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

Summary.  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 250 × 10⁹ 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 μg mL⁻¹ collagen, 6 μM epinephrine, 1.6 mM arachidonic acid and 1.0 μM 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.     

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.     

Prospective evaluation of a pediatric bleeding questionnaire and the ISTH bleeding assessment tool in children and parents in routine clinical practice

Summary. Background: Diagnosing mild bleeding disorders (BDs) in children is difficult. Bleeding scores (BSs) have been proposed for obtaining standardized quantitative histories. Objectives: To compare the Canadian pediatric bleeding questionnaire (PBQ) with the new ISTH bleeding assessment tool (ISTH BAT) for the determination of BS in a routine pediatric outpatient setting. Methods: One hundred children with a suspected BD were enrolled in this cross‐sectional study. Bleeding scores were calculated for all children and their natural parents. For all children, extensive laboratory investigations were performed. Results: Based on laboratory tests, 56 children were diagnosed as having no BD, 11 were diagnosed with possible VWD, 12 with VWD 1, 11 with VWD 2, five with possible platelet defects, and five with mild factor deficiencies. Both questionnaires were able to discriminate between no BD and VWD (P = 0.0001), but the area under the receiver characteristics curve to detect any mild BD was only 0.76. Despite the inherited nature of the BD, a family score did not increase the ability to discriminate between no BD and VWD (P = 0.2052). There was no significant difference between the two tools used (P = 0.3253) or simple qualitative criteria, such as yes/no questions regarding bleeding (P = 0.3477). Conclusions: The two tools translated into German did not differ substantially. Both were able to discriminate between no BD and a possible BD with acceptable accuracy. A BS of < 2 makes a BD unlikely. Simple qualitative criteria were similar; however, to allow comparison of studies and follow‐up in patients over time, we recommend the ISTH BAT.     

Application of the ISTH bleeding score in hemophilia

Background

Hemophilia is an inherited bleeding disorder. With proper treatment and self-care, persons with hemophilia can maintain an active, productive lifestyle. Hemophilia can be mild, moderate, or severe, depending on the degree of plasma clotting factor deficiency. The aim of the study was to assess the utility of ISTH-BAT in diagnosis, determining severity of the bleeding condition in newly diagnosed and known hemophilia patients, compare the bleeding score (BS) in adult and pediatric groups and investigate its association with plasma factor levels.

How to estimate bleeding risk in mild bleeding disorders

Summary.  The concept of mild bleeding disorders (MBD) has evolved in contrast to severe hemophilia A and B to indicate less severe disorders, characterized by the presence of more frequent and/or more prominent bleeding symptoms than in the normal population. These symptoms occur mostly after a recognizable challenge and do not lead to major discomfort or organ damage, even in the absence of specific medical intervention. However, it has become clear that, from the most severe to the mildest hemostatic disorders, there is a continuous spectrum of bleeding manifestations, which overlap with the occasional bleeding occurring in people without any identifiable hemostatic abnormality. By reviewing the principal hemorrhagic disorders we have tried to identify those entities that could fit a diagnosis of MBD and result, at the same time, in a net benefit for treatment or prophylaxis of patients rather than being simply accurate. This goal can usually be achieved by comparing the patient's phenotype with known nosological entities. However, limitations of this approach are evident, considering the paucity of clinical data and the biases of most published reports on the different disorders. In addition, in a partial deficiency of a clotting factor, a reliable relationship between the residual activity and bleeding severity is not invariably found. Molecular characterization of the defects is also generally useless. Accordingly, an accurate bleeding history in the propositus and his/her family remains of major importance. For this purpose, new standardized and possibly quantitative tools are being developed in several institutions. Innovative approaches, combining into a single probability phenotypic and genetic data, could possibly estimate better the bleeding risk in specific disorders.     

Laboratory diagnosis of inherited platelet function disorders

Platelets respond to vessel wall injury by forming a primary hemostatic plug to arrest blood loss. Hemostatic plug formation is complex, and involves platelet adhesion to the subendothelium that results in platelet activation and ultimately, aggregation. If any of these processes are deficient, primary hemostasis is impaired. Inherited platelet function disorders (IPFDs) are a heterogeneous group of defects in these processes, with patients experiencing mainly mucocutaneous bleeding symptoms that can range from very mild to life threatening, depending on the specific disorder. Here, we review the approach to an initial patient assessment required to inform laboratory testing, and the frequently used clinical laboratory assays for diagnostic evaluation of IPFDs. Newer testing approaches that may improve laboratory diagnosis in the near future are described.     

International collaboration as a tool for diagnosis of patients with inherited thrombocytopenia in the setting of a developing country

Summary. Background: Inherited thrombocytopenias (ITs) are heterogeneous genetic disorders that frequently represent a diagnostic challenge. The requirement of highly specialized tests for diagnosis represents a particular problem in resource‐limited settings. To overcome this difficulty, we applied a diagnostic algorithm and developed a collaboration program with a specialized international center in order to increase the diagnostic yield in a cohort of patients in Argentina. Methods: Based on the algorithm, initial evaluation included collection of clinical data, platelet size, blood smear examination and platelet aggregation tests. Confirmatory tests were performed according to diagnostic suspicion, which included platelet glycoprotein expression, immunofluorescence for myosin‐9 in granulocytes and platelet thrombospondin‐1 and molecular screening of candidate genes. Results: Thirty‐one patients from 14 pedigrees were included; their median age was 32 (4–72) years and platelet count 72 (4–147) × 10⁹ L^?1. Autosomal dominant inheritance was found in nine (64%) pedigrees; 10 (71%) had large platelets and nine (29%) patients presented with syndromic forms. A definitive diagnosis was made in 10 of 14 pedigrees and comprised MYH9‐related disease in four, while classic and monoallelic Bernard–Soulier syndrome, gray platelet syndrome, X‐linked thrombocytopenia, thrombocytopenia 2 (ANKRD26 mutation) and familial platelet disorder with predisposition to acute myelogenous leukemia were diagnosed in one pedigree each. Conclusions: Adoption of an established diagnostic algorithm and collaboration with an expert referral center proved useful for diagnosis of IT patients in the setting of a developing country. This initiative may serve as a model to develop international networks with the goal of improving diagnosis and care of patients with these rare diseases.     

Hemorrhagic symptoms and bleeding risk in obligatory carriers of type 3 von Willebrand disease: an international, multicenter study

OBJECTIVES: We undertook an international, multicenter study to describe the clinical picture and to estimate the bleeding risk in a group of obligatory carriers of type 3 von Willebrand disease (VWD). PATIENTS AND METHODS: Obligatory carriers (OC) of type 3 VWD were identified by the presence of offspring with type 3 VWD or by being an offspring of a type 3 patient. Normal controls were age- and sex-matched with the obligatory carriers. A physician-administered standardized questionnaire was used to evaluate hemorrhagic symptoms at presentation. A score system ranging from 0 (no symptom) to 3 (hospitalization, replacement therapy, blood transfusion) was used to quantitate bleeding manifestations. Odds ratios were computed for each symptom. RESULTS: Ten centers participated to the study, enrolling a total of 35 type 3 VWD families, with 70 OC. A total of 215 normal controls and 42 OC for type 1 VWD were also included. About 40% of type 3 OC had at least one bleeding symptom compared to 23% of normal controls and 81.8% of type 1 OC (P < 0.0001 by chi-squared test), showing that type 3 OC clearly represent a distinct population from type 1 OC. The clinical situations associated with an increase of bleeding risk in type 3 OC were epistaxis [odds ratio 3.6; 90% confidence intervals (CI) 1.84-21.5], cutaneous bleeding (odds ratio 5.5; 90% CI 2.5-14.1) and postsurgical bleeding (odds ratio 16.3; 90% CI 4.5-59). The severity of bleeding score correlated with the degree of factor (F) VIII reduction in plasma. CONCLUSIONS: OC for type 3 VWD represent a distinctive population from type 1 OC. These patients, however, present with more frequent bleeding symptoms in comparison to normal controls, especially in case of significantly low FVIII. Desmopressin and/or tranexamic acid might be useful to prevent or treat bleeding in these cases.     

Implementation of buffy‐coat‐derived pooled platelet concentrates for internal quality control of light transmission aggregometry: a proof of concept study

Essentials

• In platelet function testing, standardized internal controls (IQC) are not commercially provided.
• Platelet function testing was performed daily on aliquoted pooled platelet concentrates.
• Pooled platelet concentrates showed stability for control purposes from Monday to Friday.
• Pooled platelet concentrates provide the necessary steadiness to serve as IQC material.

     

Platelet function to estimate the bleeding risk in autoimmune thrombocytopenia

BACKGROUND: Knowledge of platelet function may assist in patient care in chronic autoimmune thrombocytopenia (cAITP). MATERIALS AND METHODS: We evaluated the association of platelet function with haemorrhage in 41 patients, median age 41 years (range 14-82 years, 24 females) with chronic autoimmune thrombocytopenia (cAITP). Samples were investigated for platelet P-selectin, and adhesion and aggregate formation under high shear conditions. Data were compared to those from 28 healthy controls (median age 39 years, range 23-70 years, 17 females) and correlated with a bleeding score of 0 (no bleeding) to 3 (overt mucosal bleedings). RESULTS: P-selectin levels were higher in patients than in controls (P < 0.0004). Compared to controls, the patients' samples responded to high shear with decreased adhesion to the polystyrene surface (P < 0.0001), but formed aggregates of normal size. P-selectin expression was neither correlated with platelet counts, nor platelet adhesion, nor the bleeding score. Only the size of formed aggregates correlated with P-selectin (P = 0.01). Platelet counts (odds ratio 0.5, 95% confidence interval 0.22-0.88; P = 0.04) and adhesion (odds ratio 0.45, 95% confidence interval 0.17-0.87; P = 0.04) were independently inversely correlated with bleeding symptoms. CONCLUSION: Platelet adhesion correlates with bleeding symptoms, while the size of aggregates that are formed under high shear correlates with in vivo platelet activation. The determination of these parameters may assist in estimating an individual bleeding risk and thus a decision for treatment.     

Usefulness of PFA-100® testing in the diagnostic screening of patients with suspected abnormalities of hemostasis: comparison with the bleeding time

BACKGROUND: Global tests of hemostasis that are used to screen patients with clinical suspicion of bleeding disorders should help the physician to identify the phase of the hemostatic system that is abnormal and guide further diagnostic workup. PATIENTS AND METHODS: We compared the performance of Platelet Function Analyzer-100 (PFA-100) closure time (CT) with bleeding time (BT), both of which are screening tests for primary hemostasis, in the diagnostic workup of 128 consecutive patients who were screened for bleeding disorders. The sensitivities of BT and PFA-100 CT for known defects of hemostasis were evaluated; in addition, we calculated their correlation with the levels of severity of the bleeding symptoms, which were recorded using a standardized questionnaire. RESULTS: The sensitivity of PFA-100 testing was 71% for von Willebrand disease (VWD) [with both collagen-adenosine diphosphate (C-ADP) and collagen-epinephrine (C-EPI) cartridges]; 58% (C-EPI) and 8% (C-ADP) for platelet function disorders (PFDs); and the sensitivity of BT was 29% (VWD) and 33% (PFD). C-EPI CT was also prolonged in about 20% of patients with abnormalities of coagulation or fibrinolysis. Only the C-EPI CT was significantly associated with the levels of severity of the patients' bleeding scores. CONCLUSIONS: BT and C-EPI are insufficiently sensitive to be recommended as hemostasis screening tests. The C-ADP cartridge, which is sensitive to VWD only, might prove useful in further diagnostic workup of defects of primary hemostasis. The association of C-EPI CT with the severity of bleeding symptoms as a useful predictor of risk of bleeding in clinical practise should be tested in properly designed studies.