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Inherited platelet disorders (IPDs) are rare bleeding disorders characterized by impaired platelet function and/or reduced blood platelet count. Their diagnosis typically relies on complex laboratory methods, including flow cytometry, aggregometry, and molecular genetic analysis. In recent years, immunofluorescence microscopy has been established as an alternative diagnostic method for IPDs. Background/Objectives: This study aims to validate a quantitative approach enhancing reproducibility through automated image analysis for diagnosing IPDs using immunofluorescence microscopy, with Bernard–Soulier Syndrome (BSS) and Glanzmann thrombasthenia (GT) as model IPDs. Methods: Native blood smears from patients with suspected BSS or GT were stained using a standardized immunofluorescence protocol targeting platelet surface glycoproteins, granules, and cytoskeletal components. The slides were analyzed using an automated fluorescence microscope, and a rule-based subpopulation analysis was implemented to quantify fluorescence signals. The results were compared to those of a healthy control group, as well as data from flow cytometry and molecular genetic testing. Results: The automated analysis successfully differentiated BSS and GT patients from healthy controls based on distinct fluorescence signal patterns. In BSS samples, CD42b (GPIbα) expression was absent or severely reduced, while GT samples showed a deficiency of CD41/CD61 (GPIIb/IIIa). The platelet size distribution confirmed macrothrombocytopenia in BSS patients. Flow cytometry and molecular genetic testing corroborated these findings, supporting the diagnostic reliability of the automated immunofluorescence microscopy approach. Conclusions: This proof-of-principle study demonstrates that automated quantitative immunofluorescence microscopy is a viable alternative for diagnosing IPDs, offering a standardized, objective, and efficient method, particularly in settings where flow cytometry is not feasible.

Glanzmann thrombasthenia (GT) is a rare, inherited autosomal recessive disorder characterized by qualitative or quantitative deficiency of integrin αIIbβ3 [glycoprotein IIb (GPIIb)/IIIa, CD41/CD61] diagnosed by absent or reduced platelet aggregation to physiological agonists, namely, collagen, adenosine-di-phosphate, epinephrine and arachidonic acid. The objective of this study was to quantitate platelet surface GPs, classify GT patients and relate the results with the severity of bleeding and platelet aggregation studies. Fifty one patients of GT diagnosed by platelet aggregation studies were evaluated for the expression of CD41, CD61, CD42a and CD42b on platelet surface by flow cytometry. The association between the clinical phenotype based on bleeding score and GT subtype on flow cytometric evaluation was assessed. Twenty four (47%) patients of GT were classified as type I (as CD41/CD61 were virtually absent, <5%), six (11.8%) patients as type II (5-20% CD41/CD61) and 21 (41.2%) as type III or GT variants as they had near normal levels of CD41 and CD61. Type III GT patients had significantly lower numbers of severe bleeders (P=0.034), but the severity of bleeding did not vary significantly in type I and II GT patients. In all GT patients, mean CD41 expression was found to be lower than mean CD61 expression (P=0.002). Type I GT was found most common in our patients and with lowered mean CD41 expression in comparison with CD61. Type III GT patients had significantly lower numbers of severe bleeders, but the severity of bleeding did not vary significantly in type I and II GT patients.

The fibrin(ogen) receptor, integrin α(IIb)β(3), has a well-established role in platelet spreading, aggregation and clot retraction. How α(IIb)β(3) contributes to platelet-dependent coagulation is less well resolved. Here, we demonstrate that the potent suppressing effect of clinically used α(IIb)β(3) blockers on tissue factor-induced thrombin generation is linked to diminished platelet Ca(2+) responses and phosphatidylserine (PS) exposure. The same blockers suppress these responses in platelets stimulated with collagen and thrombin receptor agonists, whereas added fibrinogen potentiates these responses. In platelets spreading on fibrinogen, outside-in α(IIb)β(3) signaling similarly enhances thrombin-induced Ca(2+) rises and PS exposure. These responses are reduced in α(IIb)β(3)-deficient platelets from patients with Glanzmann's thrombasthenia. Furthermore, the contribution of α(IIb)β(3) to tissue factor-induced platelet Ca(2+) rises, PS exposure and thrombin generation in plasma are fully dependent on Syk kinase activity. Tyrosine phosphorylation analysis confirms a key role of Syk activation, which is largely but not exclusively dependent on α(IIb)β(3) activation. It is concluded that the majority of tissue factor-induced procoagulant activity of platelets relies on Syk activation and ensuing Ca(2+) signal generation, and furthermore that a considerable part of Syk activation relies on α(IIb)β(3) signaling. These results hence point to a novel role of Syk in integrin-dependent thrombin generation.

BACKGROUND: Two congenital bleeding diatheses have been identified in Thoroughbred horses: Glanzmann thrombasthenia (GT) and a second, novel diathesis associated with abnormal platelet function in response to collagen and thrombin stimulation. HYPOTHESIS/OBJECTIVES: Platelet dysfunction in horses with this second thrombasthenia results from a secretory defect. ANIMALS: Two affected and 6 clinically normal horses. METHODS: Ex vivo study. Washed platelets were examined for (1) expression of the αIIb‐β3 integrin; (2) fibrinogen binding capacity in response to ADP and thrombin; (3) secretion of dense and α‐granules; (4) activation of the mammalian target of rapamycin (mTOR)‐protein kinase B (AKT) signaling pathway; and (5) cellular distribution of phosphatidylinositol‐4‐phosphate‐3‐kinase, class 2B (PIK3C2B) and SH2 containing inositol‐5′‐phosphatase 1 (SHIP1). RESULTS: Platelets from affected horses expressed normal amounts of αIIb‐β3 integrin and bound fibrinogen normally in response to ADP, but bound 80% less fibrinogen in response to thrombin. α‐granules only released 50% as much Factor V as control platelets, but dense granules released their contents normally. Protein kinase B (AKT) phosphorylation was reduced after thrombin activation, but mTOR Complex 2 (mTORC2) and phosphoinositide‐dependent kinase 1 (PDK1) signaling were normal. SH2‐containing inositol‐5'‐phosphatase 1 (SHIP1) did not localize to the cytoskeleton of affected platelets and was decreased overall consistent with reduced AKT phosphorylation. CONCLUSIONS AND CLINICAL SIGNIFICANCE: Defects in fibrinogen binding, granule secretion, and signal transduction are unique to this thrombasthenia, which we designate as atypical equine thrombasthenia.

Perioperative hemostatic management is a challenge in patients with Glanzmann thrombasthenia (GT). The standard means of preventing surgical bleeding in GT patients is platelet transfusion. However, GT patients often possess alloantibodies against GPIIb/IIIa and/or HLA, which cause resistance to platelet transfusion. HLA-matched platelet transfusion, plasmapheresis, or recombinant human-activated factor VII (rFVIIa) are alternative interventions in such cases. Monitoring of hemostasis is also critical in the management of GT patients who undergo surgery. Here, we report the case of a 56-year-old female GT patient with anti-HLA antibodies, who underwent a right total mastectomy without significant blood loss under HLA-matched platelet transfusion. Bleeding at the surgical site, which occurred on the 18th postoperative day, was successfully treated by immediate bolus administration of rFVIIa and subsequent HLA-matched platelet transfusion. The perioperative hemostatic state was monitored in combination with bleeding time, platelet aggregation assay, and flow cytometric analysis of GPIIb/IIIa expression. Although a flow cytometric analysis is not a functional assay, it enabled the estimation of transfused platelet counts, and helped to inform the decision regarding whether to perform the surgery. Thus, perioperative hemostasis was successfully managed in our GT patient by HLA-matched platelet transfusion, rFVIIa administration, and the close monitoring of hemostasis.

Glanzmann's thrombasthenia (GT) is an autosomal recessive inherited bleeding disorder due to a defect in platelet function. The hallmark of this disease is severely reduced/absent platelet aggregation in response to multiple physiological agonists. Bleeding signs in GT include epistaxis, bruising, gingival hemorrhage, gastrointestinal hemorrhage, hematuria, menorrhagia, and hemarthrosis. Homozygous or compound heterozygous mutations in the genes of GPIIb and GPIIIa lead to GT. A patient with GT, with no possible causative mutations in GPIIb and GPIIIa genes, may harbor defects in a regulatory element affecting the transcription of these 2 genes. GT occurs in high frequency in certain ethnic populations with an increased incidence of consanguinity such as in Indians, Iranians, Iraqi Jews, Palestinian and Jordanian Arabs, and French Gypsies. Carrier detection in GT is important to control the disorder in family members. Carrier detection can be done both by protein analysis and direct gene analysis.

To delineate the critical features of platelets required for formation and stability of thrombi, thromboelastography and platelet aggregation measurements were employed on whole blood of normal patients and of those with Bernard-Soulier Syndrome (BSS) and Glanzmann's Thrombasthenia (GT). We found that separation of platelet activation, as assessed by platelet aggregation, from that needed to form viscoelastic stable whole blood thrombi, occurred. In normal human blood, ristocetin and collagen aggregated platelets, but did not induce strong viscoelastic thrombi. However, ADP, arachidonic acid, thrombin, and protease-activated-receptor-1 and -4 agonists, stimulated both processes. During this study, we identified the genetic basis of a very rare double heterozygous GP1b deficiency in a BSS patient, along with a new homozygous GP1b inactivating mutation in another BSS patient. In BSS whole blood, ADP responsiveness, as measured by thrombus strength, was diminished, while ADP-induced platelet aggregation was normal. Further, the platelets of 3 additional GT patients showed very weak whole blood platelet aggregation toward the above agonists and provided whole blood thrombi of very low viscoelastic strength. These results indicate that measurements of platelet counts and platelet aggregability do not necessarily correlate with generation of stable thrombi, a potentially significant feature in patient clinical outcomes.

Results of the laboratory evaluation included: white blood cell count, 8.2 X 109/L [reference interval (RI),3 4 X 109/Lto 10 X 109/L]; hemoglobin, 10.7 g/dL (RI, 11-15 g/dL); platelet count, 142 X 109/L (RI, 100 X 109/L to 300 X 109/L), prothrombin time, 11.5 s (RI, 9-13 s); activated partial thromboplastin time, 31.2 s (RI, 26-39 s); and fibrinogen, 2.5 g/L (RI, 2.0-4.0 g/L). Molecular testing can identify the pathogenic gene quickly and accurately. [...]the disease can be diagnosed earlier, thereby allowing proper treatment. The hemorrhagic diathesis in GT is notable for its variability and for the lack of a correlation between the biochemical platelet abnormalities and clinical severity (1). [...]factors other than the platelet defect itself appear to play an important role in determining the risk of bleeding.

Immune thrombocytopenia (ITP) is an autoimmune condition primarily induced by the loss of immune tolerance to the platelet glycoproteins. Here we develop a novel flow cytometry approach to analyze integrin α β functioning in ITP in comparison with Glanzmann thrombasthenia (GT) (negative control) and healthy pediatric donors (positive control). Continuous flow cytometry of Fura-Red-loaded platelets from whole hirudinated blood was used for the characterization of platelet responses to conventional activators. Calcium levels and fibrinogen binding were normalized to ionomycin-induced responses. Ex vivo thrombus formation on collagen was observed in parallel-plate flow chambers. Platelets from all ITP patients had significantly higher cytosolic calcium concentration in the quiescent state compared to healthy donors (15 ± 5 nM vs. 8 ± 5 nM), but calcium increases in response to all activators were normal. Clustering analysis revealed two subpopulations of ITP patients: the subgroup with high fibrinogen binding (HFB), and the subgroup with low fibrinogen binding (LFB) (8% ± 5% for LFB vs. 16% ± 3% for healthy donors in response to ADP). GT platelets had calcium mobilization (81 ± 23 nM), fibrinogen binding (5.1% ± 0.3%) and thrombus growth comparable to the LFB subgroup. Computational modeling suggested phospholipase C-dependent platelet pre-activation for the HFB subgroup and lower levels of functional integrin molecules for the LFB group.

A sensitive and specific sandwich ELISA was developed for the diagnosis of Glanzmann’s thrombasthenia (GT) and the heterozygote carriers of the disease using whole blood platelets. The assay used anti-CD36 antibody to capture platelets from platelet-rich plasma which was subsequently treated with a bioengineered disintegrin/alkaline phosphatase hybrid protein specific for GP IIb/IIIa. The test allows large number of samples to be typed and can also be used on stored samples. The assay correctly diagnosed 40 normal healthy individuals, 10 GT cases, 10 heterozygotes, 3 Bernard–Soulier syndrome cases and 2 type 3 GT cases. ELISA plates were stable at room temperature up to 3 weeks without any loss of activity. This novel and simple test can be widely used for heterozygote detection besides diagnosing GT cases without using a sophisticated flow cytometer or a platelet aggregometer and has wide applicability in countries like India where many of these cases remain undiagnosed due to the lack of diagnostic facilities.