The utility of flow cytometric platelet forward scatter as an alternative to mean platelet volume.

The use of mean platelet diameter (MPD) to classify inherited thrombocytopenia (IT) has been demonstrated in several studies. Alternatively, the mean platelet volume (MPV) may be used, but in macrothrombocytopenia this may not be available. We hypothesized that platelet forward scatter (FSC) measurements using flow cytometry may be used for the size-based classification of IT. The study aimed to assess the ability of platelet FSC to measure platelet size and whether it could be used as an alternative to the MPD or MPV.Blood samples were obtained from individuals undergoing investigation for inherited platelet function disorders (IPFD, n = 40) or platelet number disorders (IPND, n = 46). A hematology analyzer was used to obtain MPV and platelet counts, flow cytometry to measure platelet FSC and ImageJ software to measure MPD from stained blood smears. The International Society of Thrombosis and Hemostasis (ISTH) Bleeding Assessment Tool (BAT) was used to calculate bleeding scores.Twenty-nine(63%) of IPND patients had an MPV that could not be reported. A significant correlation to platelet FSC was found to the MPD (p < .0001) and MPV (p < .0001) and an inverse correlation with platelet count (p < .0001). No significant correlation was found between FSC and bleeding history. In conclusion, platelet FSC is an alternative to MPV and may be used in macrothrombocytopenia where the MPV is not recorded.

Diagnostic work up of patients with increased bleeding tendency.

INTRODUCTION: The diagnostic trajectory of patients with increased bleeding tendency can be very costly and time-consuming. In addition, previous studies have shown that half of these patients remain without final diagnosis despite all efforts. AIM: This study aimed to improve insight into the current diagnostic process of these patients. METHODS: A total of 117 adult patients, referred to an academic hospital because of being suspected to have an increased bleeding tendency, were included. Different parameters were compared between patients receiving final diagnosis, patients without final diagnosis but a high Tosetto bleeding assessment tool (BAT) score (classified as bleeding of unknown cause, or BUC) and a control group consisting of patients without final diagnosis and a low BAT score. RESULTS: The BAT score was significantly higher in patients in the BUC group as compared to patients reaching final diagnosis (8.1 vs 4.9). Interestingly, the two subcategories most prevalently increased were surgery and post-partum haemorrhage-associated bleeding (surgery: 2.1 vs 1.1; post-partum haemorrhage: 0.7 vs 0.0). Laboratory screening results were more often abnormal in patients reaching final diagnosis compared to patients remaining without diagnosis and a high BAT score (n = 32 (78%) vs n = 14 (46%), 95% CI 1.5-12), especially concerning the PFA (=27 (66%) vs n = 10 (33%), 95% CI 1.4-10) and von Willebrand factor activity levels (n = 11 (27%) vs n = 1 (3%), 95% CI 1.3-91). CONCLUSION: Isolated high bleeding score on surgical or post-partum bleeding correlates with a lower chance of receiving final diagnosis. Withholding extensive haemostatic testing should be considered. Better screening and confirmative haemostatic assays are still needed.

Severity and Features of Epistaxis in Children with a Mucocutaneous Bleeding Disorder.

OBJECTIVE: To use standardized bleeding questionnaires to compare the severity and patterns of epistaxis in children with a mucocutaneous bleeding disorder and control children. STUDY DESIGN: The epistaxis sections of the Pediatric Bleeding Questionnaire (PBQ) administered to pediatric patients with von Willebrand disease or a platelet function disorder and healthy control children were reviewed. Scores and features of epistaxis (frequency, duration, onset, site, seasonal correlation, and need for medical/surgical intervention) were recorded. A PBQ epistaxis score ≥2 was defined as clinically significant. The Katsanis epistaxis scoring system was administered to eligible patients, ie, with ≥5 episodes of epistaxis per year. RESULTS: PBQ epistaxis scores were obtained for 66 patients, median age 12 years (range 0.6-18.3 years), and 56 control children. The median PBQ epistaxis score in patients was 2 vs 0 in control children (P <.0001). All of the features of epistaxis, except spontaneous onset, occurred in a significantly greater proportion of patients than control children with epistaxis. A total of 50% of the patients were graded as having severe epistaxis by the Katsanis epistaxis scoring system, and 30 of these (91%) had a clinically significant PBQ epistaxis score. CONCLUSION: Standardized bleeding questionnaires are useful in the assessment of epistaxis severity and pattern and may help to distinguish children with and without a mucocutaneous bleeding disorder.

Challenges on the diagnostic approach of inherited platelet function disorders: Is a paradigm change necessary?

Inherited platelet function disorders (IPFD) have been assessed for more than 50 years by aggregation- and secretion-based tests. Several decision trees are available intending to standardize the investigation of IPFD. A large variability of approaches is still in use among the laboratories across the world. In spite of costly and lengthy laboratory evaluation, the results have been found inconclusive or negative in a significant part of patients having bleeding manifestations. Molecular investigation of newly identified IPFD has recently contributed to a better understanding of the complexity of platelet function. Once considered "classic" IPFDs, Glanzmann thrombasthenia and Bernard-Soulier syndrome have each had their pathophysiology reassessed and their diagnosis made more precise and informative. Megakaryopoiesis, platelet formation, and function have been found tightly interlinked, with several genes being involved in both inherited thrombocytopenias and impaired platelet function. Moreover, genetic approaches have moved from being used as confirmatory diagnostic tests to being tools for identification of genetic variants associated with bleeding disorders, even in the absence of a clear phenotype in functional testing. In this study, we aim to address some limits of the conventional tests used for the diagnosis of IPFD, and to highlight the potential contribution of recent molecular tools and opportunities to rethink the way we should approach the investigation of IPFD.

The Role of Platelets and ε-Aminocaproic Acid in Arthrogryposis, Renal Dysfunction, and Cholestasis (ARC) Syndrome Associated Hemorrhage.

Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is a rare disorder associated with platelet abnormalities resembling gray platelet syndrome. Affected patients have normal platelet numbers but abnormal morphology and function. Bleeding symptomatology ranges from postprocedural to spontaneous life-threatening hemorrhage. We report a patient with ARC syndrome and compound heterozygous mutations in VPS33B (vacuolar protein sorting 33B) who presented with significant bleeding requiring numerous admissions and transfusions. She was treated with prophylactic platelet transfusions and ε-aminocaproic acid. This was well-tolerated and significantly decreased transfusion requirements and admissions for bleeding. Our experience provides support for consideration of prophylactic measures in these patients as well as the possibility of using prophylaxis in related disorders.

Haemostatic disorders in reproductive age women with menorrhagia and effects on quality of life.

The aim of this study was to determine the frequency of haemostatic abnormalities in women with menorrhagia and to evaluate their effect on quality of life (QoL). The study population was composed of patients with menorrhagia seen in the outpatient clinic, having a score of >185 with a pictorial blood assessment chart. Structured questionnaires were used in the assessment of demographic characteristics and QoL, and patients were tested for bleeding disorders. Ninety women were recruited for the study. Bleeding disorders were detected in 40% of them: 11.1% had von Willebrand disease, 2.2% had low von Willebrand factor and 26.7% had platelet function disorders (PFD). In 22 (91.6%) cases with PFD the, defect was non-specific and impaired aggregation response to ristocetine (37.5%) was the most commonly detected problem. Bleeding disorders were not associated with any significant reduction in QoL (p > .05). Hereditary bleeding disorders may be the cause of unexplained menorrhagia even in the middle-aged women, but they had no prominent effect on QoL.

Clinical Dilemma, Bernard Soulier Syndrome versus Immune Thrombocytopenic Purpura: A Case Report.

BACKGROUND: Bernard Soulier Syndrome (BSS) is a rare autosomal recessive disorder due to deficiency or dysfunction of the glycoprotein GPIb-V-IX complex on the platelet surface. It is also known as hemorrhagiparous thrombocytic dystrophy or congenital hemorrhagiparous thrombocytic dystrophy. The patient usually presents with severe and prolonged bleeding along with characteristics of giant blood platelets and low platelet counts. Manifestations of BSS include epistaxis, gum bleeding, purpuric rashes, menorrhagia, rarely melena, and hematemesis. On the other hand, immune thrombocytopenic purpura (ITP) is an acquired autoimmune disorder in which there is accelerated platelet destruction and reduced platelet production. Isolated thrombocytopenia without fever, lymphadenopathy, and organomegaly usually lead to the diagnosis of immune thrombocytopenia. CASE PRESENTATION: A 20 years old female presented with complaints of recurrent episodes of epistaxis since childhood and menorrhagia during menarche. She was misdiagnosed as ITP elsewhere. Later, based on thorough clinical examination and investigation, the diagnosis was confirmed as BSS. CONCLUSION: BSS should always be taken in the differential diagnosis of ITP, especially when persistent, refractory, and treated unsuccessfully with steroids or splenectomy.

A new decade awaits sticky platelet syndrome: where are we now, how do we manage and what are the complications?

INTRODUCTION: Sticky platelet syndrome is a less known platelet function disorder with a familiar occurrence and likely genetic background. Clinically, it is characterized by an increased risk of venous and arterial thromboembolic events and obstetric placenta-mediated complications. The increased aggregation after low-dose ADP and/or epinephrine is its distinctive laboratory feature. Though described for almost 40 years, several issues regarding its etiology, involved pathomechanisms, genetic background, optimal diagnostic and treatment approach remain controversial. AREAS COVERED: The work aims to summarize published studies, the actual definition of the syndrome, and point out its drawbacks. A literature search on Medline, Embase, and archives from EHA congresses was performed (terms: 'sticky platelet syndrome' - 'platelet hyperreactivity' - 'platelet hyperaggregability'). The authors added in their unpublished data. The introductory overview of the present understanding is followed by the discussion of the pathophysiologic, diagnostic, and therapeutic problems. EXPERT OPINION: Despite the growing evidence provided by case reports and series, the lack of robust studies limits the decision-making on diagnostics and management. The diagnostic issues, particularly the standardization of light transmission aggregometry, represent the crucial problem for the broader acceptance of the syndrome.

PLAIN LANGUAGE SUMMARY Sticky platelet syndrome is aplatelet function disorder. It is associated with an increased risk of venous thromboembolism, arterial thrombosis, and obstetric placenta-mediated complications. Increased aggregation after low-dose ADP and/or epinephrine is the defining laboratory feature. Furthermore, afew studies report the familiar occurrence with possible genetic background. Several issues regarding the syndrome remain controversial: its exact etiology, genetics, optimal diagnostic, and treatment approach. These uncertainties provide ground for debate of the syndrome as aunique clinical entity. The review has two goals. Firstly, it summarizes the published studies and the actual definition of the syndrome. Secondly, it tries to point out the open pathophysiologic, diagnostic, and therapeutic problems.

Approach to the Patient with Bleeding.

Approach to the patient with bleeding begins with a thorough bleeding, medical, and family history to determine the nature of bleeding and severity of bleeding symptoms. Use of a Bleeding Assessment Tool allows the clinician to obtain a comprehensive bleeding history and ultimately determine the individual bleeding score that reflects bleeding severity and is classifiable as either normal or abnormal. In the absence of significant findings within patient history or presenting symptoms clearly pointing to a specific bleeding pathology, an approach to laboratory investigation is presented that proceeds through first-line, second-line, and third-line testing.

Bernard-Soulier syndrome or idiopathic thrombocytopenic purpura: A case series.

BACKGROUND: Bernard-Soulier syndrome (BSS) is a rare, autosomal recessive platelet function disorder which is commonly mistaken for idiopathic thrombocytopenic purpura (ITP).The report includes seven cases of BSS that have been diagnosed and treated as ITP for a long time. METHODS: Between 2006 and 2016, data of seven BSS patients who have long been diagnosed and treated as ITP were collected and analyzed. RESULTS: Two patients were males and 5 were females. The patient's age range was between one day and four years at the onset of symptoms. Easy bruising, nose bleeds and mucocutaneous bleeding were the most frequent symptoms. Bleeding attacks of the gum, gastrointestinal tract and menorrhagia also occurred and in one case bleeding in the injection site of the first vaccination was reported. In 6 patients, parents were relatives and in three cases, there was a family history of low platelet counts. Variable thrombocytopenia, prolonged bleeding time (BT), and large platelets with increased bone marrow megakaryocyte were seen in all cases. Most patients were treated with steroids, Intravenous immunoglobulin (IVIG), and some with IV anti-D, Azathioprine, Danazol, Rituximab. Splenectomy was performed in one case. In supplementary tests the platelet aggregation to ristocetin was absent and GPIb expression level by flow cytometry method was lower than 10%. CONCLUSION: BSS should always be considered in differential diagnosis of ITP especially in persistent and refractory ITP.

Use of Targeted High-Throughput Sequencing for Genetic Classification of Patients with Bleeding Diathesis and Suspected Platelet Disorder.

Inherited platelet disorders (IPD) form a rare and heterogeneous disease entity that is present in about 8% of patients with non-acquired bleeding diathesis. Identification of the defective cellular pathway is an important criterion for stratifying the patient's individual risk profile and for choosing personalized therapeutic options. While costs of high-throughput sequencing technologies have rapidly declined over the last decade, molecular genetic diagnosis of bleeding and platelet disorders is getting more and more suitable within the diagnostic algorithms. In this study, we developed, verified, and evaluated a targeted, panel-based next-generation sequencing approach comprising 59 genes associated with IPD for a cohort of 38 patients with a history of recurrent bleeding episodes and functionally suspected, but so far genetically undefined IPD. DNA samples from five patients with genetically defined IPD with disease-causing variants in WAS , RBM8A , FERMT3 , P2YR12 , and MYH9 served as controls during the validation process. In 40% of 35 patients analyzed, we were able to finally detect 15 variants, eight of which were novel, in 11 genes, ACTN1 , AP3B1 , GFI1B , HPS1 , HPS4 , HPS6 , MPL , MYH9 , TBXA2R , TPM4 , and TUBB1 , and classified them according to current guidelines. Apart from seven variants of uncertain significance in 11% of patients, nine variants were classified as likely pathogenic or pathogenic providing a molecular diagnosis for 26% of patients. This report also emphasizes on potentials and pitfalls of this tool and prospectively proposes its rational implementation within the diagnostic algorithms of IPD.

Recognition and management of platelet-refractory bleeding in patients with Glanzmann's thrombasthenia and other severe platelet function disorders.

Patients with rare qualitative platelet disorders or platelet function disorders (PFDs) may present to the hospital physician with severe bleeding episodes or excessive surgical bleeding. Although standard treatment consists of platelet transfusions, repeated transfusions may result in the development of antiplatelet antibodies (APA) or clinical refractoriness, rendering further platelet therapy ineffective. In such settings, an approved treatment option for patients with Glanzmann's thrombasthenia (GT), one of the well-known rare PFDs, is recombinant activated coagulation factor VII (rFVIIa). Data regarding the efficacy of rFVIIa in patients with GT and platelet refractoriness are available from a large patient registry, an international survey, and multiple case reports and demonstrate efficacy in patients with and without refractoriness or APA. This article reviews the rFVIIa clinical data in patients with GT and platelet refractoriness and discusses clinical implications relevant to the hospital-based physician. Because uncontrolled bleeding can be life-threatening, hospital physicians should be alert to the signs of platelet refractoriness, be able to recognize continued internal or external bleeding, and know how to adapt treatment regimens for the effective management of bleeding. The management of patients who receive rFVIIa should occur in consultation with a hematologist with experience in PFDs, and patients with suspected platelet refractoriness should be referred to such a hematologist as early as possible. A critical unmet need is the development of a definition of an adequate response to platelet transfusion, which would facilitate early recognition of platelet refractoriness in patients with PFDs who exhibit a normal platelet count.

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.

Current and emerging approaches for evaluating platelet disorders.

Platelets play a central role in physiological hemostasis and also in pathological thrombosis. It is well established that congenital or acquired abnormalities of platelet function are associated with a heightened risk of bleeding of variable severity and excessive hemorrhage after surgery or trauma. Several kinds of different platelet function tests have been developed over the years to identify or diagnose platelet function disorders. The use of these tests for the assessment of thrombotic risk or for monitoring the effects of drugs inhibiting platelet function is not well established. Light transmission aggregometry (LTA) is the gold standard for the study of patients with defects of platelet function. Its results are affected by several pre-analytical and analytical variables. The Subcommittee on Platelet Physiology of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis published official guidelines for the standardization of the variables affecting LTA, which should be followed to harmonize the procedures across different laboratories worldwide. The lumi-aggregometer, a modification of LTA that measures platelet secretion in parallel with aggregation, is preferable to LTA for diagnosing inherited defects of platelet function, because it is more sensitive to the most common disorders, which are characterized by abnormalities of platelet secretion. LTA (or lumi-aggregometry) is useful as a first screening test of patients with the clinical suspicion of defects of platelet function, because it helps to provide an interim diagnostic hypothesis, which can then be confirmed or discounted using appropriate and specific tests.

The evolution and value of bleeding assessment tools.

A personal history of excessive mucocutaneous bleeding is a key component in the diagnosis of a number of mild bleeding disorders, including von Willebrand disease (VWD), platelet function disorders (PFD), and coagulation factor deficiencies. However, the evaluation of hemorrhagic symptoms is a well-recognized challenge for both patients and physicians, because the reporting and interpretation of bleeding symptoms is subjective. As a result, bleeding assessment tools (BATs) have been developed and studied in a variety of clinical settings. This work has been pioneered by a group of Italian researchers, and the resultant 'Vicenza Bleeding Questionnaire' stands as the original BAT. In this review, we will discuss the modifications of the Vicenza Bleeding Questionnaire that have taken place over the years, as well as the validation studies that have been published. Other BATs that have been developed and published will be reviewed, as will the special situations of assessing pediatric bleeding as well as menorrhagia. Lastly, the clinical utility of BATs will be discussed including remaining challenges and future directions for the field.