Assay for ADAMTS-13 Activity with Flow Cytometric Readout

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) is a metalloprotease that regulates the size of circulating von Willebrand factor (vWF) multimers. Severe lack of ADAMTS-13 activity [<10% of normal (0.1 IU/mL)] leads to thrombotic thrombocytopenic purpura (TTP), a specific type of thrombotic microangiopathy (TMA). Timely determination of plasma ADAMTS-13 activity is essential to discriminate TTP from other types of TMA with respect to adequate treatment. Identification of the minimal substrate motif for ADAMTS-13 within the A2 domain of vWF (vWF73) as well as the generation of monoclonal antibodies (mAbs) that specifically recognize the ADAMTS-13 cleavage site enabled the development of a variety of methods for determination of plasma ADAMTS-13 activity. In order to further extend the range of analytical platforms applicable for quantitative determination of plasma ADAMTS-13 activity, a specific, vWF/mAb-based assay with flow cytometric readout was developed and validated. Basic assay characteristics include a total assay time of 80 to 90 min, a near linear dynamic range from 0.005 (lower limit of quantification) to 0.2 IU/mL, and intra- and interassay coefficients of variation below 5 and 30% at input plasma ADAMTS-13 activities of 0.015 and ≤0.050 IU/mL, respectively. When compared to the results obtained with a commercially available quantitative ADAMTS-13 activity ELISA, analysis of 18 plasma samples obtained from patients with suspected TTP revealed full agreement of results with respect to the clinical 0.1 IU/mL TTP threshold. Based on these data, it is assumed that the described assay principle can be successfully transferred to virtually all laboratories that have a flow cytometer available.


Labeling and characterization of the anti-human vWF-A2 (ADAMTS-13 cleaved)-specific mAb (clone #490628)
In order to apply the anti-human vWF-A2 (ADAMTS-13 cleaved)-specific mAb for flow cytometry, the R-Phycoerythrin (PE) Conjugation Kit (cat. no. ab102918) Abcam, Berlin, Germany) was used according to the manufacturer's instructions. Since there is only a minor change of input volume of the sample and also no purification step needed when using this kit, the concentration of the labeled antibody was set according to the original input concentration (500 µg/ml = 3,33 µM). For preparation of a working solution, the labeled antibody was further diluted 1 in 10 using DPBS pH 7.4, 0.1% BSA, 2 mM NaN 3 and stored at 4°C until use.
Assay optimization included testing of different quantities of the PE-labeled antibody per reaction. With respect to best possible assay sensitivity, corresponding results indicated an optimal signal-to-noise ratio (S/N, 1% / 0% plasma ADAMTS-13 activity) when using 2 µl of the PE-labeled mAB (0.66 pmol/reaction, Table S2). This amount of PE-mAb was therefore used during all experiments described in the paper.
In order to assess the degree of labeling when using the (PE) Conjugation Kit, another batch of the anti-human vWF-A2 (ADAMTS-13 cleaved)-specific mAb was labeled along with an antiprotein C antibody and non-reducing sodium-dodecyl-sulfate polyacrylamide-gelelectrophoresis (SDS-PAGE) followed by fluorescence readout were performed. Silver staining was used for further characterization. In brief, samples containing one µg (6.67 pmol) of either non-conjugated mAb or PE-mAb were mixed with the same volume of 2x Laemmli buffer and run on 4-15% Mini-PROTEAN TGX Stain-Free Gels (BioRad, Munich, Germany). Detection of PE fluorescence and visualization of silver staining (SilverXpress sliver staining kit, ThermoFisher Scientific, Darmstadt, Germany) was done using a GelDoc Imaging System (Bio-Rad, Munich, Germany).
As shown in Figure S1, a significant proportion of the anti-human vWF-A2 (ADAMTS-13 cleaved)-specific mAb introduced remained unlabeled. Most of the PE-conjugated mAbs obviously exhibited one PE-molecule,Evaluation of the novel PE-mAb preparation revealed good performance in the low plasma ADAMTS-13 activity range, indicating reproducible and sufficient performance of the applied PE-labeling kit with respect to the assay described here. However, the assay may be further improved by using a PE-mAb preparation with higher degree of labelling.

Evaluation of assay specificity
Pierce Protease Inhibitor Mini Tablets, EDTA-free (ThermoFisher Scientific, Cat. No. A32955) were used to evaluate the specificity of the assay. The tablets contain broad-spectrum protease inhibitors (AEBSF, aprotinin, bestatin, E-64, leupeptin, and pepstatin A) and were dissolved in the used substrate buffer (10 mM Tris-HCl, pH 9.0, 5 mM BaCl 2 , 0.015% Tween 20) according to the manufacturers recommendations. Due to the EDTA-free formulation, the protease inhibitor mix is not effective against metalloproteases. Analysis of plasma calibrators and patient samples (< 0.2 IU/ml ADAMTS-13 activity) was done in parallel, in the absence and presence of the protease inhibitor mix. Obtained results are shown in Figure S4.

Minimum Information about the Flow Cytometry Experiment (MIFlowCyt)
1. Experiment Overview

Purpose
The purpose of the study is to establish a flow cytometric method that allows the determination of ADAMTS-13 activity in citrated plasma samples.

Experiment Variables
An N-terminally biotinylated minimal peptide motive for ADAMTS-13 (vWF73) was used as substrate for determination of ADAMTS-13 activity in citrated plasma samples. Cleavage rates of the substrate under optimized assay conditions were determined by incubation of (cleaved) vWF73 with Alexa Fluor 647-labeled Dynabeads M-280 Streptavidin and a PE-labeled monoclonal antibody (mAb) that specifically recognizes the ADAMTS-13 cleavage site. After incubation, the reaction mixture was analyzed by flow cytometric readout, with Alexa Fluor 647-labeled beads gated by specific fluorescence pattern (FL6) and the amount of bound mAB determined by PE (FL2) mean fluorescence intensities (MFI). Assay calibrators were prepared by dilution of the WHO international standard for plasma ADAMTS-13 antigen and activity (WHO 1 st International Standard ADAMTS13 Plasma, NIBSC code: 12/252) into heat-inactivated citrated pooled normal plasma (PNP, in-house preparation). Plasma calibrators were prepared by dilution of characterized PNP into heat-inactivated PNP. Validation of assay characteristics included determination of the lower limit of quantification (LLOQ) as well as assay reproducibility and precision. Plasma samples (citrate [10.5 mM]) of patients with suspected TTP were assayed to verify the clinical applicability of the proposed method.

Conclusions
The assay performance characteristics evaluated demonstrated a sufficient LLOQ as well as acceptable assay reproducibility and precision. Compared to the results obtained with a commercially available S11 quantitative ADAMTS-13 activity ELISA, analysis of 18 citrated plasma samples from patients with suspected TTP revealed full agreement of results with respect to clinical decision limits. Thus, the assay appears to be applicable for routine clinical analysis.

Quality Control Measures
The flow cytometer optical alignment and fluidics system were calibrated / checked using Flow-Check Pro Fluorospheres (Beckman Coulter) according to the manufacturers´ instructions. Plasma ADAMTS-13 controls above and below the clinical decision limit (0.1 IU/ml plasma ADAMTS-13 activity) were included in each run. Citrated pooled normal plasma (PNP) was prepared from citrate-anticoagulated whole blood obtained from 4 healthy blood donors who gave informed written consent. PNP was frozen in aliquots of 1 ml at -40°C until use. A portion of the PNP were heat inactivated to eliminate all ADAMTS-13 activity in order to allow for preparation of plasma ADAMTS-13 calibrators and controls.

Flow Sample / Specimen Details
Whole blood from 18 patients with suspected TTP was drawn from an antecubital vein into citrate-tubes (Sarstedt, Nümbrecht, Germany; final sodium citrate concentration: 10.5 mM) and transferred to our laboratory for routine plasma ADAMTS-13 activity analysis. Aliquots of citrated plasma prepared by centrifugation were stored at ≤ -40°C to be used for additional investigation by the flow cytometric assay described here.