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Keywords:

  • diagnostic testing;
  • blood disorders;
  • platelet disorders;
  • bleeding disorder investigations

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Perspectives on the Importance of Advancing Blood Disorder Diagnosis
  5. Basic Research Leading to New Diagnostic Tests: the Tale of Quebec Platelet Disorder
  6. Interchanges on Practices as a Mean to Improving Blood Disorder Diagnostic Testing
  7. Research on Current Diagnostic Tests for Bleeding Disorders
  8. Launch of a Quality Assurance Program for Platelet Function Disorder Tests
  9. Current and Future Challenges
  10. Acknowledgements
  11. References

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.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Perspectives on the Importance of Advancing Blood Disorder Diagnosis
  5. Basic Research Leading to New Diagnostic Tests: the Tale of Quebec Platelet Disorder
  6. Interchanges on Practices as a Mean to Improving Blood Disorder Diagnostic Testing
  7. Research on Current Diagnostic Tests for Bleeding Disorders
  8. Launch of a Quality Assurance Program for Platelet Function Disorder Tests
  9. Current and Future Challenges
  10. Acknowledgements
  11. References

As the recipient of the 2013 Berend Houwen award, I am dedicating this article to the memory of Berend Houwen. Berend Houwen was instrumental to creating the discipline of laboratory hematology and was a catalyst for the formation of the International Society of Laboratory Hematology [1, 2]. He served as editor of the Society's journal, fostered the sharing and translation of technical advances at Society meetings and other venues, and promoted the development of guidelines to standardize measurement and testing, in collaboration with the International Council for Standardization in Hematology [1, 2].

This article reviews the author's experiences with research to improve blood disorder diagnosis, including research studies on Quebec platelet disorder, other bleeding disorders, and coagulation laboratory practices.

Perspectives on the Importance of Advancing Blood Disorder Diagnosis

  1. Top of page
  2. Summary
  3. Introduction
  4. Perspectives on the Importance of Advancing Blood Disorder Diagnosis
  5. Basic Research Leading to New Diagnostic Tests: the Tale of Quebec Platelet Disorder
  6. Interchanges on Practices as a Mean to Improving Blood Disorder Diagnostic Testing
  7. Research on Current Diagnostic Tests for Bleeding Disorders
  8. Launch of a Quality Assurance Program for Platelet Function Disorder Tests
  9. Current and Future Challenges
  10. Acknowledgements
  11. References

Hematology laboratories are vital for the diagnosis of many benign and malignant blood disorders and for the monitoring of some therapies, including anticoagulation. The launch of new diagnostic tests for blood disorders often requires many years of knowledge generation, translation, and extensive validation before assays can be used for clinical diagnostic purposes. Efforts to improve the laboratory testing for blood disorder diagnosis can be directed at a number of levels, as outlined in Table 1. Such efforts are essential for meeting the evolving needs of patients and clinical services and many essentials of total quality management.

Table 1. Important considerations for improving diagnostic testing
ConsiderationRationaleImportance
Patient needsIdentify disorders and healthcare problems faced by patients who fail to be diagnosed due to limits in knowledge on pathogenesis or limitations in technologies, methodologies, knowledge translation, decision making, and/or implementationEnsures that diagnostic testing is meeting patient needs, which is essential to quality health care
Laboratory service needsIdentify procedures that are suboptimal for diagnosis and/or therapy monitoring and are amenable to quality improvementEssential for quality management and improvement
External evaluation of qualityIdentify whether performance is at the level of peers and/or whether it needs further quality evaluation and/or improvementEssential for quality management and improvement by providing comparisons to peers
Identification of issues requiring knowledge generation and translationIdentify questions that need pursuing to generate new discoveries and improve practice, including advanced knowledge on pathogenesis but also the development and comparisons of diagnostic methodsEssential for ensuring new discoveries are made and are translated into practice improvements

Presently, in many regions of the world, there are significant gaps in hematology laboratory diagnostic testing, due to limitations in healthcare funding, variances in local priorities, and inadequate knowledge, limited knowledge translation, and other factors. There is a need for healthcare professionals in laboratory hematology, and total quality management strategies, to address the gaps proactively.

From a patient's perspective, failures and delays in diagnosing a blood disorder can have devastating impacts, whereas knowing their diagnosis can lead to improved health outcomes and enabling effects on the attainment of life goals. An illustrative example is provided in reference [3]. Accordingly, it is tremendously important to consider both patient and clinical service needs for diagnostic testing in practice and to collaborate with other health professionals in promoting education for proper test utilization.

Patient-oriented research is important for blood disorder diagnosis and assessment and for advancing the field of laboratory hematology. Researchers, research trainees, research agencies, academic institutions, industry, and healthcare organizations are all important for supporting this research agenda. While a minority of medical discoveries are successfully translated into useful diagnostic assays, promising discoveries require additional investments for translation into clinical laboratory practice. This translation often requires extensive assay characterization, validation, and sometimes comparisons of new to old methods, before new or improved tests are ready for implementation in diagnostic laboratories. Like other fields of medicine, laboratory hematology increasingly relies on evidence generated from research to guide decisions on appropriate practice. Healthcare professionals have an important role in generating and appraising the evidence to maintain, optimize, and update practices in blood disorder diagnostics.

The sections that follow provide some examples of research that has improved blood disorder diagnosis, sometimes triggered by uncertainties about best practices, a desire to address hypothesis-driven questions or questions arising from serendipity, another ‘building block of discovery’ [4].

Basic Research Leading to New Diagnostic Tests: the Tale of Quebec Platelet Disorder

  1. Top of page
  2. Summary
  3. Introduction
  4. Perspectives on the Importance of Advancing Blood Disorder Diagnosis
  5. Basic Research Leading to New Diagnostic Tests: the Tale of Quebec Platelet Disorder
  6. Interchanges on Practices as a Mean to Improving Blood Disorder Diagnostic Testing
  7. Research on Current Diagnostic Tests for Bleeding Disorders
  8. Launch of a Quality Assurance Program for Platelet Function Disorder Tests
  9. Current and Future Challenges
  10. Acknowledgements
  11. References

My participation in this research began with serendipity, with initial discoveries quickly stimulating hypothesis-driven research questions (summarized in Table 2).

Table 2. Chronology of discoveries related to Quebec platelet disorder. Years are indication for key publications and their impacts on diagnostic testing for this disorder, which had been designated as factor V Quebec prior to discovering that the disorder alters many platelet proteins
YearPublished observations and its impact on diagnostic testing, if any
  1. uPA, urokinase plasminogen activator.

1984Factor V Quebec is reported as a unique bleeding disorder with abnormal platelet but not plasma factor V, with defective platelet prothrombinase function in the absence of plasma factor V (in research laboratories) [11]
1993Platelets are identified to contain factor V-binding proteins [9]
1995Factor V is reported to be stored in platelets complexed to multimerin 1, the major factor V-binding protein in platelets that is not normally detectable in plasma [10]
1996Factor V Quebec is identified to be associated with proteolysis of a number of α-granule proteins and platelet multimerin 1 deficiency [12, 13]. Additional features of the disorder are identified, including reduced platelet counts (by about 50%) and absent secondary aggregation with epinephrine [12]. Assays for platelet protein degradation become the preferred diagnostic test for the disorder (in research laboratories). Laboratories begin considering the diagnosis in patients with absent secondary aggregation with epinephrine and normal-to-reduced platelet counts
1997Quebec platelet disorder is demonstrated to selectively degrade megakaryocyte-synthesized and plasma-derived proteins stored in α-granules, without defective targeting of α-granule proteins [14]. Platelet but not plasma fibrinogen is identified to show a unique pattern of degradation, resulting in normal plasma D-dimer levels but high levels of fibrinogen degradation products, detectable by polyclonal antibodies, in serum but not in plasma [18]. The commercial polyclonal antibody assay for fibrinogen degradation products, that detected these abnormalities, was later discontinued
2001Quebec platelet disorder is identified to markedly increase the levels of uPA in platelets, consuming intraplatelet stores of plasminogen activator inhibitor 1, without major changes in plasma uPA levels [20]. Diagnostic testing for the disorder was expanded to include assays for increased platelet uPA (in research laboratories)
2003The unique pattern of Quebec platelet disorder α-granule proteolysis is attributed to uPA-triggered, intracellular generation of plasmin (from the plasminogen within platelets), without systemic fibrinolysis [21]
2004Quebec platelet disorder is reported to increase risks for joint bleeds, delayed-onset bleeding after surgery and dental extraction, and for large bruises and to be associated with episodic, spontaneous hematuria [17]. Pregnancy outcomes are reported to be good, with no treatments required for uncomplicated deliveries [17]
2006Quebec platelet disorder is reported to cause a unique gain-of-function defect in fibrinolysis, from release of platelet uPA during clot formation [19]. Thromboelastography is excluded as a possible diagnostic screening test [19]
2008Quebec platelet disorder is reported to have minimal effect on urinary uPA [24]
2009Quebec platelet disorder is linked to PLAU (uPA gene) with increased expression of uPA, by the linked allele, during megakaryocyte differentiation [22]. uPA is demonstrated to be targeted to α-granules in the disorder [23]. α-granule proteins are identified to be stored undegraded in Quebec platelet disorder megakaryocytes grown in culture, which do not contain plasminogen, confirming that their proteolysis in the disorder reflects intraplatelet plasmin generation [23]
2010Quebec platelet disorder mutation is associated with a highly specific mutation: tandem duplication of a 78-kb genomic segment on chromosome 10 that includes PLAU [25]. This knowledge is translated into diagnostic laboratory practice, leading to the diagnosis of new cases in multiple provinces of Canada and in the United States [15, 16, 26]. Genetic testing becomes the recommended diagnostic test for Quebec platelet disorder [15, 16, 25]
2011Lumiaggregometry is found to falsely normalize aggregation findings for Quebec platelet disorder, and some other platelet function defects, due to an aggregation potentiating effect of the luciferin/luciferase reagent [26]. Laboratories cautioned not to exclude the disorder based on lumiaggregometry responses to epinephrine [26]

The tale starts at the point when I was in the final stages of completing doctoral studies after hematology training. For my thesis, I was characterizing multimerin 1, a massive, soluble, homopolymeric protein, found in platelets and endothelium, but not in plasma, that had unknown functions [5-8]. At the same time, Dr. Michael Nesheim and colleagues in Kingston were working to isolate platelet proteins that bind coagulation factor V to better understand how platelets influence blood coagulation [9]. When I saw him present on this research at an international meeting [9], I was struck that on reduced gels, the major protein that they had isolated appeared to have the mobility of multimerin 1. The possibility that multimerin 1 was the major factor V-binding protein led our paths to cross; after a visit to Kingston, our collaborations led to the discovery that multimerin 1 is a factor V-binding protein [10]. While this discovery has led to other basic science investigations, it also led to questions about the pathogenesis of an unusual bleeding disorder.

While visiting in Kingston, I had the opportunity to chat with Dr. Alan Giles, who told me that when he sees patients with a bleeding problem of unknown cause, he considers the diagnosis of factor V Quebec, a moderate-to-severe bleeding disorder with an unexplained functional defect in platelet, but not plasma, factor V [11]. The curious feature of abnormal platelet but not plasma factor V in this condition led me to postulate that perhaps multimerin 1 was abnormal in factor V Quebec. I then contacted Dr. Georges E. Rivard, who was enthusiastic to explore this possibility. Together, we did find multimerin 1 abnormalities in this disorder, but it turned out to be for an unexpected reason: patients with factor V Quebec have multimerin 1 deficiency as part of a biochemical defect that causes abnormal proteolytic degradation of multiple platelet proteins stored in α-granules [12-14].

In reviewing the laboratory findings of persons with this disorder with Dr. Rivard, we identified that the platelet counts of persons with factor V Quebec were approximately 50% lower than their unaffected relatives, and curiously, their platelets showed absent secondary platelet aggregation with epinephrine, with or without impaired aggregation responses to adenosine diphosphate and/or collagen [12-14]. The recognition that this disorder had complex platelet abnormalities, involving but not restricted to platelet factor V, led to its redesignation as Quebec platelet disorder [12, 14-16].

I remained fascinated by this disorder, and as an independent investigator, I continued my collaborations with Dr. Rivard. Together, we identified more than 40 cases with Quebec platelet disorder, in Canada and the United States, with the majority residing in the Canadian Provinces of Quebec, British Columbia, and Ontario [15-17]. Although it took time, we eventually did find a link between the first two families that we identified had this disorder [15, 16].

We questioned: Why individuals with Quebec platelet disorder had rather unique abnormalities in clinical laboratory tests of fibrinolysis? We found that the abnormalities reflected degradation of the platelet but not plasma pool of fibrinogen, resulting in normal levels of D-dimer in plasma but significantly elevated levels of fibrinogen degradation products, recognized by polyclonal antibody assays, in serum from whole blood, but not in serum made from platelet-poor plasma [18]. The euglobulin clot lysis times and whole blood clot lysis times are normal in the disorder [15, 16, 18, 19].

We postulated that the disorder resulted from a protease defect involving the α-granule compartment of platelets, based on the sparing of outer membrane and cytosolic platelet proteins, striking proteolysis of diverse, plasma-derived and megakaryocyte-synthesized α-granule proteins, and the normal targeting of these proteins to α-granules [14]. I decided to test for a protease defect, with a simple experiment and question: Do the supernatants of activated platelets contained excessive protease activity in Quebec platelet disorder? Because fibrinogen was one of the proteins degraded, I used fibrinogen substrate gels, prepared with and without added plasminogen [20]. The hypothesis was confirmed: platelets from individuals with Quebec platelet disorder contained fibrinogen-degrading activity, not evident in normal samples, that was enhanced by plasminogen, indicating that the protease was a plasminogen activator [20]. Dr. Walter Kahr, who was a postdoctoral fellowship in my laboratory at that time, investigated the identity of the protease, which he determined was urokinase plasminogen activator (uPA) [20]. The quantitative analysis, by enzyme-linked immunosorbent assays, indicated that uPA increased more than 100-fold in the platelets, but curiously not in the plasma, of persons with Quebec platelet disorder [20]; this offered an explanation for why persons with Quebec platelet disorder had degraded platelet proteins but not systemic fibrinolysis [20]. These observations gave us other options for diagnosing Quebec platelet disorder in research laboratories, as Western blots were a very time-consuming way to look for platelet protein proteolysis.

For his Master's project, Prameet Sheth studied α-granule protein degradation in Quebec platelet disorder ex vivo, illustrating that excess uPA can trigger a similar pattern of platelet protein degradation and implicating intraplatelet plasmin generation as the mechanism of degradation [21].

The potential to explore relationships between bleeding risks and laboratory findings for Quebec platelet disorder led us to undertake another study. To gather evidence on the phenotype of Quebec platelet disorder, we compared the medical histories of individuals with this disorder, with their unaffected relatives, using a standardized, bleeding history assessment tool that was specifically designed for assessing Quebec platelet disorder. We used the diagnostic findings for platelet uPA and α-granule protein degradation assays to define the affection status of family members [17]. We found that having Quebec platelet disorder markedly increases a person's risks for bleeding after surgery or dental extraction [17]. Additionally, every affected person who had been challenged by surgery or a dental extraction reported experiencing clinically significant bleeding, sometimes needing blood transfusion, which typically occurred 1–3 days after the procedure, unless they were treated with a fibrinolytic inhibitor drug [17]. We found that the disorder also increases risks for experiencing abnormally large bruises and/or bruises that track downward, joint bleeding and episodic, spontaneous hematuria [17]. Although our study was not designed to evaluate causes of death, none of the affected individuals in the family had ever experienced a thrombotic stroke or myocardial infarction, although some had experienced intracranial bleeding and, in some family members, the intracranial bleeding had been fatal [17].

As fibrinolytic inhibitors are the only treatment that has ever controlled bleeding in Quebec platelet disorder, we explored the disorder's effect on fibrinolysis, ex vivo. Platelet uPA levels are several orders of magnitude higher than normal in Quebec platelet disorder, and this results in a unique, platelet-mediated, gain-of-function defect in fibrinolysis, which accelerates the lysis of forming and preformed fibrin clots [19]. Although we were unable to detect abnormalities by thromboelastography, the detection of increased fibrinolysis by this method requires more uPA than is found in the blood in Quebec platelet disorder [19].

My doctoral students Maria Diamandis and Kika Veljkovic explored complementary questions about the pathogenesis of Quebec platelet disorder: its genetic cause; how megakaryocyte differentiation from hematopoietic stem cells into platelets alters the levels of uPA message and protein in the disorder; and when and where α-granule proteins are degraded in Quebec platelet disorder. Maria Diamandis linked Quebec platelet disorder to PLAU (uPA gene), the candidate gene, and together with Kika Veljkovic illustrated that it causes selective overexpression of uPA by the disease chromosome as hematopoietic stem cells differentiate into megakaryocytes and produce α-granule proteins, without dysregulating the adjacent genes [22, 23]. Additionally, Kika Veljkovic showed that uPA is targeted to α-granules in Quebec platelet disorder, and in the absence of stored platelet plasminogen, it does not trigger α-granule protein degradation (this requires plasmin generation) [23]. They also found that Quebec platelet disorder has minimal if any impact on uPA levels in urine [24]. As the exhaustive search for PLAU sequence changes in Quebec platelet disorder failed to identify a mutation [22], we began considering whether the cause was a mutation that might be difficult to detect by sequencing. We found that persons with Quebec platelet disorder have a gain-of-copy-number mutation: they have three rather than two copies of PLAU due to tandem duplication of a 78-kb region on chromosome 10 that contains PLAU, its regulatory elements, and large flanking regions [25]. With this discovery, Quebec platelet disorder became the very first bleeding disorder recognized to result from a gain-of-copy-number mutation [25]. The knowledge of the disease-specific mutation transformed diagnostic testing for Quebec platelet disorder: in both Hamilton and Montreal, we began testing for the disorder in the clinical laboratory, leading to the diagnosis of new cases of Quebec platelet disorder in Canada and the United States [15, 16]. Affected parents were grateful that genetic testing allowed their newborns to be tested for the disorder, using routinely collected, cord blood samples [15, 16].

Recently, serendipity contributed to another observation on Quebec platelet disorder. We did testing for the disorder as part of investigations for a platelet disorder in an Ontario family with an unexplained, autosomal dominant bleeding disorder that suggested the possibility of Quebec platelet disorder. This family did turn out to have Quebec platelet disorder. Their diagnostic testing unexpectedly led to the observation that lumiaggregometry (which a number of laboratories perform to simultaneously evaluate for aggregation and dense granule release defects) falsely normalizes epinephrine aggregation in Quebec platelet disorder and in some other human platelet function disorders [26]. As absent secondary aggregation with epinephrine has many different causes, we caution against using aggregation to screen for Quebec platelet disorder. If lumiaggregometry is performed, we strongly recommend that epinephrine aggregation responses be assessed without simultaneously measuring dense granule release as the luciferin/luciferase reagent that is used to measure dense granule adenosine triphosphate release normalizes epinephrine aggregation in Quebec platelet disorder [26].

The discoveries from our research on Quebec platelet disorder have changed our approach to the laboratory diagnosis of this disorder, which can now be performed on shipped blood samples from any part of the world. Molecular testing is rarely used in the initial investigations of a blood disorder, but we view it as the most appropriate and only necessary test for diagnosing Quebec platelet disorder [15, 16]. The genetic test is useful for determining affection status for relatives of individuals with Quebec platelet disorder (e.g., newborns, children of affected persons, siblings of affected persons) and for individuals who are not known to be part of the Quebec platelet disorder family when their bleeding history is suggestive of Quebec platelet disorder (e.g., a history of parents or siblings with bleeding, and a personal and/or familial history of delayed-onset bleeding with challenges, bleeding that is responsive only to fibrinolytic inhibitor therapy, with or without joint bleeds, large ecchymoses, spontaneous hematuria) [15-17, 25]. Indeed, this strategy has led to diagnosing the disorder in individuals who are not known to be related to the family [26].

We are now working on solving an important mystery: How does having one extra copy of PLAU lead to >100-fold increased uPA in platelets, without marked changing uPA levels in urine or plasma? My doctoral student Jessica Blavignac is exploring another important issue: whether the gain-of-function defect in fibrinolysis extends to other hematopoietic lineages in Quebec platelet disorder. We are also testing the hypothesis that Quebec platelet disorder leads to overexpression of C10orf55, a gene of unknown function that is transcribed by opposite strand at the same locus.

My experiences with researching this disorder have taught me that serendipity, and hypothesis-driven questions, advance our knowledge on the pathogenesis and diagnosis of blood disorders.

Interchanges on Practices as a Mean to Improving Blood Disorder Diagnostic Testing

  1. Top of page
  2. Summary
  3. Introduction
  4. Perspectives on the Importance of Advancing Blood Disorder Diagnosis
  5. Basic Research Leading to New Diagnostic Tests: the Tale of Quebec Platelet Disorder
  6. Interchanges on Practices as a Mean to Improving Blood Disorder Diagnostic Testing
  7. Research on Current Diagnostic Tests for Bleeding Disorders
  8. Launch of a Quality Assurance Program for Platelet Function Disorder Tests
  9. Current and Future Challenges
  10. Acknowledgements
  11. References

As head of a specialized coagulation laboratory, and as a physician who evaluates patients referred to a bleeding disorder clinic, one of my biggest challenges in diagnostic evaluation has been platelet function testing. Like other coagulation laboratories, our laboratory was uncertain about the best procedures for testing platelet function, including which agonists and agonist concentrations to use for diagnostic purposes. In the 1980s, we did not have a standardized approach, or reference intervals, to evaluate patients by aggregometry or dense granule release assays, which made the interpretation of the findings very subjective. We consulted with experts on best approaches, including Dr. Harvey Weiss, who suggested that we should add a low concentration of collagen to our light transmission platelet aggregometry (LTA) panel.

After observing patients who had been tested at different centers with discrepant findings, and even interpretations that suggested very different diagnoses, we decided to engage the North American Specialized Coagulation Laboratory Association (NASCOLA) in a fact-finding mission. Our goal was to find out exactly what laboratories were doing, rationalizing that sharing information on practices might help identify the more common approaches, as the first step in standardization. My colleague Karen Moffat led the patterns-of-practice survey initiative to compare what NASCOLA laboratories were actually doing to diagnose platelet function disorders [27]. The data collected were so surprising that we felt obligated to do a second survey, for a more thorough check on final agonist concentrations before reporting the findings [27]. This was the first of a number of surveys that identified how variable the testing for platelet function disorders is in practice [28]. As part of a preguideline development initiative of the Platelet Physiology Subcommittee of the International Society on Thrombosis and Haemostasis (ISTH), we had the opportunity to evaluate worldwide practices, which confirmed the need to standardize platelet aggregation testing [28].

Through these, other surveys and quality assurance exercises [26, 28-35], we learned that participation in such activities is useful to promote practice improvements. For example, our NASCOLA study to determine whether laboratories were following the lupus anticoagulant testing guidelines resulted in more laboratories complying with guideline recommendations by the time of a second survey [35]. Similarly, the sharing of experiences and evidence over time led to many more laboratories testing for platelet secretion defects and for dense granule deficiency [27, 34]. Importantly, the sharing of information and evidence among laboratories participating in the proficiency testing offered by the Quality Management Program–Laboratory Services (QMP-LS) in Ontario, and by NASCOLA, stimulated the development of consensus guidelines on how to perform and interpret LTA for bleeding disorder investigations [32]. Currently, these are the only published platelet function testing guidelines to be developed by consensus of the diagnostic laboratories that do such testing [32]. These guidelines are also the first to offer recommendations on how to interpret LTA findings [32].

It is encouraging that practices for platelet function testing have improved over time [34], and the involvement of diagnostic laboratories in the consensus building [32] likely facilitated the improvements. Our most recent practice survey of NASCOLA laboratories has identified some gaps that need to be addressed for diagnostic testing [34]. For example, the cutoffs used to diagnose von Willebrand disease in laboratories are inconsistent, and only a minority of laboratories have adopted the National Heart Lung and Blood Institute recommendations for diagnosing type 1 von Willebrand disease [34, 36].

The collaborative sharing of information on practice has been helpful to explore other issues relevant to hematology laboratories, such as the critical values that laboratories use for coagulation tests [37] and the investigations that are typically performed to assess bleeding disorders [34].

Research on Current Diagnostic Tests for Bleeding Disorders

  1. Top of page
  2. Summary
  3. Introduction
  4. Perspectives on the Importance of Advancing Blood Disorder Diagnosis
  5. Basic Research Leading to New Diagnostic Tests: the Tale of Quebec Platelet Disorder
  6. Interchanges on Practices as a Mean to Improving Blood Disorder Diagnostic Testing
  7. Research on Current Diagnostic Tests for Bleeding Disorders
  8. Launch of a Quality Assurance Program for Platelet Function Disorder Tests
  9. Current and Future Challenges
  10. Acknowledgements
  11. References

The ideal hematology laboratory test is a convenient, cost-effective, precise, reproducible, well-standardized assay that provides clinically relevant information, is easy to interpret, and has well-characterized sensitivity and specificity, with findings evaluated for a full range of subjects and an established utility in relevant patient populations [38]. As many commonly performed investigations for bleeding disorders have uncertain sensitivity and specificity for bleeding disorders, we decided to address the gaps by retrospective and prospective studies.

After developing reference intervals for LTA in large numbers of subjects, by valid statistical methods [39], we explored the diagnostic usefulness of LTA as a test for bleeding disorders [40]. We identified that LTA is a useful test, with moderate sensitivity and high specificity for common platelet function disorders [40]. We also identified the agonists that are useful to detect common platelet function disorders by LTA [40]. Additionally, we found that LTA abnormalities with single agonists (except with collagen and ristocetin) are likely to represent false-positive findings [40].

Dr. Menaka Pai led our efforts to similarly evaluate the usefulness of platelet dense granule release assays, identifying that such assays have high specificity and moderate sensitivity for platelet function disorders [41]. Importantly, release assays help detect platelet function abnormalities, even when LTA findings are normal, and they are often used to determine whether a platelet function disorder has features of a platelet secretion defect [41]. Some caution is warranted in interpreting dense granule release findings with weak agonists, as the amount of release is quite variable [41].

To address controversies that emerged about whether LTA should be done with native or platelet count-adjusted samples for bleeding disorder diagnosis (discussed in [42]), we initiated a prospective, noninferiority study, using areas under receiver operator curves to objectively compare the two approaches. Our end-point was the diagnosis of a bleeding disorder by aggregometry, and the study was carried out by parallel testing of native and platelet count-adjusted samples from both patient and healthy controls [42]. This study, led by Dr. Jean Francois Castilloux, represents the first published noninferiority study of a diagnostic test performed in hematology laboratories [42]. While the study validated that both sample types detect platelet function abnormalities due to bleeding disorders, platelet count-adjusted samples were superior for diagnosing a bleeding disorder [42]. The study also confirmed, in a different cohort, that single-agonist abnormalities in LTA often represent false positives [42], an issue that many laboratories fail to recognize when interpreting the findings [29].

We had questions about the usefulness of the tests that many laboratories include in bleeding disorder panels, as other studies found many of these tests are not valuable for detecting bleeding problems in preoperative settings (reviewed in [43]). We evaluated data for patients referred to our center for bleeding disorder assessments, who had been tested by a standardized approach [43]. We found that the tests in bleeding disorder panels have high specificity and that LTA and von Willebrand disease screens have much higher sensitivity for common bleeding disorders than screening tests of coagulation [43]. We identified that even among patients referred for a bleeding disorder assessment, coagulation screening test abnormalities are often false positives [43]. The study generated helpful evidence on which tests are commonly helpful for initial bleeding disorder investigations [43]. Additionally, we observed that simultaneous testing for von Willebrand disease and platelet function disorders avoided some potential false negatives [43].

Launch of a Quality Assurance Program for Platelet Function Disorder Tests

  1. Top of page
  2. Summary
  3. Introduction
  4. Perspectives on the Importance of Advancing Blood Disorder Diagnosis
  5. Basic Research Leading to New Diagnostic Tests: the Tale of Quebec Platelet Disorder
  6. Interchanges on Practices as a Mean to Improving Blood Disorder Diagnostic Testing
  7. Research on Current Diagnostic Tests for Bleeding Disorders
  8. Launch of a Quality Assurance Program for Platelet Function Disorder Tests
  9. Current and Future Challenges
  10. Acknowledgements
  11. References

External quality assurance programs are essential to total quality management in hematology laboratories. After a successful pilot exercise [30], we postulated that the launch of regular external quality assurance exercises for platelet dense granule electron microscopy (EM) (the predominant method that North American laboratories use to diagnose platelet dense granule deficiency). Similarly, we postulated that external quality assurance exercises on the interpretation of platelet LTA would be useful to diagnostic laboratories and stimulate quality improvement [29]. The dense granule EM exercises were designed to include two clinical samples (one normal and one dense granule deficient), on grids, to be examined by EM at the participating sites, in addition to EM images of similar samples for the sites to quantify dense granules and indicate which structures they would, or would not, count as a dense granule [29, 30]. The LTA exercises, which were distributed to participants from NASCOLA and from the ECAT (External quality Control of diagnostic Assays and Tests) Foundation, consisted of aggregation tracings and worksheets for five actual cases per exercise, chosen to represent important and rare findings that clinical laboratories should be able to assess to properly diagnose platelet function disorders [29]. For the EM exercises, there was excellent performance in pilot and subsequent exercises [29, 30], with almost all participants successfully recognizing which were the normal and dense granule-deficient samples [29]. There was also good consensus on which structures in platelets were classified as a dense granule [29, 30]. For the LTA exercises, we identified significant problems in the interpretation of findings by NASCOLA and ECAT Foundation participants [29]. Diagnostic laboratories often misclassified normal variants, and case interpretations were also problematic, particularly for common findings [29]. There were some common errors. For example, a number of the participants reported that the aggregation findings for an individual with proven dense granule deficiency showed an aspirin-like defect [29]. Interestingly, this had actually happened in real life to the subject whose aggregation findings were used for an exercise. The experiences led us to publish on the performance of diagnostic laboratories in LTA interpretation and the common errors to avoid when reporting on aggregation findings [29]. We suspect that the North American guidelines on LTA interpretation [32] will improve practices over time, as following the recommendations would prevent laboratories from making errors when interpreting aggregation findings [29]. NASCOLA has recently surveyed participants of its platelet quality assurance program, who are enthusiastic about the program and feel that the exercises are quite valuable to their practice (unpublished). It was a pleasure to lead the NASCOLA platelet quality assurance program over a 5-year period.

Current and Future Challenges

  1. Top of page
  2. Summary
  3. Introduction
  4. Perspectives on the Importance of Advancing Blood Disorder Diagnosis
  5. Basic Research Leading to New Diagnostic Tests: the Tale of Quebec Platelet Disorder
  6. Interchanges on Practices as a Mean to Improving Blood Disorder Diagnostic Testing
  7. Research on Current Diagnostic Tests for Bleeding Disorders
  8. Launch of a Quality Assurance Program for Platelet Function Disorder Tests
  9. Current and Future Challenges
  10. Acknowledgements
  11. References

In hematology laboratories, the conditions that we cannot diagnose, including the blood disorders of uncharacterized pathogenesis, are a major challenge that can only be addressed by research. Our own investigations of bleeding disorders confirm that many individuals extensively evaluated for bleeding problems end up with completely normal test findings, yet have significant bleeding problems that mimic von Willebrand disease and platelet function disorders [40, 43]. I strongly believe that we need to improve the diagnosis of uncharacterized platelet function disorders and ‘undefined’ blood disorders by exploring the pathogenesis and translating discoveries into diagnostic tests for blood disorders.

The impacts of a blood disorder diagnosis on patients are often underestimated. On September 28, 2012, I received an affirmation that scientific advances very much matter to patients. The message came from a person with Quebec platelet disorder who had volunteered many times to donate blood for our research studies. It read: ‘Subject: I will be missing you. Dear Catherine, I am dying from my cancer. Another 1-2 weeks and I'll be gone. I want to thank you sincerely and all your team for making our life and our descendants easier for the future……Adios’. To paraphrase what he said earlier, when we found the genetic cause of their disorder and developed a diagnostic test: ‘I used to be so worried if I needed to have an operation or a tooth pulled. The bleeding was pretty horrible. Now there is a test, I know what I have, if my children have it, and why it causes bleeding. So I take tranexamic acid [a fibrinolytic inhibitor drug] when I need an operation and I am normal!’

I am grateful to the many wonderful individuals who have supported our scientific journey and discoveries. I look forward to future discoveries that improve the diagnosis and understanding of blood disorders.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Perspectives on the Importance of Advancing Blood Disorder Diagnosis
  5. Basic Research Leading to New Diagnostic Tests: the Tale of Quebec Platelet Disorder
  6. Interchanges on Practices as a Mean to Improving Blood Disorder Diagnostic Testing
  7. Research on Current Diagnostic Tests for Bleeding Disorders
  8. Launch of a Quality Assurance Program for Platelet Function Disorder Tests
  9. Current and Future Challenges
  10. Acknowledgements
  11. References

Catherine Hayward is a career investigator of the Heart and Stroke Foundation of Ontario and is supported by operating grants from the Canadian Institutes of Health Research (MOP 97942), the Heart and Stroke Foundation of Ontario, and the Canadian Hemophilia Society. We are grateful for the contributions of trainees, collaborators, and colleagues to our research studies, which would not have been possible without the support of the Quebec platelet disorder family and other participants in our research studies.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Perspectives on the Importance of Advancing Blood Disorder Diagnosis
  5. Basic Research Leading to New Diagnostic Tests: the Tale of Quebec Platelet Disorder
  6. Interchanges on Practices as a Mean to Improving Blood Disorder Diagnostic Testing
  7. Research on Current Diagnostic Tests for Bleeding Disorders
  8. Launch of a Quality Assurance Program for Platelet Function Disorder Tests
  9. Current and Future Challenges
  10. Acknowledgements
  11. References
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