Measurement of von Willebrand factor-cleaving protease (ADAMTS-13) activity in plasma: a multicenter comparison of different assay methods
Professor Bernhard Lämmle, Central Hematology Laboratory, University Hospital Inselspital, CH-3010 Bern, Switzerland.
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Summary. A severely deficient ADAMTS-13 activity (<5%) is a key laboratory finding confirming the diagnosis of thrombotic thrombocytopenic purpura (TTP), whereas a mildly or moderately decreased activity is found in various other conditions. Laboratory tests for ADAMTS-13 activity must reliably identify a severe deficiency and detect inhibitory antibodies against ADAMTS-13. We carried out a multicenter comparison of different assays for ADAMTS-13 activity in plasma, including the quantitative immunoblotting of degraded von Willebrand factor (VWF) substrate, the residual collagen binding activity and ristocetin cofactor activity of degraded VWF, and an immunoradiometric assay. The main goal was to investigate whether all assays concordantly identified severe ADAMTS-13 deficiency and detected inhibitory antibodies. ADAMTS-13 activity was determined by five laboratories in 30 plasma samples of patients with hereditary and acquired TTP and other conditions. ADAMTS-13 activity values of the samples ranged from <3% to > 100%. Concerning the identification of a severe ADAMTS-13 deficiency, good interassay and interlaboratory agreement was observed with only one false-negative and two false-positive results by two laboratories using a collagen binding assay. For samples with normal or mildly to moderately reduced ADAMTS-13 activity, results were less concordant. There was good agreement for the detection of strong inhibitors. We conclude that all assays investigated are useful as a screening test in suspected TTP. Further assay improvement is needed, however.
ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type 1 domains 13) is a metalloprotease [1–5] that specifically cleaves the von Willebrand factor (VWF) subunit in vivo at the peptide bond Tyr1605–Met1606 (Tyr842–Met843 in mature subunit numbering) [6,7], generating fragments of 176 kDa and 140 kDa.
A severely deficient ADAMTS-13 activity (<5% of that in normal plasma), caused either by mutations of the ADAMTS-13 gene [3,8–10] or by inhibitory antibodies against ADAMTS-13 [11–13], was found to be linked to [3,12–14] and specific for [12,13,15] a thrombotic microangiopathy commonly labeled thrombotic thrombocytopenic purpura (TTP). The specificity of ADAMTS-13 deficiency for TTP has been challenged by several authors claiming that a reduced ADAMTS-13 activity may also be found in other thrombocytopenic or inflammatory conditions [16–18], and even in healthy subjects . However, in two of these studies ADAMTS-13 activities were mostly lowered to about 15–50% but in no case to <5% [17,18], while the third study did not give quantitative values for ADAMTS-13 activity . Veyradier et al.  and Remuzzi et al.  found a severe ADAMTS-13 deficiency not only in TTP, but also in a few patients clinically diagnosed as hemolytic uremic syndrome (HUS). This probably reflects the difficulty in clinically distinguishing between these two conditions . A severely deficient ADAMTS-13 activity is therefore a specific laboratory finding for a thrombotic microangiopathy most often diagnosed as TTP . However, the sensitivity of severe ADAMTS-13 deficiency for TTP is only between 33% and 100% according to five large cohort studies [12,13,19,22,23].
Laboratory assays must reliably identify severe ADAMTS-13 deficiency and distinguish it from moderately or mildly decreased levels found in various inflammatory conditions, liver disease, pregnancy , disseminated neoplasia , disseminated intravascular coagulation , sepsis, and heparin-induced thrombocytopenia . They should also be capable of detecting inhibitory antibodies against ADAMTS-13, to differentiate between hereditary and acquired ADAMTS-13 deficiency.
Several methods have been described for the determination of ADAMTS-13 activity in plasma, such as the quantitative immunoblotting of VWF substrate degraded by BaCl2-activated ADAMTS-13 under mildly denaturing conditions (1.5 m urea, low ionic strength) [11,14], the determination of 350-kDa disulfide-linked carboxyterminal proteolytic VWF fragments generated from guanidinium-treated VWF substrate by diluted plasma [13,25], assays measuring the residual collagen binding activity  or ristocetin cofactor activity  of degraded VWF, and an immunoradiometric assay (IRMA) using monoclonal antibodies to the C- and N-terminal VWF subunit .
In order to evaluate whether the presently available assays for ADAMTS-13 activity give concordant results, we performed a pilot multicenter comparison, inviting several laboratories performing different assays to participate. The main goal was to investigate whether all laboratories concordantly identified severe ADAMTS-13 deficiency and detected inhibitory antibodies against ADAMTS-13.
Materials and methods
Four research laboratories and one industry laboratory from Switzerland, Germany, France, and Austria agreed to participate in this study. The Hemostasis Research Laboratory in Bern (laboratory 1) used a quantitative immunoblotting assay, the Laboratory Keeser and Arndt in Hamburg (laboratory 2) and Baxter BioScience in Vienna (laboratory 3) an assay measuring the residual collagen binding activity of degraded VWF, the Department of Hemostaseology in Frankfurt (laboratory 4) an assay measuring its residual ristocetin cofactor activity, and the INSERM U.143 in Le Kremlin-Bicetre (laboratory 5) an IRMA. The study was approved by the responsible ethical committee (Kantonale Ethische Kommission, Bern, Switzerland).
Identical aliquots of 30 citrated plasma samples of patients with hereditary and acquired TTP and other conditions were prepared (by J.-D.S.) and supplied to all laboratories. Diagnoses and sample descriptions are given in Table 1. ADAMTS-13 activity of the samples ranged from <3% to > 100%. Two samples (nos 10 and 17) were mixtures of a citrated normal human plasma pool from healthy donors (NHP) and plasma of a patient with severe hereditary ADAMTS-13 deficiency (<3%), adjusted to 50% and 25% ADAMTS-13 activity, respectively. Samples with an ADAMTS-13 activity of <20% were screened for inhibitors. An identical NHP standard defined to contain 100% ADAMTS-13 activity was supplied to all laboratories for assay calibration. All samples were stored and shipped in frozen condition. Preliminary experiments, performed at the Hemostasis Research Laboratory in Bern, showed that prolonged storage for up to 6 years at − 20 °C or − 70 °C, as well as repeated thawing, did not alter the ADAMTS-13 activity of the plasma samples.
Table 1. ADAMTS-13 activity values in 30 plasma samples, as determined by the participating laboratories
|1||<3||<3||<3||<6.25||<5||122||TTP, probably acquired|
|3||55||68||64||86||53||139||Acquired TTP in remission|
|6||25||22||<3||24||10||318||Acquired TTP (1 h after infusion of 4 U FFP)|
|8||30||17||20||20||34||220||Suspected relapse of TTP, after splenectomy|
|10||45||45||27||50||61||138||1 : 1 mixture of NHP and congenitally |
ADAMTS-13 deficient plasma
|12||8||6||5||13||7||62||TTP, probably hereditary (24 h after infusion of 3 U FFP)|
|15||5||7||<3||<6.25||5||69||TTP, probably hereditary (48 h after infusion of 3 U FFP)|
|17||20||15||4||34||19||155||1 : 3 mixture of NHP and congenitally |
ADAMTS-13 deficient plasma
|18||100||58||84||114||110||192||Suspected antiphospholipid antibody syndrome|
|22||8||<3||<3||10||<5||165||TTP, probably acquired|
|24||50||48||47||62||62||268||Atypical pediatric HUS|
|25||8||<3||<3||12||<5||168||Suspected atypical HUS vs. TTP?|
|26||100||88||112||100||85||89||Acquired TTP in remission|
|27||<3||<3||<3||<6.25||<5||173||TTP, probably acquired|
|28||50||64||24||59||32||387||TMA with disseminated neoplasia|
All assays were performed as described elsewhere [11,14,26–28] with the following modifications.
Quantitative immunoblotting assay [11,14]
ADAMTS-13 activity was determined by comparison with a calibration curve obtained with NHP dilutions in 0.01 m Tris, 0.15 m NaCl2, 1 mm Pefabloc SC (Roche, Mannheim, Germany), pH 7.4 (1 : 20, 1 : 40, 1 : 80, 1 : 160, 1 : 320, 1 : 640, and buffer control). Dilutions of 1 : 40 and higher were prepared with dilution buffer containing 2 g L−1 bovine serum albumin. For inhibitor detection, plasma samples were heated for 30 min at 56 °C and centrifuged for 15 min at 15 000×g The supernatant was mixed 1 : 1 (v : v) with NHP, incubated for 2 h at 37 °C , and the mixture diluted 1 : 10 before BaCl2 activation and addition of VWF substrate. For inhibitor assay calibration, NHP was mixed 1 : 1 (v : v) with dilution buffer, incubated for 2 h at 37 °C, and the mixture diluted 1 : 10, 1 : 20, 1 : 40, and buffer control.
Residual collagen binding activity of degraded VWF 
The Laboratory Keeser and Arndt (laboratory 2) used recombinant instead of plasma-derived VWF as substrate. Baxter BioScience (laboratory 3) applied slight modifications as described .
Residual ristocetin cofactor activity of degraded VWF 
Imidazole buffer was replaced by heat-inactivated NHP (30 min at 60 °C, centrifuged for 15 min at 13 000×g) to dilute NHP for assay calibration.
The assay was performed with slight modifications .
Analysis of results
All laboratories were unaware of the results of the other participants until completion of the study.
Categories for ADAMTS-13 activity were arbitrarily defined as [15,23]: <5%, severe deficiency; 5–9%, borderline severe deficiency; 10–25%, moderate decrease; > 25% to <50%, mild decrease; ≥ 50%, normal activity. As the detection limit of the assay measuring the residual ristocetin cofactor activity of degraded VWF is 6.25%, values <6.25% were considered to reflect severe ADAMTS-13 deficiency for this assay.
Inhibitors were classified as: +, definite inhibitor; (+), uncertain/low titer inhibitor; 0, no inhibitor.
The correlation between the different methods was assessed by Spearman rank order correlation, and Passing–Bablock test was used for calculation of the slopes and intercepts of the regression lines.
All laboratories identified a severely deficient ADAMTS-13 activity in nine samples from patients with the diagnosis of constitutional or acquired TTP (nos 1, 2, 5, 9, 11, 13, 19, 23, 27) (Table 1). In three samples (nos 15, 22, 25) some laboratories found a severe deficiency, while the others measured an activity between 5% and 12%. However, concerning the identification of a severe ADAMTS-13 deficiency, one false-negative (no. 30) and two false-positive (nos 6, 17) diagnoses were made by two laboratories using a collagen binding assay (laboratories 2 and 3). The first laboratory measured an activity of 44% in a sample of a patient with confirmed hereditary TTP (no. 30), while all others diagnosed a severe deficiency. The second laboratory diagnosed a severe deficiency in two samples (nos 6, 17), while all others found an only mild to moderate decrease (10–25% and 15–34%, respectively). Sample no. 6 was obtained from a patient with acquired TTP 1 h after infusion of 4 units of fresh frozen plasma (FFP); no. 17 was a mixture of NHP and congenitally deficient plasma adjusted to 25% ADAMTS-13 activity.
Results were less concordant in samples with normal or mildly to moderately reduced ADAMTS-13 activity (Table 1). Nevertheless, Spearman rank order correlation coefficients between each two methods were Rs = 0.89–0.97 (P <0.001), and Passing–Bablock test between each two methods revealed slopes of the regression lines between 0.75 and 1.33 and y-intercepts between − 1.40% and 1.51%. These calculations showed good general agreement between the assays and did not indicate a systematic deviation for any assay.
For none of the assays was a correlation of ADAMTS-13 activity with VWF antigen concentration (VWF:Ag) (Table 1) observed (Rs between − 0.13 and 0.31, P > 0.05; severe ADAMTS-13 deficiencies were excluded for this correlation analysis). This shows that even very high VWF:Ag concentrations do not influence the assay results.
Strong inhibitors [>1 Bethesda unit (BU) mL−1] were identified by all laboratories in four samples from patients with acute acquired TTP (nos 5, 9, 11, 13) (Table 2). All laboratories diagnosed absence of inhibitors in two samples from patients with hereditary TTP (nos 2, 30). Discrepancies occurred in nine samples which had mostly been classified as uncertain or low-titer inhibitors (nos 1, 6, 12, 15, 19, 22, 23, 25, 27).
Table 2. Inhibitory antibodies against ADAMTS-13, as detected by the participating laboratories
The assays compared in this study have certain advantages and disadvantages. The immunoblotting assay is very sensitive and reproducible in the lowest activity range. It permits a reliable differentiation between 3% and 0% activity  and, depending on the quality of the VWF substrate used, even between 1% and 0%. Its disadvantage is the time-consuming and labour-intensive procedure which limits its application to the specialized laboratory. All other assays can be performed faster, and should therefore be suitable for a wider range of laboratories.
Concerning the identification of a severe ADAMTS-13 deficiency, we found generally good agreement between the different assays and laboratories. However, occasional erroneous results were observed for the collagen binding assay. The deviations were inconsistent among the two laboratories using this assay, suggesting that there is no systematic error but that this test is very delicate, subject to disturbance, and should be improved further.
Although results were less concordant in plasma samples with normal or mildly to moderately reduced ADAMTS-13 activity, the overall interassay and interlaboratory comparability was surprisingly good considering the complexity of these bioassays. There was also good agreement for the identification of high-titer inhibitors. All assays allowed the differentiation between hereditary and acquired protease deficiency, provided the inhibitor titer was > 1 BU mL−1.
We conclude that all assays investigated are useful as a screening test in suspected TTP patients.
Recently, Dong and coworkers  showed that ADAMTS-13 cleaves ultralarge VWF multimers on the endothelial cell (EC) surface within seconds to minutes under flow conditions. This suggests that ADAMTS-13 binds to EC  and locally cleaves these extremely adhesive multimers. An EC-dependent assay  might therefore reflect ADAMTS-13 function in a more physiological way, and in addition shorten the assay procedure. As hypothesized by Moake , there might be defects of the anchoring of ADAMTS-13 to EC, for instance due to anti-CD36 antibodies  or to hypothetical structural defects of the ADAMTS-13 molecule. In such situations a severe dysfunction of ADAMTS-13 may only be detectable by an EC-based assay but not by the presently available methods measuring ADAMTS-13 activity in plasma. This could explain some cases of clinically clear-cut TTP with normal plasma activity of ADAMTS-13 [22,23].
This work was supported by a grant from the Swiss National Foundation for Scientific Research (32-66756.01 to B.L.).