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.

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.

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.

Effect of standardized perioperative dabigatran interruption on the residual anticoagulation effect at the time of surgery or procedure.

UNLABELLED: ESSENTIALS: Anticoagulants need to be stopped preprocedure so there is little or no remaining anticoagulant effect. We assessed the residual anticoagulant effect with standardized interruption for patients on dabigatran. With this protocol, 80-86% of patients had no residual anticoagulant effect at the time of a procedure. A standardized perioperative dabigatran protocol appears to be safe, but requires further study. BACKGROUND: In patients taking dabigatran who require treatment interruption for a surgery/procedure, a sufficient interruption interval is needed so that there is little or no residual anticoagulant effect at the time of the surgery/procedure. METHODS: A prospective cohort study of patients receiving dabigatran (110 mg or 150 mg twice daily) who required an elective surgery/procedure and received a standardized dabigatran interruption protocol based on surgery/procedure bleeding risk and renal function was performed. Before the surgery/procedure, a blood sample was taken for measurement of the prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and dilute thrombin time (dTT). We determined the proportion of all patients and those having a high bleeding risk surgery/procedure with normal coagulation test results at the time of the surgery/procedure. The APTT and dTT were considered to be most likely to reflect a dabigatran anticoagulant effect. Patients were followed up for 30 days postprocedure to assess for bleeding and thromboembolism. RESULTS: One hundred and eighty-one patients were studied: 118 with low bleeding risk, and 63 with high bleeding risk. For all patients, the proportions with normal PT, APTT, TT dTT levels were 92.8%, 79.6%, 33.1%, and 80.7%, respectively. In patients with high bleeding risk, the proportions with normal PT, APTT, TT dTT levels were 93.7%, 85.7%, 57.1%, and 87.3%, respectively. During follow-up, there was one (0.6%) major bleed, there were nine (5.0%) minor bleeds, and there was one (0.6%) transient ischemic attack. CONCLUSIONS: In patients receiving dabigatran who require an elective surgery/procedure, a standardized interruption protocol yielded 80-86% of patients with no residual anticoagulant effect at the time of surgery/procedure, and with a low incidence of bleeding.

Internal Quality Control Practices in Coagulation Laboratories: recommendations based on a patterns-of-practice survey.

INTRODUCTION: Internal quality control (IQC) procedures are crucial for ensuring accurate patient test results. The IQMH Centre for Proficiency Testing conducted a web-based survey to gather information on the current IQC practices in coagulation testing. METHODS: A questionnaire was distributed to 174 Ontario laboratories licensed to perform prothrombin time (PT) and activated partial thromboplastin time (APTT). RESULTS: All laboratories reported using two levels of commercial QC (CQC); 12% incorporate pooled patient plasma into their IQC program; >68% run CQC at the beginning of each shift; 56% following maintenance, with reagent changes, during a shift, or with every repeat sample; 6% only run CQC at the beginning of the day and 25% when the instruments have been idle for a defined period of time. IQC run frequency was determined by manufacturer recommendations (71%) but also influenced by the stability of test (27%), clinical impact of an incorrect test result (25%), and sample's batch number (10%). IQC was monitored using preset limits based on standard deviation (66%), precision goals (46%), or allowable performance limits (36%). 95% use multirules. Failure actions include repeating the IQC (90%) and reporting patient results; if repeat passes, 42% perform repeat analysis of all patient samples from last acceptable IQC. CONCLUSION: Variability exists in coagulation IQC practices among Ontario clinical laboratories. The recommendations presented here would be useful in encouraging standardized IQC practices.

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.

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.

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.

Vitamin D receptor expression, 24-hydroxylase activity, and inhibition of growth by 1alpha,25-dihydroxyvitamin D3 in seven human prostatic carcinoma cell lines.

Although prostatic cancer is often viewed as an androgen-dependent malignancy, a number of other hormones including 1alpha, 25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] are now recognized to modulate its growth and differentiated phenotype. Seven different continuous human prostatic carcinoma cell lines were examined for the presence of biologically active receptors for 1alpha,25(OH)2D3. All seven lines were found to contain mRNA for the vitamin D receptor using an RNase protection assay. Six of the seven cell lines were found to have high-affinity saturable binding sites for 1alpha,25(OH)2D3. The seventh line was found to contain vitamin D receptors by sucrose gradient analysis. All seven lines were found to express 24-hydroxylase activity by a HPLC assay that measures the conversion of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3. 24-Hydroxylase activity was up-regulated in all seven cell lines by preincubation with 1alpha,25(OH)2D3. In the presence of fetal bovine serum, the growth of four of the seven cell lines was inhibited. In the majority of cell lines growth inhibition was related not only to the number of receptors per cell, but also in inverse proportion to the 24-hydroxylase activity of each cell line. The ubiquitous presence of vitamin D receptor and 24-hydroxylase activity in human prostatic carcinoma cells suggests new alternatives for the pharmacological treatment of advanced prostatic cancer and implies that chemoprevention strategies could also make use of this endocrine axis.