Testing for platelet function using whole blood

Dr. Reyes Gil said her laboratory always runs a normal control with the patient for the same agonist during whole blood platelet aggregation. In this case, the low-dose ADP release was normal for the control and at acceptable limits for the patient. The high-dose ADP release result was useful, she said, because “if it’s abnormal at low dose but then normal at high dose, we call it normal because we can overcome the deficiency.”

In this case, the ADP release improved on the patient at a higher dose and was considered normal. The pattern for collagen was also normal.
For arachidonic acid, the control result was normal, but the patient’s result revealed no aggregation for arachidonic acid and no ATP release. (For thromboxane A2 analogue, for control and patient: normal aggregation and ATP release.) “So there was a problem with the response to arachidonic acid: zero aggregation, zero ATP release,” Dr. Reyes Gil said. (Von Willebrand studies were normal.)

“So the interpretation of this is that the only abnormality was absent aggregation and ATP release with arachidonic acid,” she said. “We always make sure patients do not take aspirin or NSAIDs” by calling when the aggregation is scheduled and on the day before aggregation when patients are reminded of their appointment and asked whether they took aspirin or NSAIDs in the prior two weeks.

“We knew this patient had not [taken aspirin or NSAIDs], and indeed she mentioned she avoids those medications as she bruises when she does. So we called it an aspirin-like defect,” she said. “This is a very rare, most times autosomal dominant condition. Most times it is a cyclooxygenase deficiency and tends to be mild bleeding and underdiagnosed.”

Aggregometry revealed no response to arachidonic acid but normal aggregation response to thromboxane A2 analogue, indicating its receptor, downstream pathway, and secretion are normal. “This shows us how useful thromboxane A2 is,” she said.

Another case is that of a 34-year-old female from Puerto Rico. The patient had excessive vaginal bleeding and a known platelet disorder in the past, though what disorder it was wasn’t known because the medical record couldn’t be accessed. She had desmopressin for her three deliveries, with no issues.

“With whole blood platelet aggregation, we can measure ATP release with thrombin. The patient has decreased thrombin ATP release when her platelets are induced with thrombin,” she said. With collagen, the patient’s platelets could aggregate, but there was no ATP release. The patient’s platelets were also able to aggregate with ADP, but the ATP release was decreased. With arachidonic acid, the patient’s platelets could aggregate, but there was no ATP release. And with thromboxane A2 analogue, she said, there was a somewhat decreased aggregation compared with the control and no ATP release.

“The pattern we’re seeing is that there is a problem with ATP release and secretion,” Dr. Reyes Gil said, adding that the patient had “a classic presentation of Hermansky-Pudlak,” for which electron microscopy is recommended for confirmation.

Hermansky-Pudlak, which is a storage pool disorder, has a “constellation of symptoms,” such as oculocutaneous albinism, Dr. Reyes Gil said. “It’s common in Puerto Rico [1/1,800 people] because of a founder HPS1 mutation in Puerto Rico but is fairly rare elsewhere [1/500,000].”

Dense granules would be visible by electron microscopy of whole mount of platelets in a normal case, she said, “and in this patient we saw no dense granules.”

The patient was given desmopressin and advised to wear sunscreen. “One of the major complications in this patient is pulmonary fibrosis, so we treated with steroids and immunomodulators.”

The third case is that of a 52-year-old female with a medical history of heavy menses and with chronic, stable thrombocytopenia since 2001. The patient presented for recent menorrhagia with symptomatic anemia requiring RBC transfusion.

The platelet aggregation study result (whole blood) with arachidonic acid was decreased compared with the normal control, as was the ATP release. “When we looked at thromboxane A2 analogue U46619, there was absolutely no response,” Dr. Reyes Gil said. The ATP release was also decreased.

“So in this patient we see absent aggregation with ADP and thromboxane A2, and decreased aggregation with collagen and arachidonic acid. However, there is normal ATP release with ADP and low-dose collagen, decreased ATP release with high-dose collagen and arachidonic acid, and normal response to ristocetin.”

Platelet electron microscopy revealed normal dense granules, so it was not a dense granule deficiency.

In looking at the thin section platelet transmission electron microscopy, the patient was seen to have normal mitochondria, tubular networks, glycogen, and dense granules. “But we see no alpha granules,” she said (Fig. 1).

“This is called gray platelet syndrome. It’s a rare congenital autosomal recessive bleeding disorder caused by a reduction or absence of alpha granules in the blood platelets.” Mutations in the NBEAL2 gene on chromosome 3p is the cause for about 90 percent of cases. The NBEAL2 gene encodes a protein containing a BEACH domain that is involved in vesicular trafficking, she said.

Case No. 4 is that of a one-year-old male with Jacobsen syndrome and persistent borderline thrombocytopenia who presented for follow-up. The patient’s screening test for coagulation was normal.

“This patient had a delayed response to arachidonic acid compared to the control,” Dr. Reyes Gil said. He had no ATP release with arachidonic acid and a somewhat delayed response to and no ATP release with thromboxane A2.

The aggregation studies were normal for other agonists, but the decreased ATP release and the delayed response to arachidonic acid and thromboxane A2 were significant. “It’s somewhat similar to what we saw before in the storage pool disorder,” she said.

“When we looked at the platelet electron microscopy, we found large, giant alpha granules” and an increased tubular network (Fig. 2).

Jacobsen syndrome is a rare disorder caused by a congenital partial deletion of chromosome 11q23. About 90 percent of JS patients have a mild bleeding disorder called Paris-Trousseau syndrome, which is a macrothrombocytopenia characterized by large platelets and mucocutaneous bleeding. The electron microscopy showed giant alpha granules and decreased numbers of dense or delta granules (Krishnamurti L, et al. Am J Hematol. 2001;66[4]:295–299).

Dr. Reyes Gil said that her laboratory at Montefiore had analyzed more than 100 cases using the thromboxane A2 analogue in whole blood studies. “We have pretty good sensitivity [94 percent with a single agonist; 100 percent with more than two agonists] and specificity [88 percent with a single agonist; 96 percent with more than two agonists] using the U46 in whole blood. It’s useful because it allows us to differentiate storage pool disorders—where U46 would be abnormal along with arachidonic acid—from aspirin-like defects, in which only arachidonic acid would be abnormal but there should be normal response to U46.”

Dr. Reyes Gil’s final case was an acquired platelet function disorder. “Most acquired conditions known to be associated with a platelet dysfunction don’t need to have a platelet aggregation,” she said. “If you know the patient to have a myeloproliferative syndrome and/or thrombocythemia, you don’t need to do platelet aggregation studies.” In some cases where the cause is unknown, though, “it may be helpful.”

In this case, a 53-year-old male was being treated with multiple chemotherapeutic agents for stage IV colon cancer. Every time he received chemotherapy infusions, his platelet count dropped significantly. The patient’s hematologist requested that the laboratory test each individual chemotherapy drug in the patient’s blood just before his next chemotherapy cycle. The platelet aggregation results showed no aggregation with the drug fluorouracil but a strong platelet aggregation with low and higher doses of the drug leucovorin, but not with the control. A large ATP release was also shown with leucovorin.

“We looked at the clot formation in the electrode that we put in the cuvette with the blood, and we were able to see a very large clot with only leucovorin in this patient but not in the control,” Dr. Reyes Gil said.

“Most times we don’t do platelet aggregation studies for acquired conditions,” she said, “but in this particular case, it was extremely useful.” The patient’s thrombocytopenia improved after withdrawal of leucovorin. “This was likely a drug-induced autoantibody that induces platelet activation in the presence of the drug. This can be confirmed by flow cytometry, but only a few laboratories offer this test.”

Amy Carpenter Aquino is CAP TODAY senior editor.