Christopher Shuford (Presenter)
Laboratory Corporation of America
Authorship: Christopher M. Shuford (1), Meghan N. Bradley (1), Russell P. Grant (1)
(1) Laboratory Corporation of America Holdings, Burlington, NC
| Short Abstract Thrombotic Thrombocytopenic Purpura (TTP) is a life-threatening disease characterized by acute and severe decrease in the activity of ADAMTS13, the enzyme responsible for cleaving von Willebrand factor. Despite the acuity of TTP, the complexity and cost of ADAMTS13 activity testing necessitate that most institutions use reference laboratories rather than in-house assays, which leads to delayed testing results that can impact patient management. To counter this delay as a reference laboratory, we are developing a simple LC-MS/MS assay capable of STAT testing through the use of historical (e.g., biweekly) calibration. Key elements in development and validation of the STAT LC-MS/MS assay will be presented. |
Long Abstract
INTRODUCTION
Thrombotic Thrombocytopenic Purpura (TTP) is a life-threatening disease characterized by acute and severe decrease in the activity of ADAMTS13, the enzyme responsible for cleaving von Willebrand factor. Both congenital and acquired (autoimmune) deficiency of ADAMTS13 activity are characterized by the presence of unusually large vWF factor multimers that are more platelet adhesive than smaller multimers found in normal plasma, resulting in the formation of platelet-rich thrombi in the microcirculation typical in TTP. As such, measurement of ADAMTS13 activity can play a role in differentiating TTP from a number of clinically similar conditions that have different underlying causes; however, given the acuity of the symptoms, life sustaining Total Plasma Exchange (TPE) is often initiated prior to ADAMTS13 activity testing when TTP is suspected based on clinical presentation.
The complexity and cost of ADAMTS13 activity testing necessitate that most institutions use reference laboratories rather than in-house assays, which leads to delayed testing results that can impact patient management It has been proposed that at turnaround time (TAT) less than 24 hours for ADAMTS13 testing would have significant implications for patient management and cost reduction for unnecessary treatment.[1] To enable such a TAT as a reference laboratory, we are developing a STAT in-house LC-MS/MS assay for the measurement of ADAMTS13 Activity. A TAT of less than 4 hours from receipt of sample by the LC-MS/MS test is accomplished using historical (e.g., bi-weekly) calibration, as opposed to conventional daily or batch calibration. Key elements in development and validation of the STAT LC-MS/MS assay will be presented.
METHODS
ADAMTS13 activity is determined by incubating patient plasma with a synthetic substrate under optimized (i.e., non-physiological) conditions. The synthetic substrate, termed vWF73, is a 73 amino acid residue peptide derived from the natural amino acid sequence of vWF residues Aspartic acid-1596 through Arginine-1668 and, thereby, possesses the ADAMTS13 cleavage site (Tyrosine-1605/Methionine-1606). Upon cleavage of vWF73 in-vitro, a 10 amino acid residue product peptide is formed from the N-terminus of the substrate having the sequence DREQAPNLVY. ADAMTS13 activity is proportional to the amount of product peptide created during a 30 min incubation, which is measured by isotope dilution following methanol precipitation using liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). The assay is externally calibrated using pooled plasma derived from ostensibly normal individuals and standardized against the WHO 1st International Standard for ADAMTS13 in Plasma (12/252). The ADAMTS13 activity, expressed in units of “percent normal activity”, is interpolated from the external calibration curve created from dilution of the pooled normal plasma to span 2 to 100% normal activity.
RESULTS
Given ADAMTS13 activity is defined using the initial velocity of the enzyme reaction, kinetic measurements by single time point analysis requires the reaction to be linear at the time of analysis. As such, the substrate:plasma ratio (i.e., substrate:ADAMTS13 ratio) was initially evaluated to ensure that the substrate was in sufficient excess to maintain pseudo-first order reaction kinetics within the desired time period of 30 minutes. A two hour time course analysis of the un-optimized reaction at room temperature using pooled normal plasma (PNP) indicated the reaction rate did not slow by more than 15% (i.e., was linear) out to 60 minutes when using 200 pmol substrate per microliter of plasma. As expected, linearity was only observed out to 30 minutes with half the substrate:plasma ratio at 100 pmol/µL, 15 minutes with 50 pmol/µL, and 7.5 minutes with 25 pmol/µL.
Next, the reaction conditions were further optimized to increase the reaction rate at 30 minutes. Various divalent cations were tested for their impact on the ADAMTS13 activity and it was observed that supplemental Ba2+, Ca2+, and Mg2+ provided a 12.3-, 28.8-, and 7.1-fold faster reaction, respectively. The concentration of supplemental Ca2+ was further titrated between 0.05 and 50 mM and demonstrated to provide optimal ADAMTS13 activity between 2.5 and 5 mM. Interestingly, the pH optimal for ADAMTS13 activity differed noticeably at different reaction temperatures. Under ambient reaction temperatures, the optimum buffer pH was observed to be 6.0 with less than ±12% difference between pH spanning 5.0 to 6.5; however, when performing the reaction at 45 °C, which provided approximately a 3-fold increase in the measured reaction rate, the optimum buffer pH was increased to between 6.5 and 7.0, with greater than a 40% decrease in the measured activity at pH 6.0 or lower.
Following these optimization studies, significant non-linearity (quadradicity) was observed in calibration curves that was not attributable to MS detector saturation (as confirmed by dilution series of neat product peptide spanning the same dynamic range of MS signal), nor due to interference in the internal standard detection in the presence of high levels product peptide (as confirmed by negative control studies). Consequently, it was concluded the non-linearity of the calibration curve was due to depletion of the substrate over the course of the 30 minute incubation period under the optimized conditions. Using only a 15 minute incubation period with the same condition in a preliminary correlation study to a predicate FRET (fluorescence resonance energy transfer) ADAMTS13 activity assay, non-linear calibration was still observed, but reasonable agreement (Passing-Bablok Slope = 0.873; R = 0.821). It was hypothesized, the relatively poor correlation was due in-part to imprecision resulting from non-uniform heating of the specimens at 45 °C, which was observed to be 24% within the same batch as determined from 11 replicates of a 1.8% activity specimen.
As such, the same correlation and precision study was repeated under ambient generation temperatures for 30 minutes. Under these conditions, improved linearity in the calibration series was observed and the within-batch imprecision was markedly reduced to only 12.6% at the same level. The improved linearity of the LC-MS/MS assay translated into improved agreement with the FRET assay (Passing-Bablok Slope = 0.991), but the decreased imprecision for the LC-MS/MS measurment did not work to improve the correlation (R = 0.837).
These results suggest the relatively poor correlation may be a result of 1) imprecision in the FRET assay, 2) pre-analytical variables (e.g., freeze-thaw), or 3) fundamental differences in the FRET and LCMS assay specificities. Nonetheless, using a clinical cut-off of 30% ADAMTS13 activity, the assays demonstrated good clinical concordance (N=36), with 93.1% negative agreement and 85.7% positive agreement in this provisional study using ambient generation conditions.
SUMMARY
A preliminary ADAMTS13 activity assay was developed and optimized for LC-MS/MS, which has demonstrated reasonable agreement with FRET-based assays in early studies. Further development will comprise isolating the source of variability between the FRET and LC-MS/MS assays. The veracity and stability of historical calibration will also be considered to enable STAT testing. Implications with respect to reagent stability, instrument stability, mode of internal standardization (i.e., pre- vs. post-incubation), and frequency of calibration will be considered for historical calibration, along with the use of the WHO 1st International Standard for ADAMTS13 in Plasma (12/252) as a longitudinal anchor for accuracy. These result will be presented along with final validation results of the STAT LC-MS/MS assay.
References & Acknowledgements:
[1] N. T. Connell, T. Cheves, and J. D. Sweeney, “Effect of ADAMTS13 activity turnaround time on plasma utilization for suspected thrombotic thrombocytopenic purpura,” Transfusion, vol. 56, no. 2, pp. 354–359, 2016.
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Laboratory Corporation of America |
| Board Member | no | |
| Stock | yes | Laboratory Corporation of America |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |