Bilirubin oxidase as a solution for the interference of hyperbilirubinemia with ADAMTS-13 activity measurement by FRETS-VWF73 assay

Authors


Bas de Laat, Department of Blood Coagulation, Sanquin Diagnostic Services, Plesmanlaan 125, 1066 CX Amsterdam, the Netherlands.
Tel.: + 31 20 512 3370; fax: + 31 20 512 3680; e-mail: b.delaat@sanquin.nl

Thrombotic thrombocytopenic purpura (TTP) is characterized by the pentad of microangiopathic hemolytic anemia, thrombo-cytopenia, fever, and neurological and renal manifestations [1]. Dr E. Moschcowitz was first to describe a clinical case resembling the clinical manifestations of TTP [2]. In same study Dr Moschcowitz suggested that TTP was caused by a poison thereby inducing increased platelet agglutination. Nowadays we know that this increase in agglutination is caused by impaired processing of unusually large von Willebrand factor (VWF) multimers. This impaired processing of VWF is probably caused by a decrease in or total absence of VWF cleaving protease, better known as the thirteenth member of a disintegrin and metalloprotease family with thrombospondin domains (ADAMTS-13) [3]. ADAMTS-13 deficiency can be caused by mutations in the ADAMTS-13 gene (hereditary TTP, Upshaw–Schulman syndrome) or by circulating antibodies that neutralize the ADAMTS-13 activity [4]. Treatment of TTP is almost solely based on plasma exchange, which is thought to supply patients with ADAMTS-13 and/or remove the neutralizing ADAMTS-13 autoantibodies [5].

As plasma exchange reduced the mortality rate from approximately 90–20%, the demand was increasing for fast and easy-to-use diagnostic tools for the measurement of ADAMTS-13 activity. In 2005, Kokame et al. [6] described an assay (FRETS-VWF73 assay) in which ADAMTS-13 activity was measured by using a substrate comprising 73 amino acids from the A2 domain of VWF. The major advantage of this assay compared with the existing assays is that the results are available within 1 h. One of the downsides of this assay, related to the use of fluorescent probes, is the unreliability of the ADAMTS-13 activity measurement in hyperbilirubinemic plasmas. Meyer et al. [7] elegantly showed that increasing amounts of bilirubin resulted in quenching of the fluorescent probe, thereby interfering with ADAMTS-13 activity measurement. As treatment strategies of TTP rely on diagnostic parameters, these falsely lowered ADAMTS-13 levels may have major consequences.

The interference of bilirubin with absorptions bands between 400 and 500 nm is well known. Because of this familiarity, several studies have been performed in order to solve this problem. One of the solutions is the addition of bilirubin oxidase to plasma samples. Bilirubin oxidase oxidizes bilirubin, thereby diminishing the interference with absorption bands between 400 and 500 nm [8]. We investigated the influence of bilirubin oxidase by adding it to 13 plasma samples of patients with suspected TTP. These 13 plasma samples could be divided into two groups: group 1 consisted of nine plasma samples that appeared ‘normal’; group 2 consisted of four samples that appeared ‘icteric/hyperbilirubinemic’ (Table 1).

Table 1.   ADAMTS-13 activity in the presence/absence of bilirubin oxidase
PatientWithout bilirubin oxidaseWith bilirubin oxidase
Dilution factorMeanCVDilution factorMeanCV
2.5×2.5×
  1. Patient plasma samples were tested for ADAMTS-13 activity in three dilutions by using the FRETS-VWF73 assay with minor modifications [6].

  2. CV, coefficient of variation.

Non-icteric plasma
 143.643.440.842.63.741.943.045.143.33.8
 263.263.963.663.60.657.359.663.460.15.1
 356.154.947.052.79.443.743.842.143.22.2
 493.995.092.293.71.583.586.191.286.94.5
 545.747.140.644.57.750.652.056.753.16.0
 632.031.832.232.00.624.725.027.525.76.0
 768.068.167.147.40.859.560.161.060.21.3
 870.273.782.675.58.561.862.265.463.13.1
 983.781.977.981.23.773.973.073.473.40.6
Mean 59.24.1 56.63.6
Icteric plasma
 1027.326.120.324.615.219.219.619.819.51.6
 1125.424.419.022.815.025.726.529.727.37.8
 1228.826.517.524.324.629.028.926.728.25.2
 1353.753.150.052.33.853.754.554.954.41.1
Mean 27.617.3 31.75.4

We performed the FRETS-VWF73 ADAMTS-13 activity assay on these 13 samples with and without 1 mm bilirubin oxidase (Sigma-Aldrich, St Louis, MO, USA) [6]. To investigate the stability and reliability of the activity measurements, the coefficient of variation (CV) was calculated as all samples were measured three times (1 × , 2.5 ×  and 5 ×  dilution). We found no difference in CV between the samples with bilirubin oxidase and those without bilirubin oxidase regarding non-icteric plasma samples (4.1% vs. 3.6%, = 0.93); however, we did find significant difference with respect to icteric plasma samples. The CV without bilirubin oxidase was 17.3% compared with 5.4% with bilirubin oxidase (< 0.01), indicating that the interference of bilirubin in the ADAMTS-13 activity can be diminished by the addition of bilirubin oxidase. We observed no influence of bilirubin oxidase on ADAMTS-13 activity in normal pool plasma (from 50 healthy volunteers). In addition, looking at the icteric plasma samples, we observed that by diluting the plasma samples the activity levels increased. This might indicate that the dilution of bilirubin results in a decreased interference in the ADAMTS-13 activity measurement.

In conclusion, we found a simple method to diminish the interference of bilirubin in the FRETS-VWF73 ADAMTS-13 activity measurement. By applying bilirubin oxidase, correct ADAMTS-13 activity levels can be measured, thereby providing medical doctors with a more stable diagnostic tool for TTP.

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

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