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.

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.

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.

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.

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.

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.

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.

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.

Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH.

Light transmission aggregometry (LTA) is the most common method used to assess platelet function. However, there is no universal standard for its performance. The Platelet Physiology Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis formed a working party of experts with the aim of producing a series of consensus recommendations for standardizing LTA. Due to a lack of investigations that directly compared different methodologies to perform LTA studies, there were insufficient data to develop evidence-based guidelines. Therefore, the RAND method was used, which obtains a formal consensus among experts about the appropriateness of health care interventions, particularly when scientific evidence is absent, scarce and/or heterogeneous. Using this approach, each expert scored as "appropriate", "uncertain" or "inappropriate" a series of statements about the practice of LTA, which included pre-analytical variables, blood collection, blood processing, methodological details, choice of agonists and the evaluation and reporting of results. After presentation and public discussion at SSC meetings, the assessments were further refined to produce final consensus recommendations. Before delivering the recommendations, a formal literature review was performed using a series of defined search terms about LTA. Of the 1830 potentially relevant studies identified, only 14 publications were considered to be actually relevant for review. Based upon the additional information, 6 consensus statements were slightly modified. The final statements were presented and discussed at the SSC Meeting in Cairo (2010) and formed the basis of a consensus document, which is the subject of the present report. This article is protected by copyright. All rights reserved.

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.

Simultaneous measurement of adenosine triphosphate release and aggregation potentiates human platelet aggregation responses for some subjects, including persons with Quebec platelet disorder.

Platelet aggregometry and dense granule adenosine triphosphate (ATP) release assays are helpful to diagnose platelet disorders. Some laboratories simultaneously measure aggregation and ATP release using Chronolume® a commercial reagent containing D-luciferin, firefly luciferase and magnesium. Chronolume® can potentiate sub-maximal aggregation responses, normalising canine platelet disorder findings. We investigated if Chronolume® potentiates human platelet aggregation responses after observing discrepancies suspicious of potentiation. Among patients simultaneously tested by light transmission aggregometry (LTA) on two instruments, 18/43 (42%), including 14/24 (58%) with platelet disorders, showed full secondary aggregation with one or more agonists only in tests with Chronolume®. As subjects with Quebec platelet disorder (QPD) did not show the expected absent secondary aggregation responses to epinephrine in tests with Chronolume®, the reason for the discrepancy was investigated using samples from 10 QPD subjects. Like sub-threshold ADP (0.75 µM), Chronolume® significantly increased QPD LTA responses to epinephrine (p<0.0001) and it increased both initial and secondary aggregation responses, leading to dense granule release. This potentiation was not restricted to QPD and it was mimicked adding 1-2 mM magnesium, but not D-luciferin or firefly luciferase, to LTA assays. Chronolume® potentiated the ADP aggregation responses of QPD subjects with a reduced response. Furthermore, it increased whole blood aggregation responses of healthy control samples to multiple agonists, tested at concentrations used for the diagnosis of platelet disorders (p values <0.05). Laboratories should be aware that measuring ATP release with Chronolume® can potentiate LTA and whole blood aggregation responses, which alters findings for some human platelet disorders, including QPD.

Results of a worldwide survey on the assessment of platelet function by light transmission aggregometry: a report from the platelet physiology subcommittee of the SSC of the ISTH.

BACKGROUND: Light transmission aggregometry (LTA) is the most common method used in clinical and research laboratories to assess platelet function. However, the method has never been standardized. OBJECTIVES: As the first step towards development of methodological guidelines, the Platelet Physiology Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH) undertook a large, detailed, global survey of LTA practices. METHODS: Members of ISTH and of External Quality Assurance in Thrombosis and Haemostasis organizations were invited to complete a 129 item, online questionnaire. Results were analyzed anonymously to participant identities. RESULTS: The online supplement for this article (http://www.isth.org/Publications/OfficialCommunications/PlateletPhysiology/LightTransmissionAggregometry/tabid/201/Default.aspx) contains the full details of the study findings. 359 (244 clinical, 115 research) laboratories from 48 countries participated in the survey. LTA was widely used to assess inherited or acquired bleeding disorders. Common practices were identified in sample collection, processing and analysis and although some are generally considered acceptable, others are not ideal. The agonist concentrations used for LTA varied, and many laboratories used ADP, collagen, epinephrine and Ristocetin, at more than one concentration, in addition to arachidonic acid. The parameters commonly used to assess LTA responses were maximal amplitude or % aggregation, which was considered particularly important, in addition to the presence of a 'secondary wave', deaggregation, shape change and a measure of the lag phase. However, many laboratories did not have appropriate reference intervals. CONCLUSIONS: This is the largest and most detailed survey of LTA practices ever undertaken. It shows a very high variability in LTA practices worldwide, and, as a consequence, methodological standardization is necessary. The information gathered in this survey will be helpful in the development of ISTH methodological guidelines for LTA.

Diagnostic utility of light transmission platelet aggregometry: results from a prospective study of individuals referred for bleeding disorder assessments.

BACKGROUND: Light transmission aggregometry (LTA) is commonly performed to assess individuals for bleeding disorders. OBJECTIVES: The goal was to evaluate the incidence and spectrum of platelet function abnormalities in a prospective cohort of individuals referred for bleeding disorder assessments after exclusion of thrombocytopenia and von Willebrand disease. PATIENTS/METHODS: Subjects were healthy controls and patients from a prospective cohort of individuals referred for bleeding disorder assessments after exclusion of thrombocytopenia and von Willebrand disease. LTA was performed by standardized methods using platelet-rich plasma adjusted to 250x10(9) platelets L(-1). Maximal aggregation data were analyzed to determine the likelihood of detecting a platelet function disorder by LTA, and the sensitivity and specificity of LTA for platelet disorders. RESULTS: The incidence of false positive LTA among subjects excluded of having bleeding disorders was similar to healthy controls. Abnormal LTA was more common in subjects with bleeding disorders and the likelihood of a bleeding disorder was significantly increased (odds ratio 32) when maximal aggregation was reduced with two or more agonists. Receiver operator curve analyses indicated that LTA had high specificity and moderate sensitivity for detecting inherited defects in platelet function and that the LTA agonists 1.25 microg mL(-1) collagen, 6 microM epinephrine, 1.6 mM arachidonic acid and 1.0 microM thromboxane analogue U44619 detected most inherited disorders with abnormal LTA. CONCLUSIONS: LTA is valuable for detecting platelet function abnormalities among individuals referred for bleeding problems, particularly when the test indicates abnormal responses to multiple agonists.

Platelet adhesion to multimerin 1 in vitro: influences of platelet membrane receptors, von Willebrand factor and shear.

BACKGROUND: Multimerin 1 (MMRN1) is a large, homopolymeric adhesive protein, stored in platelets and endothelium, that when released, binds to activated platelets, endothelial cells and the extracellular matrix. OBJECTIVES: The goals of our study were to determine if (i) MMRN1 supports adhesion of resting and/or activated platelets under conditions of blood flow, and (ii) if MMRN1 enhances platelet adhesion to types I and III collagen. PATIENTS/METHODS: Platelet adhesion was evaluated using protein-coated microcapillaries, with or without added adhesive proteins and receptor antibodies. Platelets from healthy controls, Glanzmann thrombasthenia (GT) and severe von Willebrand factor (VWF)-deficient donors were tested. RESULTS: MMRN1 supported the adhesion of activated, but not resting, washed platelets over a wide range of shear rates. At low shear (150 s(-1)), this adhesion was supported by integrins alphavbeta3 and glycoprotein (GP) Ibalpha but it did not require integrins alphaIIbbeta3 or VWF. At high shear (1500 s(-1)), adhesion to MMRN1 was supported by beta3 integrin-independent mechanisms, involving GPIbalpha and VWF, that did not require platelet activation when VWF was perfused over MMRN1 prior to platelets. MMRN1 bound to types I and III collagen, independent of VWF, however, its enhancing effects on platelet adhesion to collagen at high shear were VWF dependent. CONCLUSIONS: MMRN1 supports platelet adhesion by VWF-dependent and -independent mechanisms that vary by flow rate. Additionally, MMRN1 binds to, and enhances, platelet adhesion to collagen. These findings suggest that MMRN1 could function as an adhesive ligand that promotes platelet adhesion at sites of vascular injury.