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Demystifying Platelet Function Testing

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September 2023

Ruth Jessen Hickman, MD

Ruth Jessen Hickman, MD, is a freelance medical and science writer based in Bloomington, Indiana.

Platelet function testing is a key part of diagnosing platelet function disorders. Yet, a single, readily available, easy-to-implement clinical test is not available to diagnose the wide spectrum of inherited platelet disorders, many of which are caused by unidentified genes. Instead, hematologists rely on a combination of tests that are not available at every institution.

“In many areas of the world, it’s difficult for patients to get access to platelet function testing. We need to have continued conversations about how to better advocate for patients and make testing more accessible,” noted Catherine P. Hayward, MD, PhD, a hematologist and professor of pathology and molecular medicine at McMaster University in Hamilton, Ontario.

ASH Clinical News spoke with Dr. Hayward and Sara J. Israels, MD, a pediatric hematologist and professor of pediatrics and child health at the University of Manitoba in Winnipeg, about the variety of platelet function tests available, their usefulness and limitations, and related topics on diagnosis and treatment.

Defining Platelet Functionality

Platelets are multifunctional anucleate cellular fragments that play key roles in hemostasis, vessel constriction and repair, inflammation, host defense, the promotion of atherosclerosis, and tumor growth in patients with cancer. However, currently available platelet function tests assess only alterations related to the hemostatic process.1

Platelets play a key and complex role in primary hemostasis, as they bind to collagen at damaged vessel walls and to von Willebrand factor through various surface receptors. Local prothrombotic factors activate platelets, which change their shape and their expression of surface proteins. They release the contents of their various granules containing serotonin, adenosine diphosphate (ADP), and various other factors which promote further platelet activation. Activated platelets extend filopodia and aggregate, ultimately forming a platelet plug.2

Although inherited platelet disorders all share a heightened risk of bleeding and excessive hemorrhage after surgery or trauma, to varying severity, they are quite heterogeneous in their different pathophysiologic mechanisms. Acquired platelet disorders, such as those from myelodysplastic syndromes, add an additional layer of complexity.

For example, platelet disorders may be due to abnormalities of the platelet receptors for various adhesive proteins, as in Bernard-Soulier syndrome or Glanzmann thrombasthenia, but they may also result from abnormalities of platelet receptors for soluble agonists, such as the thromboxane A2 receptor. They can result from abnormalities of delta-platelet granules, as with Chediak-Higashi syndrome, or alpha-platelet granules, such as in Quebec platelet disorder, as well as from abnormalities of signal transduction.3

This heterogeneity is part of what makes it difficult to develop a single, easily used test to assess platelet function and diagnose specific platelet disorders. “There has been a great appetite to see if we could find a global test that would identify those with a platelet disorder, but unfortunately, we haven’t succeeded yet,” Dr. Israels explained.

Work-up for Suspected Platelet Function Disorder

Patients with a history of excess mucocutaneous bleeding warrant work-up for a potential platelet disorder. Standardized bleeding questionnaires can help quantify information from the patient history, and the exam and family history lend important clues for potential syndromic and familial disease.

Initial investigations for a potential bleeding disorder should include a complete blood count, a review of the blood film, international normalized ratio/prothrombin time, activated partial thromboplastin clotting time, thrombin time, fibrinogen, and a screen for von Willebrand disease (VWD).4 Bleeding times, formerly used as part of initial work-up of platelet function disorders, are no longer recommended.

Dr. Hayward noted that turnaround times for platelet function tests tend to be quick. Delays are more likely to come from the time needed to get the requisite consultation appointment and related testing.

Point-of-Care Platelet Function Testing

As a replacement for bleeding time, some institutions use automated global hemostasis tests. The platelet function analyzer (PFA)-100 or PFA-200 evaluates platelet function by measuring the time required for whole blood under high shear stress to occlude holes in a membrane coated with collagen plus epinephrine or ADP; the time to close the hole is reported in seconds. Thus, it provides some measure of both platelet adhesion and aggregation.3

However, Dr. Israels noted, “You can’t use it as a screening test. It does not differentiate between VWD and a platelet function abnormality.”

“Your center has to make a choice about whether or not you want an ancillary test. If it’s abnormal, you have to go further for diagnosis. If it’s normal, you have to go further because you haven’t excluded common defects,” Dr. Hayward added.

Aggregometry Studies and LTA

The gold standard for testing platelet function is light transmission aggregometry (LTA). “When performed and interpreted properly, LTA probably tells us more about platelet function than anything else we have available in the clinical lab,” Dr. Israels noted. Depending on institutional availability, it might be performed as part of an initial bleeding disorder work-up, but it is not available at all centers.

The core principles of LTA have not changed since its independent development by Gustav Born and John R. O’Brien in 1962. The technique uses platelet-rich plasma between a light source and a photocell. Initially, the cloudy platelet suspension allows little light to pass through. Platelet responses to multiple agonists such as epinephrine, ADP, and collagen are tested, with platelet aggregate formation causing the sample to become clearer, and the increased light transmission is recorded over time.5

Various inherited and acquired platelet disorders tend to display different patterns that convey whether their response to various agonists is normal, decreased, or absent; specific patterns suggest particular diseases. However, Dr. Israels said, “Many people with mild or moderate abnormalities may have a poor response to one or two agonists without a classic pattern that can help us with a specific diagnosis.”

LTA is time-consuming and technically challenging to perform. “To some degree, it’s always a laboratory-developed test,” Dr. Hayward said. Various pre-analytical conditions, like the type of anticoagulant, can affect the results, as can different procedural conditions, such as different concentrations of agonists.1 In recent years, experts have collaboratively developed guidelines to improve the standardization and reliability of the technique among labs.5,6

“LTA requires somebody with considerable expertise to evaluate the findings, but with the guidelines that we published on how to perform LTA and interpret findings, people have a good framework,” Dr. Hayward explained.

One challenge of LTA and other related platelet function tests is that blood can’t be sent away for remote analysis. “Patients may need to travel to a lab that performs it. Samples need to be fresh, in the lab in a few hours, and they need to be collected properly,” Dr. Israels said.

Another challenge is that a somewhat large volume of blood needs to be collected for proper LTA testing, which may be challenging for small children. Dr. Israels noted children may also be at higher risk of having traumatic blood draws, which can prematurely activate platelets and confuse the interpretation of LTA and other platelet function studies. Partly because of this possibility, it is useful to repeat testing for individuals with abnormal LTA results to confirm the findings before diagnosis in children and adults.

LTA is more challenging to perform and interpret in the context of thrombocytopenia, and some labs even decline to run LTA in such patients. However, Dr. Hayward noted, “Around 11% of our patients that come in for testing have a low platelet count sample. They can have important conditions like Bernard-Soulier syndrome that are very hard to diagnose without starting with an aggregation test.”

Adjustments in procedures, including interpretation, may be needed for LTA in the context of thrombocytopenia. Experts in the area are developing new recommendations for laboratories.

An alternative to LTA that provides similar but not identical information is impedance aggregometry, which works on the principles of impaired electrical conductance. One advantage is that some applications use whole blood with a semi-automated system.

Dr. Israels explained that most clinical labs would choose to employ either LTA or impedance aggregometry but not both, and most North American labs have opted for LTA. Although it may be used to diagnose severe platelet function disorders, it appears to be inferior to LTA, especially for identifying mild disorders.7

Granule Analysis

Hemostasis labs often perform assays that measure delta (dense)-granule secretion in addition to LTA, for complementary information on an additional aspect of platelet function.

Several different assays are available. One common approach is lumi-aggregometry, performed while aggregation data are collected via classical LTA. A luciferin-luciferase reagent and an agonist are added to a sample of platelet-rich plasma. Adenosine triphosphate released from the platelet granules reacts with the reagent, and the resulting light emission is quantified relative to a standard. However, lumi-aggregometry cannot distinguish between delta-granule deficiency and primary secretion defects, and it does not address secretion of alpha granules.7

To differentiate between delta-granule deficiency and a secretion defect, delta granules can be counted by whole mount electron microscopy (EM), not available at all clinical hemostasis laboratories. Trained laboratory staff count the average number of electron dense granules per platelet to determine if delta-granule deficiency is present.

At her institution, Dr. Hayward screens with whole mount EM at the time of LTA, using the same sample. “Platelet dense granule deficiency is almost as common as VWD, and LTA and lumi-​aggregometry aren’t adequate to screen for it,” she said.

Transmission EM can also be helpful for a select subset of patients with a suspected ultrastructural abnormality, such as absent alpha granules in gray platelet syndrome. “If you want to look at the ultrastructure of platelets, then they need to be fixed, embedded, and cut into thin sections. This requires access to specialized equipment and staff and is more labor intensive,” Dr. Israels said.

Art by Arif Biswas/

Flow Cytometry

Flow cytometry can be helpful to analyze platelet surface glycoprotein and platelet activation markers.7 Dr. Hayward noted that flow cytometry can be especially helpful for diagnosing inherited disorders with glycoprotein deficiencies, such as Glanzmann thrombasthenia and Bernard-Soulier syndrome, as the lack of receptors can be detected, even in those with a low platelet count.

Researchers have also been exploring whether flow cytometry could be used to assess platelet activation and function instead of LTA, but this approach is not widely used in the clinical setting.7

“It has the advantage of using smaller volumes of whole blood compared to other assays measuring activation. It has a lot of promise as a clinical tool in the future,” Dr. Israels said.

Diagnostic Challenges and Impacts

Doctors are often unable to diagnose a specific cause when a patient has a suspected platelet disorder, even in patients who have undergone extensive testing. In some cases, genetic tests may be helpful, especially if family history or other medical context suggests a specific platelet disorder. But because platelet disorders are so heterogenous, with the underlying genes in many cases unknown, genetic panel testing often comes up negative.

Dr. Hayward explained that patients are relieved when they do finally get a diagnosis of a bleeding disorder. She noted the more common inherited platelet disorders tend to respond to the same types of treatment as mild VWD, such as desmopressin. Patients with acquired platelet disorders can also be diagnosed by the same laboratory approach, although the bleeding may not respond as well to desmopressin treatment. It’s also helpful to know that a patient has a platelet problem in case platelet transfusion is required.

Sometimes knowing the specific platelet disorder influences management. For example, Dr. Hayward noted that Quebec platelet disorder requires a different treatment approach, such as fibrinolytic inhibitors and not desmopressin or platelet transfusions. Some platelet disorders also have specific syndromic effects, like lung problems or increased blood cancer risks. Having an official diagnosis can benefit the holistic management of patients and their families.

For a pragmatic guideline related to diagnosing inherited platelet function disorders, Dr. Israels highlighted one from the British Society of Haematology, which includes a variety of case studies showing how a clinician might approach sequential testing in specific clinical contexts.8 She also recommends that clinicians reach out to their local hemostasis lab director for insight on difficult cases.

Dr. Israels noted that platelet function testing can be a challenge for some clinical hematologists because of limitations in their local testing resources. “They will often do von Willebrand testing as the initial work-up and then stop.” At least for select groups of patients with negative von Willebrand testing and significant mucocutaneous bleeding, she encourages clinicians to refer patients to centers where they can get a comprehensive assessment of platelet function.


  1. Paniccia R, Priora R, Liotta AA, Abbate R. Platelet function tests: a comparative review. Vasc Health Risk Manag. 2015;11:133-148.
  2. Periayah MH, Halim AS, Mat Saad AZ. Mechanism action of platelets and crucial blood coagulation pathways in hemostasis. Int J Hematol Oncol Stem Cell Res. 2017;11(4):319-327.
  3. Podda G, Femia EA, Cattaneo M. Current and emerging approaches for evaluating platelet disorders. Int J Lab Hematol. 2016;38 Suppl 1:50-58.
  4. Hayward CPM. How I investigate for bleeding disorders. Int J Lab Hematol. 2018;40 Suppl 1:6-14.
  5. Cattaneo M, Cerletti C, Harrison P, et al. Recommendations for the standardization of light transmission aggregometry: A consensus of the working party from the Platelet Physiology Subcommittee of SSC/ISTH. J Thromb Haemost. 2013;10.1111/jth.12231.
  6. Hayward CP, Moffat KA, Raby A, et al. Development of North American consensus guidelines for medical laboratories that perform and interpret platelet function testing using light transmission aggregometry. Am J Clin Pathol. 2010;134(6):955-963.
  7. Bourguignon A, Tasneem S, Hayward CP. Screening and diagnosis of inherited platelet disorders. Crit Rev Clin Lab Sci. 2022;59(6):405-444.
  8. Gomez K, Anderson J, Baker P, et al. Clinical and laboratory diagnosis of heritable platelet disorders in adults and children: A British Society for Haematology guideline. Br J Haematol. 2021;195(1):46-72.


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