Is it possible to make a common reference standard for D-dimer measurements? Communication from the ISTH SSC Subcommittee on Fibrinolysis

Abstract

Background: D-dimer antigen is a heterogeneous mixture of fibrin degradation products that when present at high levels in plasma indicate ongoing coagulation and fibrinolysis. The heterogeneous nature of the target D-dimer antigen and the variety of assay systems means that it is difficult to compare results from different methods.

Objectives: To identify a universally agreed D-dimer standard that could help harmonize results from different methods.

Methods: A pool of patient plasma with high D-dimer levels was freeze-dried and investigated as a long-term World Health Organization international standard for D-dimer. Fibrin degradation products from clot lysis reactions were also freeze-dried in various formulations and investigated in commutability studies with patient plasma.

Results: Problems of instability of D-dimer plasma emerged suggesting loss of reactivity after freeze-drying and storage at -20°C of 10%-18% per year. Freeze-dried fibrin degradation products added to plasma were also unstable, but the sugar trehalose was found to improve stability. However, this preparation was not suitable as a standard in widely used assay platforms. Previous studies suggest fibrin degradation products are prone to structural rearrangements and amyloid formation, which may explain the instability of candidate D-dimer standards.

Conclusions: The known difficulties of D-dimer standardization are compounded by instability of D-dimer antigen after freeze-drying, described in this report. Fibrin degradation products added to plasma and stabilized by trehalose are not suitable as a standard for D-dimer measurement harmonization. Trehalose stabilization of pooled patient plasma containing high D-dimer levels may produce a useful standard, but this requires confirmation.

Keywords: D-dimer; amyloid; fibrin degradation; protein aggregation; standardization.

FIGURE 1

Estimates for D‐dimer concentrations over time in freeze‐fried plasma stored at elevated temperatures. A, Observed data for activity at each temperature of the time period of storage. B, Fitted data from the Degtest program assuming a first order reaction that obeys the Arrhenius equation

FIGURE 2

Analysis of fibrin degradation product (FDP) generation from clotting and lysis of fibrin over time using four methods. Concentrations of D‐dimer refer to the initial reaction mixture of 2.5 mg/ml fibrinogen clotted with thrombin in the presence of activated factor XIII (FXIIIa) and either tissue plasminogen activator (tPA; T) or urokinase (uPA; U) with glu‐plasminogen (G) or lys‐plasminogen (L). Time is presented on a log scale to see changes more easily at the four time points chosen

FIGURE 3

D‐dimer measurements over time in samples of SS444, freeze‐dried fibrin degradation product (FDP) in four formulations (A–D), stored at various temperatures at the times shown. The panels correspond to results for 10 µg/ml FDP added to 10 mM HEPES buffer, pH 7.4, containing 0.15 M NaCl, with additions: (A) 10 mg/ml trehalose, (B) 10 mg/ml sucrose, (C) plasma, and (D) plasma + 10 mg/ml trehalose. Note: no measurements were made after 1.33 years for samples A and B or the 45°C sample at 1.65 years because of insolubility

FIGURE 4

Observed versus predicted percent activity remaining in ampoules of SS444 stored at elevated temperatures over time

FIGURE 5

Correlation of results between methods A, B, and C as pairwise comparisons. Samples shown are an internal standard (green circle), and freeze‐dried candidate standards SS258 (red square) and SS523 (blue triangle) with 54 plasma samples (crosses) containing various levels of D‐dimer. Two groups of inconsistent samples are highlighted in the box (plasmas 31, 35, 45, 46) and in the oval (48, 49, 52, 53), along with sample 47 highlighted with the arrow. Log scales are used for presentation purposes

FIGURE 6

Development of cross‐beta amyloid structures in freeze‐dried proteins stored at elevated temperatures. Various proteins undergoing accelerated degradation studies were incubated with thioflavin T (ThT) and increasing fluorescence indicated the development of cross‐beta structures after storage at the higher temperatures. Tukey’s method for P‐value adjustment for a family of estimates was used to calculate significance levels of differences between results from each storage temperature. P‐values for all combinations were <0.05 where no ns is indicated