Diagnosis Of Glanzmann's Thrombasthenia Research Articles

Flow cytometric analysis of platelet surface glycoproteins in the diagnosis of thirty-two Turkish patients with Glanzmann thrombasthenia: a multicenter experience

Background/aimGlanzmann thrombasthenia (GT) is a rare autosomal recessively inherited bleeding disorder characterized by the quantitative (type 1 and type 2) or qualitative (type 3) deficiency in platelet membrane glycoprotein (GP) IIb/IIIa (CD41a/CD61) fibrinogen receptors. In type 1, 2, and 3, CD41a/CD61 expression is 5%, 5%–20% and above 20%, respectively. In this study, diagnosis of GT was confirmed and subgroups were identified in 32 Turkish patients by flow cytometry analysis.Materials and methodsCD41a/CD61 expression levels in platelet-rich plasma (PRP) obtained from peripheral venous EDTA blood samples were analyzed with a BD FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). GT subgroup analysis was performed by counting 50,000 events in the BD FACSDiva Software v6.1.3 program of the instrument.ResultsIn the present study, in blood samples of 32 patients from 23 families with GT and 22 healthy controls, co-expression levels of CD41a and CD61 in PRP was analyzed. 12 out of 23 families were consistent with type 1 GT (52.2%), 4 were consistent with type 2 GT (17.4%), and 7 were consistent with type 3 GT (30.4%).ConclusionEspecially due to consanguineous marriages, GT with various glycoprotein levels may be detected. As a result of the flow cytometry analysis of the present study with the highest GT patient population in Turkey, type 1 GT patients were the most common subgroup. In the determination of the GT subgroups; especially in the detection of type 3 GT, flow cytometry is the most sensitive glycoprotein analysis method. In addition to light transmission aggregometry, CD41a/CD61 study by flow cytometer confirms diagnosis when mutation analysis cannot be performed.

A novel missense variant in the RASGRP2 gene in patients with moderate to severe bleeding disorder

Inherited platelet function disorder-18 (IPD-18) is a relatively new non-syndromic autosomal recessive bleeding disorder. It is characterized by deficient or dysfunctional CalDAG-GEFI protein. The distinctive feature of the disease is impaired platelet aggregation in response to multiple physiologic agonists. We here report a family with a platelet-type bleeding disorder and a novel mutation in the RASGRP2 gene. The overall bleeding score for the affected individuals was 15 and 12. Based on the initial diagnosis of Glanzmann thrombasthenia, targeted sequencing of integrin subunit alpha 2b and integrin subunit beta 3 encoding genes ITGA2B and ITGB3 were carried out in both affected members of a family. Sequence alignment failed to identify the disease-causing variant(s) in both genes. Therefore, whole exome sequencing in one affected individual was performed. Data analysis detected a novel homozygous missense variant (c.956C>T; p.Pro319Leu) in the exon 9 of the RASGRP2 gene. Five additional individuals of a family including both parents, an affected individual and two asymptomatic individuals were Sanger sequenced for the variant (c.956C>T). The variant segregates in the family in an autosomal recessive manner. Several in silico tools predicted the variant as pathogenic. Protein modeling studies suggest that the mutation (p.Pro319Leu) cause a conformational change in the loop structure of the RasGEF domain of the CalDAG-GEFI protein. Reported variants in the RasGEF domain impair expression and/or nucleotide exchange activity of CalDAG-GEFI protein and thus inhibit the activation of Rap1 protein required for platelet adhesion and hemostatic plug formation.

Heavy Menstrual Bleeding as a Common Presenting Symptom of Rare Platelet Disorders: Illustrative Case Examples

Heavy menstrual bleeding (HMB) is a common symptom in patients who present to the obstetrician-gynecologist or adolescent medicine specialist and might result from an underlying inherited bleeding disorder. Whereas relatively common bleeding disorders such as von Willebrand disease are often included in standard laboratory assessments, rarer platelet function disorders can be challenging to diagnose. Additionally, HMB can be a particularly difficult symptom to manage in adolescents with platelet function disorders, and it is associated with decreased quality of life. We review the diagnostic and management issues of patients with platelet function disorders through the presentation of 2 patient case reports, with a focus on a diagnosis of Glanzmann thrombasthenia, an inherited qualitative disorder that affects platelet function. Whereas the first patient presented to the emergency department before the diagnosis of a bleeding disorder and required a hematologic referral and extensive laboratory assessments, the second patient had been diagnosed with Glanzmann thrombasthenia as a child but experienced severe management challenges at the onset of menarche. In both patients, collaboration between the obstetrician-gynecologist or adolescent medicine specialist and the hematologist was critical for achieving acute management of the bleeding symptoms and for ensuring optimal long-term disease management. Together, these cases highlight the importance of properly identifying females with HMB who might have an undiagnosed bleeding disorder and of consulting with a hematologist to determine an appropriate management plan throughout all life stages.

Diagnosis of Glanzmann thrombasthenia by whole blood impedance analyzer (MEA) vs. light transmission aggregometry.

Glanzmann thrombasthenia (GT) is a rare inherited platelet disorder that is characterized by spontaneous or postprocedural bleeding. The diagnosis of GT depends on identifying the dysfunction of the platelets. The aim of this study was to compare a whole blood impedance Multiplate analyzer (MEA) with the standard method, light transmission aggregometry (LTA) in diagnosis of GT. Fifteen patients with GT were assessed on MEA and LTA using arachidonic acid (ASPI: 15 mm), (TRAP: 1 mm), collagen (100 μg/mL), ADP (0.2 mm), and ristocetin (Risto: 10 mg/mL). Whole blood samples were collected in sodium citrate and hirudin vacuum, blood collection tubes and tested within 4 h. Platelet-rich plasma was used for LTA using platelet agonists (ristocetin 1.5 mg/mL) (arachidonic acid 0.5 mg/mL) (ADP 2.5 mg/mL) and (collagen 1 mg/mL). The platelet count and PFA-100 results were (average and SD) 319 ± 93 × 10(9) L and 252 ± 34 s, respectively. Flow cytometry analysis showed that all samples are positive for CD42a and CD42b, whereas 9/15 samples were negative for CD61 and CD41. The other six patients had either partial or full expression of CD61/CD41. Aggregation analysis using both methods showed that all samples had no aggregation response to any of the agonists used apart from six samples which, using only the MEA, showed minimal aggregation in response to collagen (average = 14.3 ± 7 μg, which may suggest ability to detect qualitative abnormality of GPIIb/IIIa). These results suggest that the MEA is sensitive for the detection of Glanzmann thrombasthenia. Furthermore, MEA may also be able to differentiate between the subtypes of Glanzmann thrombasthenia.

Conduta no perioperatório de paciente pediátrico com trombastenia de glanzmann durante adenoidectomia

JUSTIFICATIVA E OBJETIVOS: Trombastenia de Glanzmann (TG) e uma doenca autossomica recessivamente hereditaria das plaquetas. Nao ha nenhum tratamento especifico. A transfusao de plaquetas e atualmente o tratamento padrao quando o sangramento nao responde a medidas locais e/ou a medicamentos antifibrinoliticos, podendo, entretanto, resultar em aloimunizacao. O fator VII recombinante ativado (rFVIIa) pode ser usado para evitar a transfusao recorrente de plaquetas. RELATO DE CASO: Apresentamos um tratamento precoce com dose baixa de rFVIIa associada a transfusao de plaquetas em um caso pediatrico (cinco anos de idade), com diagnostico de TG e apresentando sangramento prolongado durante adenoidectomia eletiva. Uma dose total de 1.200 mg (60 µg.kg-1) de rFVIIa obteve sucesso em estancar o sangramento, o que pode ser aceito como uma dose baixa. CONCLUSOES: Relatos de casos podem encorajar o uso de tratamento precoce com baixas doses de rFVIIa em hemorragias graves que nao estacam a despeito da transfusao de plaquetas e na prevencao de sangramento em procedimentos cirurgicos em pacientes com TG. Estudos adicionais sao necessarios para definir a dose minima eficaz. Portanto, as tentativas para determinar a dose eficaz mais baixa desse composto devem ser incentivadas consideando o resultado deste caso em face de restricoes financeiras no sistema de saude.

Perioperative Management of a Pediatric Patient with Glanzmann's Thrombasthenia during Adenoidectomy

Glanzmann's thrombasthenia (GT) is an autosomal recessively inherited platelet disorder. There is not any specific treatment. Platelet transfusion is currently the standard treatment when bleeding does not respond to local measures and/or antifibrinolytic treatment, although it may result in alloimmunization. Recombinant activated factor VII (rFVIIa) might be used to avoid recurrent platelet transfusion. We present early treatment with low-dose rFVIIa additional to platelet transfusion in a 5-year-old pediatric case with diagnosis of GT who developed prolonged bleeding under an elective adenoidectomy surgery. A total dose of 1,200μg (60μg.kg(-1)) rFVIIa could successfully stop bleeding, what can be accepted as low dose usage. Such case reports may encourage the use of early treatment with low doses of rFVIIa in severe bleeds that did not stop despite of platelet transfusion, as well as in preventing bleeding in surgical procedures in patients with GT. Actually, additional studies are needed to define the minimal effective dose and attempts to determine the lowest effective dose may be encouraged by the result of this case, considering financial restrictions in the health care system.

A novel ELISA for diagnosis of Glanzmann’s thrombasthenia and the heterozygote carriers

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.

A Novel Mutation (Q694X) in the ITGB3 Gene Causing Glanzmann's Thrombasthenia from the Sultanate of Oman

Background: Glanzmann Thrombasthenia (GT) results from mutations in the genes ITGA2B and ITGB3, located on chromosome 17q21–23 which encodes the platelet glycoprotein αIIbβ3 complex, namely GPIIb (αIIb) and GPIIIa (β3), the fibrinogen receptors on platelets, which play an important role in platelet aggregation. Patients with GT can require frequent hospitalization and can be a burden on the nation's health resources. The possibility that GT could be cured by gene replacement therapy makes it essential to study the molecular basis of the GT patients in a particular family or kindred.Objectives: Our aim was to identify the underlying mutations responsible for GT in Omani patients in order to establish a strategy for genetic counseling and carrier detection to prevent the occurrence of the homozygous state by prenatal diagnosis.Methods: GT was diagnosed in a 17 year old Omani female at the Sultan Qaboos University Hospital. The diagnosis of GT was based on clinical features, platelet aggregometry and biochemical studies. Platelet surface expression of GPIIb/IIIa was also studied by flowcytometry. Molecular studies performed at Medical Genetics Department, Tsukuba University, Japan, include DNA sequencing of all exons and exon-intron junctions of ITGA2B and ITGB3 of the two genes by the ABI 3100 Genetic Analyzer®. [Applied Biosystems, Foster City, CA, USA]. Genomic DNA was also analyzed by Illumina Human-1 Bead Chip Illumina® (Illumina Inc., San Diego, CA, USA) to exclude the whole region of the two genes that could produce an apparent homozygous state.Results: We have identified a novel nonsense causative mutation (Q694X) by sequencing the ITGB3 gene. [Figure 1a & b]. In addition, sequencing ITGB3 gene also revealed 2 SNPs (rs 3809863; IVS14+9C/T, rs 3809865; 3383T/A). The Micro-Array assay using Illumina Human-1 Bead chip excluded the possibility of deletion of these genes in chromosome 17 in this patient.Summary/Conclusion: A stop codon was found in exon 13 of ITGB3 gene causing the translated protein to be abnormally shortened. It is hypothesized that the altered form of ITGB3 gene is both extremely unstable and rapidly degraded after its biosynthesis, leading to a loss of function of the protein. Further RNA expression studies, transfection tests and cDNA sequencing are ongoing to elucidate the molecular mechanisms responsible for GT. [Display omitted]

Sustained engraftment post bone marrow transplant despite anti‐platelet antibodies in Glanzmann thrombasthenia

Patients with Glanzmann thrombasthenia (GT) have normal platelet counts but abnormal platelet aggregation and carry the risk of life-threatening bleeding. We report three patients who received bone marrow transplantation (BMT) for type I GT and discuss the risk and management of anti-platelet antibodies. Diagnosis of GT was made through abnormal platelet aggregation studies or the absence of GPIIb/IIIa by flow cytometry. All patients had severe bleeding requiring multiple red blood cell transfusions. One patient received an unrelated donor transplant and two received matched sibling donor transplants following conditioning therapy with busulfan, cyclophosphamide, and fludarabine. Two patients developed an anti-platelet antibody, treated in one with intravenous immune globulin (IVIG). Engraftment of white blood cells and platelets was achieved on day +13 to +14 and +17 to +25, respectively. Complete donor chimerism and GPIIb/IIIa+ platelets are sustained at +22 to +30 months post transplant. In summary, patients with GT and history of severe hemorrhage can be cured with BMT, but the presence of anti-platelet antibodies should be sought and platelet transfusions minimized prior to transplant. IVIG may be helpful in cases of refractory immune thrombocytopenia related to anti-platelet antibodies. Improvement in transplant-related complications with current transplant regimens allows consideration of BMT for life-threatening non-malignant disorders such as GT.

A point mutation in the cysteine-rich domain of glycoprotein (GP) IIIa results in the expression of a GPIIb-IIIa (αIIbβ3) integrin receptor locked in a high-affinity state and a Glanzmann thrombasthenia–like phenotype

AbstractThis article reports a Glanzmann thrombasthenia (GT) patient, N.M., with a point mutation in the third cysteine-rich repeat of β3-integrin or platelet glycoprotein (GP) IIIa, leading to the expression of a constitutively activated fibrinogen receptor. The diagnosis of GT was based on a severely reduced platelet-aggregation response to a series of agonists and approximately 20% of surface-expressed GPIIb-IIIa. The patient's GPIIb-IIIa constitutively expressed epitopes recognized by antibodies to ligand-induced binding sites (LIBS) and also spontaneously bound the fibrinogen-mimetic antibody, PAC-1. Furthermore, significant amounts of bound fibrinogen were detected on his platelets ex vivo. No signs of platelet activation were observed on sections of unstimulated platelets from N.M. by electron microscopy. Immunogold labeling highlighted the presence of surface-bound fibrinogen but revealed platelet heterogeneity with regard to the surface density. When the patient's platelets were stimulated by thrombin-receptor activating peptide, amounts of surface-expressed GPIIb-IIIa increased and the aggregation response improved, although it failed to normalize. Platelets from N.M. were able to adhere and spread on immobilized fibrinogen. Sequence analysis of genomic DNA from N.M. revealed a homozygous g1776T>C mutation in GPIIIa, leading to a Cys560Arg amino acid substitution. A stable Chinese hamster ovary (CHO) cell line was prepared expressing surface GPIIb-Arg560IIIa. Like platelets from the patient, GPIIb-Arg560IIIa–transfected CHO cells constitutively bound LIBS antibodies and PAC-1. They also showed an enhanced ability to adhere on surface-bound fibrinogen. Overall, these data demonstrate that a gain-of-function mutation can still be associated with a thrombasthenic phenotype even though platelets show spontaneous fibrinogen binding.