A 25-year-old woman with a 2-week history of an upper respiratory infection presents to the emergency department of your hospital with fever, severe headache, and confusion. A CT scan does not reveal any acute intracranial process. However, she has a number of concerning laboratory abnormalities, including marked anemia (hematocrit 19%, reference interval 34–47%) and thrombocytopenia (15 k/μL, reference interval 180–405 k/μL), markedly elevated lactate dehydrogenase (1050 U/L, reference interval 100–253 U/L), absent haptoglobin (<10 mg/dL, reference interval 30–200 mg/dL), and increased total plasma bilirubin (3.1 mg/dL, reference interval 0.2–1.4 mg/dL), with the indirect fraction representing 2.1 mg/dL.
In addition, this patient has negative direct and indirect antiglobulin (Coomb’s) testing, normal plasma creatinine, and normal coagulation parameters, including prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and only minimally elevated d-dimer. Her peripheral blood smears show severe thrombocytopenia and frequent schistocytes, accounting for 1–2% of red blood cells (also identified as 4+ schistocytes by the automated hematology analyzer), confirming microangiopathic hemolytic anemia (MAHA).
Due to the constellation of clinical and laboratory findings, the working diagnosis for this patient is thrombotic thrombocytopenic purpura (TTP)—a medical emergency. Her physician orders ADAMTS13 (A Disintegrin and Metalloprotease with ThromboSpondin type 1 motifs 13) testing and hospitalizes her to start emergency therapeutic plasma exchange (TPE). What assays would you use, and how would you interpret results for this woman presumed to have TTP?
This review examines the use of ADAMTS13 testing in evaluating patients with suspected TTP, a rare thrombotic microangiopathy (TMA) most commonlydue to acquired ADAMTS13 autoantibodies causing severe ADAMTS13 deficiency.
ADAMTS13, a protease also known as von Willebrand factor (VWF) cleaving protease, regulates the size of VWF multimers, and is an important mediator of platelet adhesion. ADAMTS13 regulates VWF size by cleaving a specific peptide bond in the A2 domain. Without this regulation, increased ultra-high-molecular-weight VWF multimers lead to microvascular platelet thrombi, destroying red blood cells and platelets and compromising blood flow to vital organs such as the brain and kidneys.
Although TTP often involves microangiopathic hemolytic anemia, thrombocytopenia, fever, neurological symptoms, and renal impairment, many patients do not exhibit the full pentad of findings. Furthermore, this same combination of abnormalities can be present in other disorders such as classic hemolytic uremic syndrome (HUS), atypical HUS (aHUS), or disseminated intravascular coagulation (DIC). With high clinical suspicion of TTP life-saving therapy must be started immediately. In fact, the disorder had a 90% fatality rate prior to the advent of modern treatments.
TTP is clinically defined as MAHA and thrombocytopenia in a patient without an alternative cause. Clinical prediction scores using readily available laboratory information (creatinine, platelet count, d-dimer, reticulocyte percentage, indirect bilirubin, etc.) have proven useful for acute decision-making. The initial therapeutic regimen for acquired TTP involves immunosuppression and TPE to suppress antibody formation, decrease antibody titer, and provide supplemental ADAMTS13. Although confirming severely decreased ADAMTS13 activity helps establish the TTP diagnosis, therapy must start even before test results are available.
Pre-Analytical Variables
The quality of ADAMTS13 testing results depends on several important pre-analytical issues. First, and crucially, diagnostic specimens have to be collected before therapy starts, since treatment alters ADAMTS13 levels. As with other coagulation testing, citrated platelet-poor plasma is the specimen of choice for ADAMTS13 testing. EDTA plasma is unacceptable since ADAMTS13 is a metalloproteinase and this anticoagulant strongly chelates metal ions that are necessary for its function. Even plasma from normal control individuals drawn into EDTA will demonstrate absent ADAMTS13 activity. Since thrombin degrades ADAMTS13, it also is best not to test serum specimens, which are prepared by allowing blood to clot. Likewise, hemolyzed plasma (particularly if the hemoglobin concentration is >2 g/L) is a concern since free hemoglobin inhibits ADAMTS13 activity. Consequently, labs should take care not to induce hemolysis in vitro during specimen collection and preparation. Since patients undergoing initial diagnostic testing can have brisk intravascular hemolysis, some degree of plasma free hemoglobin may be unavoidable. Plasma that cannot be tested within 4 hours of collection, should be frozen until the time of testing. Multiple freeze-thaw cycles should be avoided.
ADAMTS13 Activity Testing
Measurement of ADAMTS13 activity is the most commonly used laboratory test in the workup of suspected TTP. In acquired TTP, the autoantibodies can inhibit function by binding to functional regions of ADAMTS13 (neutralizing), by causing accelerated ADAMTS13 clearance (non-neutralizing), or through both neutralizing and non-neutralizing actions. Activity methods are a first-line choice since they are sensitive to all of these defects. Laboratories rarely use antigen assays due to their insensitivity to purely neutralizing inhibitors.
Although technically complex and time-consuming, labs initially used laboratory-developed tests (LDT) to gauge ADAMTS13 activity. Now, however, a number of commercial kits are available, but none have been Food and Drug Administration–approved. The commercial assays utilize synthetic VWF peptides that include the ADAMTS13 cleavage site. Although these assays are straightforward to perform on widely available laboratory equipment, and generate results within 1–3 hours, many centers refer testing to regional or national reference laboratories since TTP treatment starts prior to result availability and testing generally must be batched to be cost-effective. Laboratories that perform ADAMTS13 testing must do so keeping the critical nature of TTP front-and-center, with optimized turnaround times a top priority.
The most widely used activity assays are fluorescent resonance energy transfer (FRET) methods utilizing a VWF peptide (VWF73) containing a fluorescent moiety and a fluorescent quencher that flank the ADAMTS13 cleavage site. Substrate cleavage by ADAMTS13 results in fluorescent emission in direct proportion to the protease activity. However, FRET methods that measure ADAMTS13 activity in solution, without capture and wash steps, can underestimate activity in icteric specimens with plasma bilirubin concentrations >100 μmol/L since bilirubin may quench the fluorescent emission. This interference is dependent upon which fluorescent substrate is used and can be minimized by testing diluted specimen.
A newer activity assay with chromogenic endpoint detection also uses the VWF73 peptide. However, rather than detecting substrate cleavage directly by fluorescent emission, peptide cleavage in this activity enzyme-linked immunosorbent assay (ELISA) exposes a specific amino acid sequence that an antibody conjugated to horseradish peroxidase (HRP) detects. A subsequent HRP reaction results in color development.
In addition to these common methods, some laboratories use LDTs to perform ADAMTS13 activity testing using available synthetic substrates. These include LDT FRET methods or more unusual methods such as detecting cleavage products by mass spectrometry.
ADAMTS13 activity results have been expressed as percentage of normal activity, units/mL, or ng/mL, depending on the kit and calibrators used.
Of note, the World Health Organization (WHO) recently established the WHO 1st International Standard ADAMTS13 plasma that may improve harmonization among laboratories. Until recently, no commercial proficiency testing survey offered ADAMTS13 testing evaluation. However, several organizations, including the College of American Pathologists and North American Specialized Coagulation Laboratory Association, have added ADAMTS13 to their coagulation surveys.
Severe deficiency, defined in the literature as <10% of normal activity, appears to have good sensitivity (about 90%) and specificity (approximately 90% or higher) for TTP, although there is some variability depending on the datasets and definitions used. Studies also have shown that severe deficiency predicts good response to TPE, with approximately 80-90% of severely deficient patients responding to first-line therapies. Clinicians determine the duration of TPE based on platelet count recovery and resolution of hemolysis and neurologic symptoms, rather than recovery of ADAMTS13 activity. Although severe ADAMTS13 deficiency predicts good response to TPE, it also portends a significant risk of disease relapse, which occurs in at least one-third of cases. Some patients demonstrate severe ADAMTS13 deficiency even while in clinical remission, which is likely highly predictive of relapse risk.
Mild to moderately decreased ADAMTS13 activity can be seen in a variety of illnesses due to decreased synthesis or increased consumption, so this finding alone is not diagnostic of TTP. Very rare cases of TTP have been reported with minimal, or even no deficiency. Conversely, severe deficiency has been rarely reported in other conditions. Labs and ordering providers should approach such unusual findings with great caution.
Tests for ADAMTS13 Antibodies
To supplement ADAMTS13 activity testing, labs increasingly are performing tests for ADAMTS13 autoantibodies. Identifying the causative antibodies may help solidify the TTP diagnosis and provide prognostic information related to response to TPE and risk of relapse. ADAMTS13 autoantibody testing also differentiates acquired TTP from rare cases of hereditary TTP caused by ADAMTS13 mutations (Upshaw-Schulman syndrome).
ADAMTS13 Bethesda assays detect antibodies that neutralize function, which are present in approximately two-thirds of TTP cases. These are similar to the traditional Bethesda assays used to detect and titer coagulation factor VIII or IX inhibitors. They involve incubating patient plasma with normal pooled plasma to determine if recovery of ADAMTS13 activity in the mixture is lower-than-expected due to a patient antibody. The amount of inhibitor that decreases residual activity to 50% of expected is defined as 1 Bethesda unit (BU). Bethesda assays are LDTs that utilize commercial activity assays to measure the residual ADAMTS13 activity in the mixtures. Substances mentioned previously that can affect the activity assays, such as hemoglobin and bilirubin, also affect antibody identification and quantification in Bethesda assays and can cause false-positive results or falsely elevated antibody titers.
Because approximately one-third of TTP cases result from antibodies that do not neutralize function, Bethesda-style assays won’t detect a subset of the pathogenic antibodies. However, ELISA assays in which the ELISA plate is coated with full-length recombinant ADAMTS13 will identify the latter. Commercial ELISA kits are available for this purpose. Interestingly, ELISAs have detected ADAMTS13 antibodies in a small percentage of healthy individuals. Because ELISAs are more sensitive, while Bethesda assays are more specific, a reflexive testing algorithm is likely the optimal approach for antibody detection.
A commonly used reflexive algorithm first measures ADAMTS13 activity, and if deficient, reflexes to the Bethesda assay. This algorithm calls for performing an ELISA antibody test only if a Bethesda assay detects no antibodies. Due both to technical limitations of the assays and certain aspects of TTP pathophysiology, there may be a subset of acquired TTP cases in which an ADAMTS13 antibody cannot be identified. For instance, the Bethesda methodology generally cannot detect antibody titers below 0.5 BU, and both Bethesda and ELISA assays are incapable of detecting antibody bound to ADAMTS13 in circulating immune complexes.
Genetic Testing
A few clinical laboratories offer ADAMTS13 gene sequencing, which can help identify the mutations responsible for hereditary TTP. In addition, absence of a mutation may help to exclude a hereditary form in complex cases, such as when antibody tests do not identify an autoantibody. Figure 1 depicts an ADAMTS-13 testing algorithm incorporating activity, antigen, and genetic tests.
Case Conclusion
Returning to our opening case, clinicians confirmed the patient’s working diagnosis of acquired TTP based on laboratory findings of severe ADAMTS13 deficiency (<5% of normal activity) and an ADAMTS13 inhibitor of 1.8 BU identified in testing from acute (pre-therapy) specimens. Her initial clinical history and laboratory results largely excluded other processes associated with microangiopathic hemolytic anemia and thrombocytopenia, like HUS, aHUS, and DIC.
ADAMTS13 activity, although not available immediately as part of the initial workup, is a useful differentiator when considering these possibilities since these other disease conditions are not associated with severe ADAMTS13 deficiency. Mild to moderate decreases in the activity could be seen in these disease conditions due to the negative effects of inflammation and severe illness on the production and/or survival of ADAMTS13. Table 1 summarizes testing useful in the evaluation of suspected TTP.
The patient responded well to TPE and immunosuppression with prednisone. Indeed, her headache and confusion completely resolved after a single exchange, and her platelet count totally recovered and her hemolysis resolved after several more exchanges. Repeat ADAMTS13 testing while in clinical remission revealed a persistently low but improved ADAMTS13 activity of 15% of normal and persistence of a low-level inhibitor of 0.6 BU. Due to the high risk of relapse in acquired TTP, she is being monitored closely for disease recurrence, particularly during times of inflammation or stress such as illness, surgery, or pregnancy.
Conclusion
ADAMTS13 laboratory testing provides valuable information contributing to the diagnosis, management, and prognosis of TTP. While ADAMTS13 activity is the mainstay of this testing, a growing body of medical literature supports the utility of antibody testing. Laboratory professionals armed with knowledge of test performance characteristics and appropriate clinical use can greatly contribute to the care of patients with this life-threatening disease.
Suggested Reading
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Kristi J. Smock, MD, is an associate professor of pathology at the University of Utah and medical director of the Hemostasis/Thrombosis Laboratory at ARUP Laboratories in Salt Lake City, Utah.+Email: [email protected]