally deleted Rbfox2 in embryonic mouse hearts and found profound defects in cardiac chamber and yolk sac vasculature formation. Importantly, our Rbfox2 conditional knockout mouse model recapitulated several molecular and phenotypic features of HLHS. To determine the molecular drivers of these cardiac defects, we performed RNA-sequencing in Rbfox2 mutant hearts and identified dysregulated alternative splicing (AS) networks that affect cell adhesion to extracellular matrix (ECM) mediated by Rho GTPases. We identified two Rho GTPase cycling genes as targets of RBFOX2. Modulating AS of these two genes using antisense oligos led to cell cycle and cell-ECM adhesion defects. Consistently, Rbfox2 mutant hearts displayed cell cycle defects and inability to undergo endocardial-mesenchymal transition, processes dependent on cell-ECM adhesion and that are seen in HLHS. Overall, our work not only revealed that loss of Rbfox2 leads to heart development defects resembling HLHS, but also identified RBFOX2-regulated AS networks that influence cell-ECM communication vital for heart development.

Bone Marrow Transplantation Rescues Monocyte Recruitment Defect and Improves Cystic Fibrosis in Mice.

Cystic fibrosis (CF) is an inherited life-threatening disease accompanied by repeated lung infections and multiorgan inflammation that affects tens of thousands of people worldwide. The causative gene, cystic fibrosis transmembrane conductance regulator (CFTR), is mutated in CF patients. CFTR functions in epithelial cells have traditionally been thought to cause the disease symptoms. Recent work has shown an additional defect: monocytes from CF patients show a deficiency in integrin activation and adhesion. Because monocytes play critical roles in controlling infections, defective monocyte function may contribute to CF progression. In this study, we demonstrate that monocytes from CFTRΔF508 mice (CF mice) show defective adhesion under flow. Transplanting CF mice with wild-type (WT) bone marrow after sublethal irradiation replaced most (60-80%) CF monocytes with WT monocytes, significantly improved survival, and reduced inflammation. WT/CF mixed bone marrow chimeras directly demonstrated defective CF monocyte recruitment to the bronchoalveolar lavage and the intestinal lamina propria in vivo. WT mice reconstituted with CF bone marrow also show lethality, suggesting that the CF defect in monocytes is not only necessary but also sufficient to cause disease. We also show that monocyte-specific knockout of CFTR retards weight gains and exacerbates dextran sulfate sodium-induced colitis. Our findings show that providing WT monocytes by bone marrow transfer rescues mortality in CF mice, suggesting that similar approaches may mitigate disease in CF patients.

Clinical Manifestations and Laboratory Findings in Patients with Leukocyte Adhesion Deficiency (LAD)

Objectives: Leukocyte Adhesion Deficiency (LAD) is a rare, inherited, immunodeficiency disease which is caused by defects in the leukocyte adhesion process. The migration of leukocytes to the blood vessel’s wall, needs multiple steps called adhesion cascade. In LAD, defects in rolling, integrin activation and firm adhesion of the leukocytes have been described. Methods: In this study, we selected 67 patients with the confirmed diagnosis of LADs, from Iranian immunodeficiency registry center. A demographic information of the clinical complications and laboratory data were obtained from all the patients to evaluate the clinical manifestations. Results: A total of 67 patients (38 male and 29 female), with a median age of 18 months old, were included in the present study. The first presentations were omphalitis in 28.35% of the cases, followed by delayed umbilical cord separation in 22.38% of the patients. The frequency of delayed umbilical cord separation was 41.8%, and was higher among other manifestations of our patients. Cellulitis and Omphalitis were observed in 40.3% and 38.8% of the patients, respectively. Regarding the laboratory findings, we found leukocytosis in 86.6 %( neutrophil dominant in 76.1%), and anemia in 77.6%, and thrombocytosis in 25.4% of the patients. Conclusion: We indicated in the present study that the most common clinical manifestations, were delayed umbilical cord separation and recurrent infection in Iranian patients with LAD disorders. In laboratory findings, we found leukocytosis in most of the patients. CD18 was decreased in more than 90 % of the patients.

Eukaryotic initiation factor 6 regulates mechanical responses in endothelial cells.

The repertoire of extratranslational functions of components of the protein synthesis apparatus is expanding to include control of key cell signaling networks. However, very little is known about noncanonical functions of members of the protein synthesis machinery in regulating cellular mechanics. We demonstrate that the eukaryotic initiation factor 6 (eIF6) modulates cellular mechanobiology. eIF6-depleted endothelial cells, under basal conditions, exhibit unchanged nascent protein synthesis, polysome profiles, and cytoskeleton protein expression, with minimal effects on ribosomal biogenesis. In contrast, using traction force and atomic force microscopy, we show that loss of eIF6 leads to reduced stiffness and force generation accompanied by cytoskeletal and focal adhesion defects. Mechanistically, we show that eIF6 is required for the correct spatial mechanoactivation of ERK1/2 via stabilization of an eIF6-RACK1-ERK1/2-FAK mechanocomplex, which is necessary for force-induced remodeling. These results reveal an extratranslational function for eIF6 and a novel paradigm for how mechanotransduction, the cellular cytoskeleton, and protein translation constituents are linked.

Periorificial lesions associated with poor prognosis in patients with localized vitiligo: A retrospective study of 126 patients

Periorificial lesions associated with poor prognosis in patients with localized vitiligo: A retrospective study of 126 patients

Mussel-inspired injectable chitosan hydrogel modified with catechol for cell adhesion and cartilage defect repair.

Repairing articular cartilage defects is a great challenge due to the poor self-regenerative capability of cartilage. Hydrogel-based tissue engineering has been considered an effective strategy. In this study, inspired by mussel chemistry, catechol-modified chitosan (CS-C) hydrogel was prepared under the catalysis of horseradish peroxidase/hydrogen peroxide (HRP/H2O2) for cartilage defect repair in a rat model. The rheological and swelling properties and biodegradation behavior of the CS-C hydrogel were investigated. Besides, the chondrogenic effect of bone mesenchymal stem cells (BMSCs) within the CS-C hydrogel was also assessed in vitro. Moreover, after injecting in rat cartilage defects, the capability of cartilage repair of the BMSC-laden CS-C hydrogel was evaluated in vivo. The results showed that the rheological property, swelling property and biodegradation behavior of the CS-C hydrogel changed with the concentration of CS-C macromolecules. Besides, the CS-C hydrogel had good biocompatibility with BMSCs and could promote the proliferation and chondrogenic differentiation of BMSCs in vitro. As for cartilage defect repair in vivo, through the evaluation of gross observation and histology, the BMSC-laden CS-C hydrogel showed better reconstruction of hyaline cartilage than the untreated group and CS-C hydrogel only. Therefore, CS-C hydrogel laden with BMSC might be a promising strategy for repairing cartilage defects.

Role of Flow Cytometry in the Diagnosis of Inborn Errors of Immunity

AbstractInborn errors of immunity (IEI) are a group of inherited heterogeneous disorders affecting the immune system characterized by increased susceptibility to infections, immune dysregulation, and lymphoproliferation. Flow cytometry (FCM) is a rapid and reliable technique for evaluation and enumeration of immune cells. It also helps in understanding the functional and signaling pathways of the immune system. Lymphocyte subset analysis is a simple and effective screening tool in suspected combined and humoral immunodeficiency patients. Qualitative phagocytic defects such as chronic granulomatous disease and leucocyte adhesion defect are easily diagnosed by FCM. Study of intracellular proteins (e.g., BTK, WASP, DOCK8), cytokine production, and signaling molecules (e.g., STAT3) by FCM is very useful but also quite challenging to establish. T and B lymphocyte interaction for normal class switching of B cells can be assessed and can help in diagnosis of combined variable immunodeficiency and hyperimmunoglobulin M syndrome. FCM is also used in posttransplant monitoring of IEI patients and also in prenatal diagnosis in suspected cases. It is also useful in validation of variants of uncertain significance obtained in exome sequencing. FCM results should always be interpreted with clinical history and, if needed, should be confirmed with molecular genetic studies before establishing the final diagnosis. Ensuring good sample quality and running parallel controls with patient samples will avoid the preanalytical and analytical errors. This review describes the applications of FCM in the diagnosis of various IEI.

Leukocyte Adhesion Defects Type III

Leukocyte adhesion defect (LAD) Type III is an extremely rare disease, which presents with severe infections and a Glanzmann thrombasthenia-like bleeding phenotype. A 2-year-old male child with LAD Type III with recurrent mucosal bleeding and relatively few infections is presented to highlight this novel presentation. The rare association of primary immunodeficiency and platelet functional disease is described.