Testing for platelet function using platelet-rich plasma

Amy Carpenter Aquino

July 2021—Identifying severe disorders of primary hemostasis is relatively straightforward for most coagulation laboratories, but the more prevalent disorders with less severe bleeding and less overt diagnostic abnormalities are trickier, and platelet function testing using platelet-rich plasma remains the gold standard.

Geoffrey Wool, MD, PhD, in an AACC virtual session last year, presented some of his laboratory’s cases to illustrate the use of light transmission aggregometry and a modification called lumi-aggregation.

“The principle of LTA is based on the idea that light passes more easily through a clear than a turbid solution. It’s pretty basic,” said Dr. Wool, medical director of the coagulation laboratory and associate professor of pathology, University of Chicago.

He compares this principle to a plateletpheresis unit seen in the blood bank, which is turbid and through which light can’t pass easily. But a rapidly centrifuged plasma sample is clearer. One is platelet-poor plasma (PPP); the other is platelet-rich plasma (PRP), “and the difference in the light passage is the underpinning of light aggregation testing.”

“We’re going to take aliquots of that PRP, add various platelet activating agents, and then measure how well or not the platelets respond,” he said. Platelet activation is measured by clumping (or aggregation). As platelets are activated, their surface receptors for fibrinogen (GpIIb/IIIa) adopt an active conformation and platelets form aggregates with fibrinogen. As the platelet aggregates form, light passage through the cuvette of PRP increases.

Additionally, when platelets are activated, their granules will be brought to the membrane surface to fuse and degranulate. Release of ATP is often used as a marker for dense granule release.

“What we’re taking advantage of in lumi-aggregation,” Dr. Wool said, “is measuring the ATP from the platelets by adding firefly luciferin and luciferase and then measuring the visible light that’s generated when the ATP is released from the dense granules.”

Light transmission aggregometry (LTA) is not a perfect test, he said. “It’s performed under nonphysiologic conditions in a stirred solution under relatively low shear conditions. In contrast, we know that platelets function most potently in vivo to bind to surfaces under high shear stress, such as a damaged surface of an artery or arteriole. That’s where they’re doing a lot of their work, so with that comparison we can see how relatively nonphysiologic LTA testing is.”

Dr. Wool and his colleagues at the University of Chicago use lumi-LTA, and one of their cases was that of a Puerto Rican woman worked up for lung transplant. She had lifelong easy bruising and prolonged bleeding with minor injuries or dental or surgical procedures. She had no children but had a brother with increased bleeding. Her platelet count, mean platelet volume, and other CBC results were normal.

Her platelet aggregation results showed mildly to moderately reduced aggregation responses to a variety of agonists, including epinephrine, arachidonic acid, thromboxane analogue, thrombin receptor activating peptide, and collagen. Her adenosine diphosphate (ADP) and ristocetin aggregation responses were normal. In marked contrast, “her ATP release was uniformly absent,” Dr. Wool said, implying a severe absence or dysfunction of her delta granules.

“So her diagnosis was a dense granular deficiency called Hermansky-Pudlak, and she has type 1,” which is more common in people of Puerto Rican ancestry, he said. “Type 1 is associated with pulmonary fibrosis as well,” explaining her need for lung transplant.

Hermansky-Pudlak is an autosomal recessive disorder characterized by dense granule deficiency and a mutation in the HPS gene, and, in addition to bleeding, it is associated with oculocutaneous albinism. “The ADP and ATP in the dense granules aren’t coming out to stimulate the other platelets in the PRP. So the aggregation responses are reduced but variably, depending on the strength of the agonist you’re using. But the ATP release will be uniformly terrible,” Dr. Wool said.

In another case, a 39-year-old woman who had childhood epistaxis, lifelong bruising predisposition, and menorrhagia had a normal platelet count and MPV. Her red and white blood cell parameters were unremarkable, and her von Willebrand disease panel was negative.

Her platelet aggregation responses were reduced to most agonists, most severely for arachidonic acid. Secretion was significantly reduced for most agonists. The strong activators—thromboxane analogue, thrombin receptor activating peptide, and the calcium ionophore A23187—revealed normal to mildly reduced responses with appropriate secretion, he said.

“If we put all of her responses together, the worst response was the arachidonic acid. The next worst were the epinephrine responses, then collagen at two micrograms per mL,” Dr. Wool said. “This is a classic pattern of absent response to arachidonic acid, poor response to epinephrine, and poor response to low concentration of collagen, but relatively normal response to a high concentration of collagen that should make you think of aspirin.” Aspirin (acetylsalicylic acid) is a cyclooxygenase inhibitor that leads to total unresponsiveness to arachidonic acid, he said, and also leads to poor responsiveness to weak platelet agonists.

The juxtaposition of a “quite terrible” response to arachidonic acid and a normal response to thromboxane analogue U44619 should “hammer home the thought that this might be aspirin related,” he added.

The cyclooxygenase metabolic pathway uses a polyunsaturated fatty acid substrate such as arachidonic acid and eventually produces the prostaglandin thromboxane A2. If normal platelets are exposed to exogenous arachidonic acid, he explained, their cyclooxygenase and thromboxane synthase enzymes will produce thromboxane and lead to platelet aggregation. In aspirinated platelets, on the other hand, “if we feed in arachidonic acid at the top of the cyclooxygenase pathway, nothing happens.” If synthesized thromboxane (or analogue) is provided, however, aspirinated platelets can still respond well.

“So the pattern of platelet responses for this patient is telling you there’s a defect in the pathway between arachidonic acid and thromboxane,” Dr. Wool said. “This should make you think the patient was taking something with aspirin until proven otherwise.”

Findings were persistent on a second lumi-aggregation study. In extensive discussions with the patient, she denied using aspirin or over-the-counter products that can contain aspirin. “We even did a salicylate screen in the chemistry lab, and she had a negative screen.”

“So this patient has what’s called an aspirin-like defect,” Dr. Wool said (Rao GHR, et al. Am J Hematol. 1981;11[4]:355–366). “She had a congenital platelet disorder of either the cyclooxygenase pathway or the thromboxane synthetase pathway that prevented her platelets from responding to arachidonic acid or making thromboxane. Once you added thromboxane or an analogue of it, the platelets responded perfectly well. But they weren’t able to make their own thromboxane.”

The case of a 56-year-old woman with excessive bleeding after a cervical electrical excision procedure illustrates the importance of patient preparation for LTA. The patient had no prior history of bleeding before the procedure and she had normal CBC results. On her current medication list were levothyroxine, liothyronine, and lansoprazole, as well as several dietary supplements, each containing many herbal ingredients.

Dr. Wool described the patient’s platelet aggregation responses as generally normal, but with a significantly deficient response to thromboxane analogue U46619: Aggregation reached only to 15 to 30 percent and then platelets completely deaggregated. This shows that the platelet clumps were poorly/weakly made and fell apart under the stirring conditions of the LTA. Responses to other agonists were generally normal.

“She seemed to have an isolated deficiency of response to thromboxane, which is not a common pattern,” Dr. Wool said. “Even more bizarrely, if she had a significantly reduced response to thromboxane, you would think the response to arachidonic acid would be correspondingly reduced, but that’s not what we saw.” The patient’s arachidonic acid response was only mildly reduced and not nearly as much as her response to thromboxane. “It was a strange aggregation pattern with absent response to thromboxane.”

The peripheral blood stain was even odder, he said. The majority of platelets lacked the blue granular staining indicating alpha granules in a Wright-Giemsa–stained blood smear. “There were nicely granular neutrophils, relatively normal red cells, and there was a variety of platelet morphologies. Some of them looked relatively normal with blue granules in them, but most of them were pale, consistent with significant reduction in the alpha granular content.”

Dr. Wool said his team was surprised to see such a severe platelet function defect, called “gray platelet syndrome.” “And the thromboxane response as a standalone abnormality was rather odd. So we asked to do this study again once the patient was not taking her dietary supplements.”