Digging into drug’s effect on aPTT-based assays

In a chromogenic FVIII activity assay using human-derived reagents, the effect of emicizumab is apparent and results in an overestimation of the patient’s FVIII activity. Thus, laboratories need to recognize this point and use a chromogenic FVIII activity assay with bovine-derived reagents (using bovine FIXa and FX components), which are insensitive to the presence of emicizumab.

Some clotting and chromogenic assays, such as fibrinogen, thrombin time, PT-based APC resistance, anti-Xa, protein C activity (chromogenic method), antithrombin activity, and plasminogen activity, are not affected by emicizumab. “Certainly the ELISA-based assays, the latex immunoturbidimetric assays that we use for von Willebrand’s and other things, are not going to be impacted by emicizumab, and obviously genetic tests would not either,” Dr. Goodwin said.

He explained why emicizumab affects the aPTT clot-based assays in particular. “Let’s say we’re monitoring a patient factor VIII activity level. We’ll take the sample, we’ll add factor VIII deficient plasma, and we’ll add our activator [with phospholipid and calcium sources]. We will run a PTT, record that clot time, and we will understand that the PTT is inversely proportional to the concentration of the patient’s factor VIII.” If the aPTT is prolonged, factor VIII is low. If it’s short, factor VIII activity is higher.

Since emicizumab causes shortening of the aPTT, “it’s no wonder that you’re going to have an overestimation of the patient’s factor VIII activity.”

Chromogenic factor VIII activity assays that use a human reagent will be sensitive to the presence of emicizumab “and in the U.S. particularly, it can act as a qualitative assessment for emicizumab activity,” he said—is emicizumab present or absent? Chromogenic factor VIII activity assays using bovine reagents provide a quantitative assessment of factor VIII activity in the patient sample due to either factor replacement therapy or the patient’s own endogenous factor VIII. “This is the one you would use if you were doing a Bethesda assay to look for potential inhibitors in the patient.”

The chromogenic assay works by adding activated factor IX, activated thrombin, and factor X inactivated, in excess, to the patient sample with phospholipid and calcium. “The patient, dependent on how much factor VIII they have, is going to bind with factor IX and activate factor X, so this assay in the first stage is going to generate factor Xa.”

The limiting factor is how much factor VIII the patient has, he added.

“We then take that patient’s plasma that now has activated X and we add a chromogenic substrate. Factor X, in its activated form, will cleave the chromogenic substrate and produce a chromogenic signal, which can then be measured at 405 nanometers. In this scenario, the amount of signal that is measured at 405 nanometers is proportional to the amount of factor VIII the patient had in their sample back in the first stage.”

Whether the reagent is human or bovine is the crucial point to remember, Dr. Goodwin said. The former is sensitive to the presence of emicizumab, the latter is not. Thus, in using a chromogenic assay in a dose-dependent manner, the human reagent will show increasing factor VIII activity based on the concentration of emicizumab. With a bovine reagent, “the only factor VIII activity that is going to be detected is what the patient is able to contribute to the assay system” (Adamkewicz JI, et al. Thromb Haemost. 2019;119[7]:1084–1093).

Drs. Unold and Goodwin completed the CAP20 presentation with hypothetical case-based scenarios to help solidify the information presented. For case No. 1, the participants were asked to choose which findings were most consistent for a patient with hemophilia A:

• 14-year-old woman with heavy menses and frequent, life-long epistaxis with normal PT, prolonged aPTT, and thrombocytopenia.
• 17-year-old man with easy bruising, frequent joint dislocations, a painful right knee joint, and visible vasculature with normal PT, aPTT, and VWF assays.
• 45-year-old man with oozing from his surgical site beginning 24 hours following a colon resection, with normal PT and aPTT, and increased lysis on a viscoelastic assay.
• two-year-old boy with multiple bruises on both his extensor and non-extensor surfaces, plus a swollen knee two days after falling out of bed, with normal PT and a prolonged aPTT, which fully corrects after immediate and incubated 50:50 aPTT mixing studies.
• 34-year-old woman with four miscarriages and a current edematous right lower extremity, with normal PT, prolonged aPTT, and a 50:50 aPTT immediate mixing study that fails to correct.

The correct answer is the fourth, the two-year-old boy. Dr. Unold explained that the scenario for the 14-year-old female patient would be more consistent with von Willebrand disease since hemophilia A affects primarily males, though females can be carriers. The patient’s prolonged aPTT could have been due to decreased factor VIII as it relates to von Willebrand disease.

The 34-year-old female patient’s history of miscarriages prompted thoughts of thrombophilia and potential antiphospholipid antibodies, and her edematous lower extremity likely represents a DVT. “The failure to mix would also indicate potential antiphospholipid antibodies, which are inhibitors essentially, rather than a factor deficiency,” he said.

The normal aPTTs of the two older male patients reduced the likelihood of hemophilia A diagnoses for them. Of the 17-year-old male, Dr. Unold said, “Certainly this clinical presentation, with easy bruising and joint dislocations, you would see in a patient with connective tissue disorder, perhaps Ehlers-Danlos.” The 45-year-old man who had oozing from the surgical site and increased lysis on a viscoelastic assay (TEG or ROTEM, for example) “makes us think of fibrinolysis, which may occur sometimes in the postoperative period in some patients, perhaps due to decreased plasminogen activator inhibitor.” But it wouldn’t be consistent with hemophilia A, Dr. Unold said.

He called the two-year-old the “prototypical patient with hemophilia A.”

“You have a young patient who’s presenting with a new bleeding or bruising. He has a swollen knee—joints are a typical target of bleeding in patients with hemophilia. And the correction of the mixing studies lets us know that we’re dealing with a factor deficiency, and in this case factor VIII.”

The patient is found to have the intron 22 inversion on the F8 gene, Dr. Goodwin said. He is started on standard therapy but fails to show appropriate therapy response three years later. A Bethesda assay shows an inhibitor to factor VIII, and the patient begins treatment with emicizumab. The F8 intron 22 inversion “often presents with a severe type of disease” and has about a 30 percent chance to develop an alloimmune inhibitor, he added.

Dr. Goodwin continues with the same patient who is now a 10-year-old who falls on a rock while hiking out of state and visits the emergency department for treatment of a significant hematoma that develops over four hours. Dr. Goodwin asked session viewers to select the correct impact of emicizumab on a series of coagulation tests ordered by the emergency physician:

• aPTT: falsely overestimate.
• aPTT clot-based factor VIII activity: falsely increased activity.
• PT: falsely underestimate.
• Thrombin time: falsely overestimate.
• Fibrinogen Clauss method: falsely underestimate.

The PTT will not falsely overestimate; it will falsely underestimate—the clot time will be shorter, he said. “Therefore, you’re going to have a PTT clot-based assay that will lead to falsely increased activity because of the shortened PTT. The PT is generally not impacted, nor are the thrombin time and fibrinogen Clauss method.”

In this case, the aPTT clot-based factor VIII activity assay result would show falsely increased activity because of the shortened PTT.

In another scenario, a 14-year-old male is admitted to a hematology consult service for a swollen left knee joint, determined to be hemarthrosis. Laboratory testing is performed with the following results: prolonged aPTT at 49 seconds, normal PT at 11.2 seconds, and factor VIII activity (human-reagent chromogenic assay) is critically low at less than one percent. A Bethesda assay (chromogenic, bovine) is performed with a result of 5.2 Bethesda units (reference interval: zero to factor VIII).

Based on the results, Dr. Unold asked, which of the following is correct:

• This patient has not developed an antibody to emicizumab as literature lacks documented cases of neutralizing antibodies.
• The chromogenic FVIII activity (human) is insensitive to the presence of emicizumab.
• The detected Bethesda units are inaccurate.
• Both the aPTT and PT results are accurate.
• The FVIII PTT clot-based activity assay is best to assess the patient’s factor VIII activity.

The answer: Both the aPTT and the PT results are accurate.

“Knowing that the human-based chromogenic activity would be affected by emicizumab, there was less than one percent factor VIII in this hemophiliac,” Dr. Unold said. “We would expect with emicizumab on board that we would actually have had a falsely increased factor VIII activity because this test is affected. But in this case, because there was less than one percent, we were thinking the emicizumab was either not on board or not affecting the assay.”

Two reasons for that result could be noncompliance with the medication or development in the patient of neutralizing antibodies against emicizumab.

“Knowing that the drug was not affecting the test led us to presume then that the aPTT was actually accurate and not falsely elevated by the drug,” Dr. Unold said. And PT is usually not affected by emicizumab, though this varies depending on the PT performed. “Detected Bethesda units would be accurate in this case, certainly because a bovine assay would not be expected to be affected by the drug, much less the fact that the drug did not seem to be either present or affecting this human-based test.”

There are instances in the literature—the HAVEN trials—showing that neutralizing antibodies can be developed to emicizumab, he said. “And remember that a PTT clot-based assay is not the best way to assess a patient’s factor VIII activity if they’re on emicizumab, because these clot-based assays will be affected by the presence of the drug.”

Amy Carpenter Aquino is CAP TODAY senior editor.