Molecular phenotype and bleeding risks of an inherited platelet disorder in a family with a RUNX1 frameshift mutation.

INTRODUCTION: Inherited defects in RUNX1 are important causes of platelet function disorders. AIM: Our goals were to evaluate RUNX1-related platelet disorders among individuals evaluated for uncharacterized, inherited platelet function disorders and test a proof of concept that bleeding risks could be quantitatively estimated for typical families with an inherited platelet function disorder. METHODS: Index cases with an uncharacterized inherited platelet function disorder were subjected to exome sequencing with confirmation of RUNX1 mutations by Sanger sequencing. Laboratory findings were obtained from medical records and persistence of platelet non-muscle myosin heavy chain IIB (MYH10), a biomarker of RUNX1 defects, was assessed by Western blotting. Bleeding histories were assessed using standardized assessment tools. Bleeding risks were estimated as odds ratios (OR) using questionnaire data for affected individuals compared to controls. RESULTS: Among 12 index cases who had their exomes sequenced, one individual from a family with eight study participants had a c.583dup in RUNX1 that segregated with the disease and was predicted to cause a frameshift and RUNX1 haploinsufficiency. Unlike unaffected family members (n = 2), affected family members (n = 6) had increased bleeding scores and abnormal platelet aggregation and dense granule release responses to agonists but only some had thrombocytopenia and/or dense granule deficiency. This family's mutation was associated with persistence of MYH10 in platelets and increased risks (OR 11-440) for wound healing problems and mild bleeding symptoms, including bleeding interfering with lifestyle in women. CONCLUSION: Inherited platelet dysfunction due to a RUNX1 haploinsufficiency mutation significantly increases bleeding risks.

Genomic approaches to bleeding disorders.

The genes encoding the coagulation factors were characterized over two decades ago. Since then, significant progress has been made in the genetic diagnosis of the two commonest severe inherited bleeding disorders, haemophilia A and B. Experience with the genetic of inherited rare bleeding disorders and platelet disorders is less well advanced. Rare bleeding disorders are usually inherited as autosomal recessive disorders, while it is now clear that a number of the more common platelet function disorders are inherited as autosomal dominant traits. In both cases, DNA sequencing has been useful since most of these disorders are due to mutations located at the coding regions or splice sites of genes encoding the abnormal protein. However, in 5-10% of patients affected with severe clotting factor deficiencies, no genetic defect can be identified and until recently, the genetic characterization of inherited platelet disorders had been confined to the more prevalent conditions such as Glanzmann disease and Bernard-Soulier syndrome. In patients with no gene mutations identified, so far, the role of next-generation sequencing as well as of other new genomic technologies will very likely have increasing importance. However, such methods require extensive bioinformatics analysis that, in turn will require critical revision of our current diagnostic infrastructure.

Laboratory issues in bleeding disorders.

Selected laboratory issues critical for the appropriate diagnosis of haemophilia A and B, von Willebrand's disease (VWD) and more rare bleeding disorders (RBD) are discussed from a worldwide perspective. The overall picture that emerges is on the whole reassuring. Even in non-Western countries like Latin America, most cases of haemophilia are appropriately diagnosed. Moreover, national and international laboratory training workshops are further improving the diagnostic capabilities also in less severe disorders. Most of the RBD can be appropriately diagnosed with relatively simple tests wherever a high clinical suspicion is present. Moreover, minimal requirements for a useful clinical diagnosis are not too far from the capabilities of majority of non-Western countries. The most needed areas concern VWD and platelet function disorders, which suffer from inadequate diagnostic standardization, hampering widespread diagnostic capability in both Western and non-Western countries.

How I investigate for bleeding disorders.

INTRODUCTION: Laboratory investigations for bleeding disorders are warranted when an individual has a personal and/or family history of bleeding, and/or laboratory findings that suggest the possibility of an inherited or acquired bleeding disorder. METHODS: This review summarizes author's experience with ordering and reporting on diagnostic investigations for common and rare bleeding disorders, with consideration of recent articles on diagnosing bleeding disorders. An updated strategy is presented for investigating common and rare, congenital and acquired bleeding disorders. RESULTS: An investigation of a suspected bleeding disorder requires a practical strategy that considers the clinical problem to be investigated, the pretest probability of true-positive and false-positive findings, the investigations can be performed locally or in a reference laboratory and limit the number of blood samples required to establish a diagnosis. It is often advantageous to simultaneously test for von Willebrand disease and platelet function disorders, and for coagulation defects, including fibrinogen disorders. An investigation for rarer bleeding disorders, including those affecting factor XIII, α2 antiplasmin, and plasminogen activator inhibitor-1, is appropriate when faced with a severe congenital or acquired bleeding problem that cannot be explained by the initial diagnostic investigations. CONCLUSION: An organized strategy for investigating bleeding disorders that consider important issues, confirms abnormal findings, encourages proper interpretation of the results, and provides a helpful framework for assessing both common and rare causes of bleeding.

Electron microscopy examination of platelet whole mount preparations to quantitate platelet dense granule numbers: Implications for diagnosing suspected platelet function disorders due to dense granule deficiency.

INTRODUCTION: Dense granule (DG) deficiency (DGD) is a feature of some platelet function disorders (PFD) with a prevalence similar to von Willebrand disease. Most laboratories assess for DGD using whole mount platelet preparations and electron microscopy (EM). We evaluated our experiences with this test and associations between DGD and bleeding. METHODS: Dense granule EM records for 2006-2017 were examined for patients and simultaneously tested controls, and for an overlapping PFD study cohort to evaluate findings and their relationship to bleeding. RESULTS: More patient than control samples had reduced DG counts (6.5% vs 0.3%, P < .01). DG counts showed no relationship to age or mean platelet volume and had acceptable within-subject variability that was higher for DGD than control participants (28% vs 12%). Repeat tests confirmed DGD in all persons with initial DG counts <4.0/platelet, but not in those with less severe reductions (4.0-4.8 DG/platelet) or normal DG counts (≥4.9 DG/platelet). Aggregometry and adenosine triphosphate release tests, respectively, had only ~52% and 70% sensitivity for DGD. Confirmed DGD by EM was associated with higher bleeding scores and a bleeding disorder. CONCLUSION: Whole mount EM is useful for the evaluation of suspected PFD due to DGD and detects abnormalities associated with bleeding.

Multimerin is found in the alpha-granules of resting platelets and is synthesized by a megakaryocytic cell line.

In this report, we describe the intracellular localization of multimerin in platelets and its biosynthesis by Dami cells, a megakaryocytic cell line. Immunoelectron microscopy was used to examine frozen thin sections of resting and activated platelets. Multimerin was localized within the platelet alpha-granule in an eccentric position. Within activated platelets, multimerin was found in the surface-connected open cannalicular system and on the external plasma membrane. Light microscopic immunocytochemistry demonstrated multimerin in normal megakaryocytes and in Dami cells after stimulation with PMA. Confirmation of multimerin biosynthesis by Dami cells was obtained by metabolic labeling studies. Both platelet and Dami cell multimerin demonstrated several subunit sizes on reduced SDS-PAGE. However, peptide mapping confirmed structural homology between the different multimerin subunits. Glycosidase digestion demonstrated that multimerin is heavily glycosylated with mainly complex, N-linked carbohydrate. In contrast to the multimerin isolated from platelets, cultured Dami cells secreted mainly smaller multimers of the protein. Biosynthesis of multimerin by a megakaryocytic cell line supports endogenous biosynthesis by megakaryocytes as the origin of this platelet alpha-granule protein.

Platelet function analyzer (PFA)-100 closure time in the evaluation of platelet disorders and platelet function.

BACKGROUND: Closure time (CT), measured by platelet function analyzer (PFA-100) device, is now available to the clinical laboratory as a possible alternative or supplement to the bleeding time test. AIM: On behalf of the Platelet Physiology Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH-SSC), a working Group was formed to review and make recommendations on the use of the PFA-100 CT in the evaluation of platelet function within the clinical laboratory. METHODS: The Medline database was searched to review the published information on the PFA-100 CT in the evaluation of platelet disorders and platelet function. This information, and expert opinion, was used to prepare a report and generate consensus recommendations. RESULTS: Although the PFA-100 CT is abnormal in some forms of platelet disorders, the test does not have sufficient sensitivity or specificity to be used as a screening tool for platelet disorders. A role of the PFA-100 CT in therapeutic monitoring of platelet function remains to be established. CONCLUSIONS: The PFA-100 closure time should be considered optional in the evaluation of platelet disorders and function, and its use in therapeutic monitoring of platelet function is currently best restricted to research studies and prospective clinical trials.

Insights into abnormal hemostasis in the Quebec platelet disorder from analyses of clot lysis.

BACKGROUND: The Quebec platelet disorder (QPD) is inherited and characterized by delayed-onset bleeding following hemostatic challenge. Other characteristics include increased expression and storage of active urokinase-type plasminogen activator (u-PA) in platelets in the setting of normal to increased u-PA in plasma. There is also consumption of platelet plasminogen activator inhibitor-1 and increased generation of plasmin in platelets accompanied by proteolysis of stored alpha-granule proteins, including Factor V. AIMS AND METHODS: Although fibrinolysis has been proposed to contribute to QPD bleeding, the effects of QPD blood and platelets on clot lysis have not been evaluated. We used thromboelastography (TEG), biochemical evaluations of whole blood clot lysis, assessments of clot ultrastructure, and perfusion of blood over preformed fibrin to gain insights into the disturbed hemostasis in the QPD. RESULTS: Thromboelastography was not sensitive to the increased u-PA in QPD blood. However, there was abnormal plasmin generation in QPD whole blood clots, generated at low shear, with biochemical evidence of increased fibrinolysis. The incorporation of QPD platelets into a forming clot led to progressive disruption of fibrin and platelet aggregates unless drugs were added to inhibit plasmin. In whole blood perfusion studies, QPD platelets showed normal adherence to fibrin, but their adhesion was followed by accelerated fibrinolysis. CONCLUSIONS: The QPD is associated with "gain-of-function" abnormalities that increase the lysis of forming or preformed clots. These findings suggest accelerated fibrinolysis is an important contributor to QPD bleeding.

Report on the International Society for Laboratory Hematology Survey on guidelines to support clinical hematology laboratory practice.

INTRODUCTION: Given the importance of evidence-based guidelines in health care, we surveyed the laboratory hematology community to determine their opinions on guideline development and their experience and interest in developing clinical hematology laboratory practice guidelines. METHODS: The study was conducted using an online survey, distributed to members of the International Society for Laboratory Hematology (ISLH) in 2015, with analysis of collected, anonymized responses. RESULTS: A total of 245 individuals participated. Most worked in clinical and/or research laboratories (83%) or industry (11%). 42% felt there were gaps in current guidelines. The majority (58%) recommended that ISLH engages its membership in guideline development. Participants differed in their familiarity with, and use of, different organizations' guidelines. Participants felt it was important to follow best practice recommendations on guideline development, including engagement of experts, statement about conflict of interests and how they were managed, systematic review and grading evidence for recommendations, identifying recommendations lacking evidence or consensus, and public input and peer review of the guideline. Moreover, it was considered important to provide guidelines free of charge. Industry involvement in guidelines was considered less important. CONCLUSIONS: The clinical laboratory hematology community has high expectations of laboratory practice guidelines that are consistent with recent recommendations on evidence-based guideline development.

Variability in platelet dense granule adenosine triphosphate release findings amongst patients tested multiple times as part of an assessment for a bleeding disorder.

INTRODUCTION: Lumi-aggregometry quantification of platelet dense granule adenosine triphosphate (ATP) release is commonly used for diagnosing platelet function disorders. As the test findings show considerable variability for healthy controls, we postulated that patient findings might also be variable and investigated patients who were assessed for dense granule ATP release defects more than once. METHODS: Analyses were performed on prospectively collected data for first and second tests for subjects tested for dense granule ATP release defects more than once by the Hamilton Regional Laboratory Program (HRLMP) between January 2007 and June 2013 (cohort I). Similar analyses were performed for subjects who were recruited to a platelet disorder study (cohort II) and were assessed for ATP release defects more than once before October 2015. RESULTS: A total of 150 unique subjects had multiple ATP release tests. Results with individual agonists were variable for many subjects. While normal findings with all tested agonists were often confirmed by the second test (cohort I: 83%; cohort II: 100%), impaired release with multiple agonists was confirmed in only some subjects (cohort I: 34%; cohort II: 54%). Inconsistent findings were common (cohort I: 36%; cohort II: 39%). ISTH bleeding scores showed no relationship to the test findings. The finding of impaired ATP release with 2 or more agonists on both tests was not associated with an increased likelihood of a definite bleeding disorder. CONCLUSION: The variability in platelet dense granule ATP release findings amongst patients assessed for diagnostic purposes suggests that the test has limited value for diagnosing platelet disorders.

Treatment outcomes in patients with adult thrombotic thrombocytopenic purpura-hemolytic uremic syndrome.

BACKGROUND: Plasma treatment has improved the outcomes in adults with thrombotic thrombocytopenic purpura (TTP)-hemolytic uremic syndrome (HUS). We reviewed our experience in treating unselected patients to determine the clinical outcomes and to evaluate the treatments given in addition to plasma. METHODS: A chart review of all cases of TTP and HUS in adults treated at the Toronto (Ontario) Hospital, the largest treatment center for adults with TTP-HUS in the province of Ontario, was conducted. RESULTS: Sixty-seven episodes of TTP-HUS in 52 consecutive adult patients were treated during a 12-year period. Plasma was the primary form of therapy, and most patients received plasma exchange. A complete hematologic remission was achieved in 65 of 67 episodes; however, two patients in remission were brain-dead. The time to complete remission varied from 3 to 58 days (median, 13 days). The death rate during the acute illness was 8%. Long-term sequelae included relapses, persisting renal impairment, hepatitis, and transfusion-associated acquired immunodeficiency syndrome. Relapses occurred in 21% of patients during a median follow-up of 1.1 years (range, 0.1 to 18 years). Analyses of the treatment given in addition to plasma did not demonstrate a significant benefit in terms of reducing the illness duration, mortality, or long-term sequelae. CONCLUSION: While most patients recovered from TTP-HUS, deaths still occurred and many patients suffered long-term complications. The role of the treatments given in addition to plasma is uncertain.

Assembly and evaluation of an inventory of guidelines that are available to support clinical hematology laboratory practice.

INTRODUCTION: Practice guidelines provide helpful support for clinical laboratories. Our goal was to assemble an inventory of publically listed guidelines on hematology laboratory topics, to create a resource for laboratories and for assessing gaps in practice-focused guidelines. METHODS: PubMed and website searches were conducted to assemble an inventory of hematology laboratory-focused guidelines. Exclusions included annual, technical, or collaborative study reports, clinically focused guidelines, position papers, nomenclature, and calibration documents. RESULTS: Sixty-eight guidelines were identified on hematology laboratory practice topics from 12 organizations, some as joint guidelines. The median year of publication was 2010 and 15% were >10 years old. Coagulation topics had the largest numbers of guidelines, whereas some areas of practice had few guidelines. A minority of guidelines showed evidence of periodic updates, as some organizations did not remove or identify outdated guidelines. CONCLUSIONS: This inventory of current practice guidelines will encourage awareness and uptake of guideline recommendations by the worldwide hematology laboratory community, with the International Society for Laboratory Hematology facilitating ongoing updates. There is a need to encourage best guideline development practices, to ensure that hematology laboratory community has current, high-quality, and evidence-based practice guidelines that cover the full scope of hematology laboratory practice.

Investigation of human platelet alloantigens and glycoproteins using non-radioactive immunoprecipitation.

Sensitive techniques to detect platelet antibodies are needed for the investigation of immune thrombocytopenic syndromes such as neonatal alloimmune thrombocytopenia and post-transfusion purpura. Radioimmunoprecipitation has proved useful in the investigation of platelet-antibody interactions; however, the requirement for a radioactive label is a disadvantage. We describe the immunoprecipitation of human platelet proteins labelled with nonradioactive NHSS-biotin and compare the results with proteins labelled with 125I. The efficiency of labelling was evaluated by immunoprecipitation using well-characterized human anti-platelet antisera and murine monoclonal antibodies. The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose and detected using streptavidin-horseradish peroxidase and a chemiluminescent substrate with exposure to X ray film. The biotinylation technique labelled glycoproteins Ia/IIa, Ib/IX, IIb/IIIa, IV, and p175 which carry all of the known platelet alloantigens and isoantigens. It was as sensitive as radiolabelling and had the advantage of labelling GPs Ib beta and IX, which were not labelled using radioiodine. Human sera containing alloantibodies to HPA-1a on GP IIIa, HPA-3a on GP IIb, HPA-5a and HPA-5b on GP Ia, Govb on p175, and the isoantibody Naka on GP IV precipitated the corresponding biotinylated proteins. Biotinylated proteins could be detected using a 30 s exposure compared to 2 days or longer for 125I. Immunoprecipitation of human platelet glycoproteins labeled with NHSS-biotin is a fast and sensitive alternative to conventional radioimmunoprecipitation for the study of human platelet antigens.

Thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura is an uncommon disorder, but it continues to be of considerable interest. The disease mechanisms are unclear and the aetiology is unknown. Perhaps most enigmatic of all, the mode of action of plasma therapy, which successfully induces remission in about two-thirds of cases, is wholly inexplicable. There are currently several areas of debate on the subject of thrombotic thrombocytopenic purpura. This paper addresses these points of contention: the definition of the disease, its distinction from haemolytic uraemic syndrome, the nature of the platelet aggregating factors in the plasma of patients with acute disease, the importance of the abnormalities of von Willebrand's factor observed in the acute and quiescent phases of the disease, the nature of the factor in normal plasma that induces remission, and the possible causes of the observed superiority of plasma exchange combined with plasma infusion, over plasma infusion alone.

Technological advances in diagnostic testing for von Willebrand disease: new approaches and challenges.

Diagnostic tests for von Willebrand disease (VWD) are important for the assessment of VWD, which is a commonly encountered bleeding disorder worldwide. Technical innovations have been applied to improve the precision and lower limit of detection of von Willebrand factor (VWF) assays, including the ristocetin cofactor activity assay (VWF:RCo) that uses the antibiotic ristocetin to induce plasma VWF binding to glycoprotein (GP) IbIXV on target platelets. VWF-collagen-binding assays, depending on the type of collagen used, can improve the detection of forms of VWD with high molecular weight VWF multimer loss, although the best method is debatable. A number of innovations have been applied to VWF:RCo (which is commonly performed on an aggregometer), including replacing the target platelets with immobilized GPIbα, and quantification by an enzyme-linked immunosorbent assay (ELISA), immunoturbidimetric, or chemiluminescent end-point. Some common polymorphisms in the VWF gene that do not cause bleeding are associated with falsely low VWF activity by ristocetin-dependent methods. To overcome the need for ristocetin, some new VWF activity assays use gain-of-function GPIbα mutants that bind VWF without the need for ristocetin, with an improved precision and lower limit of detection than measuring VWF:RCo by aggregometry. ELISA of VWF binding to mutated GPIbα shows promise as a method to identify gain-of-function defects from type 2B VWD. The performance characteristics of many new VWF activity assays suggest that the detection of VWD, and monitoring of VWD therapy, by clinical laboratories could be improved through adopting newer generation VWF assays.

Evaluation of an automated method for measuring von Willebrand factor activity in clinical samples without ristocetin.

INTRODUCTION: The development of an automated, von Willebrand factor (VWF) activity assay, Innovance(®) VWF Ac (VWF:Ac), which measures VWF binding to the platelet receptor glycoprotein Ibα without ristocetin, led us to evaluate the assay for diagnosing von Willebrand disease (VWD) and monitoring therapy. METHODS: After validating that the assay could be performed on an instrument from a different manufacturer, we compared VWF:Ac to VWF ristocetin cofactor activity (VWF:RCo) findings, including ratios of activity/antigen, for 100 healthy controls and 262 consecutive clinical samples from 217 patients (197 adults, 64 children, n = 1 age unknown) referred for VWF testing. RESULTS: There was excellent correlation (R(2) = 0.96) between VWF:Ac results run at two different sites on two different instruments. VWF:Ac had greater precision and sensitivity to low levels of VWF than the VWF:RCo method. Although there was good correlation between VWF:Ac and VWF:RCo results among healthy controls and patient subjects, VWF:Ac results were undetectable and/or significantly lower than VWF:RCo among patients who had types 2A, 2B, or 2M VWD. Additionally, a higher proportion of patient samples were classified as showing qualitative defects using the VWF:Ac compared with VWF:RCo method. While most samples drawn on VWD therapy had similar VWF levels by VWF:Ac and VWF:RCo, a type 2B VWD subject on replacement had much lower activity estimated by VWF:Ac. CONCLUSION: We conclude that Innovance(®) VWF Ac is suitable for the diagnosis, classification, and monitoring of VWD, and that it has a number of advantages over VWF:RCo method.

Diagnosis of suspected inherited platelet function disorders: results of a worldwide survey.

BACKGROUND: Diagnosis of inherited platelet function disorders (IPFDs) is important for appropriate management and to improve epidemiologic and clinical knowledge. However, there remains a lack of consensus on the diagnostic approach. OBJECTIVES: To gain knowledge on the current practices for the diagnosis of IPFD worldwide. METHODS: A 67-item questionnaire was distributed to the ISTH members and to the members of several national hemostasis and thrombosis societies. RESULTS: A total of 202 laboratories from 37 countries participated in the survey. The most frequent criterion to define patients with a suspected IPFD was a history of mucocutaneous bleeding and no acquired cause, but heterogeneity on the identification criteria was evident. Only 64.5% of respondents performed a direct clinical interview. On average, each laboratory studied 72 patients per year. The most commonly used laboratory equipment were the light-transmission aggregometer, the Platelet Function Analyzer-100, and the flow cytometer. Screening tests were platelet count, peripheral blood smear, light-transmission aggregometry, and Platelet Function Analyzer-100. Second-step tests were flow cytometry, molecular genetic analysis, and electron microscopy. Methodologies varied widely. In total, ~ 14,000 patients were investigated yearly and 60% turned out to not have a defect. Of the remaining 40%, only 8.7% received a diagnosis at a molecular level. CONCLUSIONS: Many laboratories worldwide are involved in the diagnosis of IPFD. A large fraction of the patients studied remain without a diagnosis. A high variability in the diagnostic approaches is evident.

Improving blood disorder diagnosis: reflections on the challenges.

Hematology laboratories have a vital role in providing diagnostic testing for a wide range of blood disorders. Improvements in hematology laboratory diagnostics are highly dependent on new discoveries on blood disorder pathology, the translation of new knowledge into assays for clinical testing purposes, and research that assesses, compares, and optimizes diagnostic practices. This article reviews the author's experiences with research leading to improved blood disorder diagnosis, including research studies on Quebec platelet disorder and other bleeding disorders, evaluations of practice, and research on the external quality assessment of diagnostic testing for platelet function disorders. The importance of research to advancing diagnostic testing for blood disorders is emphasized.

Laboratory testing for bleeding disorders: strategic uses of high and low-yield tests.

Laboratory testing is essential for diagnosing bleeding disorders. The tests and panels that laboratories currently use for bleeding disorder evaluation are not standardized, although most offer coagulation screening tests in bleeding disorder panels. Some tests for bleeding disorders, including von Willebrand factor multimer assays and tests for rarer disorders, are not widely available. Accordingly, clinicians and laboratories need tailored strategies for evaluating common and rare bleeding disorders. Coagulation screening tests have high specificity, however, false positives and false negatives do occur among subjects evaluated for bleeding disorders and more specific tests (e.g., factor assays) are required to further assess abnormalities. Tests for defects in primary hemostasis have similar high specificity but much greater sensitivity for common bleeding disorders than coagulation screening tests. Nonetheless, extensive testing fails to establish a diagnosis in a significant number of individuals considered to have significant bleeding problems. Rare bleeding disorder investigations are important to diagnose some conditions, particularly those with delayed-onset bleeding, such as factor XIII deficiency, α2 antiplasmin deficiency, plasminogen activator inhibitor-1 deficiency, and Quebec platelet disorder. These issues need careful consideration when assessing patients for congenital and acquired bleeding problems.