Results of an External Proficiency Testing Exercise on Platelet Dense-Granule Deficiency Testing by Whole Mount Electron Microscopy

Abstract

Performance on specialized diagnostic tests for platelet disorders, including dense-granule deficiency, is rarely evaluated by external quality assessment (EQA). Members of the North American Specialized Coagulation Laboratory Association that evaluate platelet dense-granule deficiency commonly use whole-mount electron microscopy (EM) methods. This observation led us to develop a pilot EQA survey with standardized EM images and clinical samples on grids from a healthy control subject and a subject with dense-granule deficiency. The survey participants were 8 centers, including 2 with no experience in platelet whole mount EM. All participants, including inexperienced sites, correctly interpreted findings for the normal and dense-granule-deficient platelets. Among experienced sites, agreement was excellent (>82%) on platelet structures to count or not count as dense granules. Participants indicated that future EQA challenges should include clinical samples on grids and standardized images. This is the first report that platelet EM can be assessed by EQA.

New developments in laboratory diagnosis and monitoring

“…As almost all platelet function tests require rapid processing of freshly collected blood samples, all platelet EQA challenges have used some type of strategy to overcome this need, ranging from performing tests on freshly collected healthy control samples spiked with or without an inhibitory addition, or by partially preparing the material required for evaluation of a platelet function disorder [24]. For example, EQA of electron microscopy assays for diagnosing platelet dense granule deficiency have used air-dried samples of platelet rich-plasma transferred onto grids, and participants have correctly identified normal and dense granule-deficient samples [28]. For LTA EQA, the North American Specialized Coagulation Association and ECAT foundation have performed post-analytical exercises, using distributed aggregation values and tracings for cases, to assess the quality of LTA interpretation.…”

Improving blood disorder diagnosis: reflections on the challenges

“…Through these, other surveys and quality assurance exercises [26,[28][29][30][31][32][33][34][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].…”

“…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].…”

“…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].…”

Super-resolution microscopy in the diagnosis of platelet granule disorders

“…Various functional assays are available, but these do not identify the structural deficit underlying a platelet disorder. Apart from the requirement for the instrumentation and associated sample preparation requirements, skilled interpretation of the images obtained is needed, as the dense granules appear on a complex background that would challenge automated image analysis [6,7]. Furthermore, the samples must be imaged on the day of preparation [8].…”