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

Brunet, Justin; Iyer, Janaki Krishnamoorthy; Badin, Matthew; Graf, Laura; Moffat, Karen A.; Timleck, Marnie; Spitzer, Ernie; Hayward, Catherine P.M.

doi:10.1111/ijlh.12801

Cited by 44 publications

(72 citation statements)

References 27 publications

(97 reference statements)

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“…The current diagnostic approach for δ‐SPD does not include a rapid screening test and could benefit from an additional test such as flow cytometry. Unlike the currently available diagnostic tools, flow cytometry is applicable in thrombocytopenic samples and requires only a small sample volume, allowing rapid exclusion of δ‐SPD in children . Our data indicate that flow cytometric analysis is very reproducible, even immediately after blood collection.…”

Section: Discussionmentioning

confidence: 80%

Flow cytometric mepacrine fluorescence can be used for the exclusion of platelet dense granule deficiency

Asten

Blaauwgeers

Granneman

et al. 2020

Journal of Thrombosis and Haemostasis

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Background δ‐storage pool disease (δ‐SPD) is a bleeding disorder characterized by a reduced number of platelet‐dense granules. The diagnosis of δ‐SPD depends on the measurement of platelet ADP content, but this test is time consuming and requires a relatively large blood volume. Flow cytometric analysis of platelet mepacrine uptake is a potential alternative, but this approach lacks validation, which precludes its use in a diagnostic setting. Objectives To evaluate the performance of platelet mepacrine uptake as a diagnostic test for δ‐SPD. Patients/Methods Mepacrine fluorescence was determined with flow cytometry before and after platelet activation in 156 patients with a suspected platelet function disorder and compared with platelet ADP content as a reference test. Performance was analyzed with a receiver operating characteristic (ROC) curve. Results Eleven of 156 patients had δ‐SPD based on platelet ADP content. Mepacrine fluorescence was inferior to platelet ADP content in identifying patients with δ‐SPD, but both mepacrine uptake (area under the ROC curve [AUC] 0.87) and mepacrine release after platelet activation (AUC 0.80) had good discriminative ability. In our tertiary reference center, mepacrine uptake showed high negative predicitive value (97%) with low positive predictive value (35%). Combined with a negative likelihood ratio of 0.1, these data indicate that mepacrine uptake can be used to exclude δ‐SPD in patients with a bleeding tendency. Conclusion Mepacrine fluorescence can be used as a screening tool to exclude δ‐SPD in a large number of patients with a suspected platelet function disorder.

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Section: Discussionmentioning

confidence: 80%

Flow cytometric mepacrine fluorescence can be used for the exclusion of platelet dense granule deficiency

Asten

Blaauwgeers

Granneman

et al. 2020

Journal of Thrombosis and Haemostasis

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“…The whole mount EM examination of platelet dense granules (DG), that is used to assess for DG deficiency (DGD), has an acceptable, within‐subject CV of 12% for control samples . Although the CV for this test is higher (28%) when platelets have reduced DG numbers, almost all moderate‐to‐severe DGD is confirmed on subsequent tests …”

Section: Resultsmentioning

confidence: 99%

“…This type of comparison is superior to comparing those with a PFD to healthy controls, although a useful test will detect more abnormalities among referred patients than controls unless the comparison is underpowered and abnormalities are rare. Impaired LTA with two or more agonists (evaluated with either native or platelet count–adjusted platelet‐rich plasma [PRP], OR: 23‐42), and confirmed DGD by EM (OR: 91), is very predictive of a bleeding disorder (note: the 95% confidence intervals [CI] for the OR estimate does not cross 1) (Table ), and DGD is also associated with increased bleeding scores . In contrast, confirmed impaired platelet dense granule ATP release is not predictive of a bleeding disorder (OR: 1.5 and the 95% CI cross 1) (Table ) or increased bleeding scores .…”

Section: Resultsmentioning

confidence: 99%

“…Ideally, all PFD tests used for diagnosing IPFD among patients referred for bleeding disorder assessment would have an estimated likelihood, or odds ratio (OR), that an abnormal result is associated with a bleeding disorder, based on prospective studies of representative patient populations. 5,27,31 , and DGD is also associated with increased bleeding scores. 5 In contrast, confirmed impaired platelet dense granule ATP release is not predictive of a bleeding disorder (OR: 1.5 and the 95% CI cross 1) (…”

Section: Update On the Predictive Value Of Platelet Function Disordmentioning

confidence: 89%

“…Platelet function disorders (PFD) are clinically important bleeding disorders that are particularly challenging for clinical laboratories to diagnose. Many PFD are associated with increased bleeding scores and increased risks for bleeding . Commonly, laboratory tests for PFD are performed to investigate unexplained bruising, heavy menstrual bleeding, prolonged and excessive bleeding from cuts, nosebleeds, and dental and surgical procedures .…”

Section: Introductionmentioning

confidence: 99%

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Update on diagnostic testing for platelet function disorders: What is practical and useful?

Hayward

Moffat

Brunet

et al. 2019

Int J Lab Hematology

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Introduction Platelet function disorders (PFD) are an important group of bleeding disorders that require validated and practical laboratory strategies for diagnosis. Methods This review summarizes the authors’ experiences, current literature, and an international survey to evaluate the practices of diagnostic laboratories that offer tests for PFD. Results Blood counts, blood film review, and aggregation tests are the most commonly performed investigations for PFD and help determine whether there is thrombocytopenia and/or defective platelet function due to a variety of causes. The performance characteristics of tests for PFD, and the level of evidence that these tests detect bleeding problems, are important issues to determine where tests are useful for diagnostic or correlative purposes, or research only uses. Platelet aggregation assays, and quantitative analysis of platelet dense granule numbers, are tests with good performance characteristics that detect abnormalities associated with increased bleeding in a significant proportion of individuals referred for PFD investigations. Lumiaggregometry estimates of platelet adenosine triphosphate release show greater variability which limits the diagnostic usefulness. Diagnostic laboratories report that fiscal and other constraints, including a lack of high‐quality evidence, limit their ability to offer an expanded test menu for PFD. Conclusion PFD are clinically important bleeding disorders that remain challenging for diagnostic laboratories to investigate. While some PFD tests are well validated for diagnostic purposes, gaps in scientific evidence and resource limitations influence diagnostic laboratory decisions on which PFD tests to offer.

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Postoperative tonsil/adenoidectomy bleeding management in patients with diagnosed bleeding disorders

Pai

Desai

Stein

et al. 2021

Laryngoscope Investig Oto

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Objective Tonsil/adenoidectomy (T/A) is a commonly performed procedure with an average post‐tonsillectomy bleed (PTB) rate between 3 and 5%. Patients with bleeding disorders (BDs) are believed to have an increased risk of PTB. We hypothesize that our medical management of BD patients using a combination of DDAVP/antifibrinolytic agents has a similar PTB rate to control patients. This study suggests a standardized protocol for patients with BDs to avoid PTB. Methods A retrospective cohort study was completed for patients with BD who underwent tonsillectomy or T/A at Promedica Toledo or Flower Hospital between 2013 and 2020. Exclusion criteria included incomplete records, diagnosis of BD after surgery, and inability to find age and sex matched control. We defined the control group as patients who underwent T/A without BD. The following variables were collected: age, sex, medical history, BD severity, medications, type of surgery, indication for surgery, estimated blood loss (EBL), pre/postoperative medications, PTB status, and post‐PTB intervention. Results A total of 164 patient charts were reviewed. There were 82 patients in both cohorts. The BDs represented were platelet function disorder (80.5%), von Willebrand disease (14.6%), and others such as Factor VII and IX deficiency (4.9%). Of the BD patients included, 13.4% had severe disease. There was no significant difference between the age, sex, EBL, and PTB rates. Of the 8 BD patients with PTB, 62% bled 9‐10 days postoperatively and none had severe disease. Conclusion Our protocol to prevent PTB in patients with BDs produced similar bleed rates to control patients in this study. Further studies are required to assess postoperative length of antifibrinolytic treatment in BD patients. Level of Evidence III.

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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

Abstract: Whole mount EM is useful for the evaluation of suspected PFD due to DGD and detects abnormalities associated with bleeding.

Cited by 44 publications

References 27 publications

Flow cytometric mepacrine fluorescence can be used for the exclusion of platelet dense granule deficiency

Flow cytometric mepacrine fluorescence can be used for the exclusion of platelet dense granule deficiency

Update on diagnostic testing for platelet function disorders: What is practical and useful?

Postoperative tonsil/adenoidectomy bleeding management in patients with diagnosed bleeding disorders

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