Research paper
Functional C1-Inhibitor diagnostics in hereditary angioedema: Assay evaluation and recommendations

https://doi.org/10.1016/j.jim.2008.06.004Get rights and content

Abstract

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of potentially life-threatening angioedema. The most widespread underlying genetic deficiency is a heterozygous deficiency of the serine protease inhibitor C1 esterase inhibitor (C1-Inh). In addition to low C4 levels, the most important laboratory parameter for correct diagnosis of HAE or angioedema due to acquired C1-Inh deficiency is reduced C1-Inh function (fC1-Inh).

No direct recommendations about the assays for fC1-Inh or sample handling conditions are available, although this would prove especially useful when a laboratory first starts to offer assays on fC1-Inh for HAE diagnosis. In the present study we evaluated the performance of fC1-Inh assays in the 15 different laboratories that are specialised in HAE diagnostics and assessed inter-laboratory variation with each laboratory using their own assays and standards. A double-blind survey was conducted using plasma/serum samples from healthy donors and HAE patients and the uniformity of HAE diagnosis was evaluated.

It can be concluded that the diagnosis of fC1-Inh deficiency was made correctly in most cases in this survey. We can recommend the chromogenic assay for the determination of fC1-Inh, while the complex ELISA needs further investigation.

Introduction

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent episodes of angioedema. Patients exhibit episodes of localized swelling in the extremities, face, gut or upper airways. If not treated properly, swelling of the airways can be fatal. As HAE is rare (incidence ~ 1:10,000 to 1:150,000) it is often misdiagnosed or remains undiagnosed altogether (Picavet and Baas, 2005, Roche et al., 2005).

The genetic defect underlying HAE is a heterozygous mutation in the serine protease inhibitor C1 esterase inhibitor (C1-Inh) gene, although recently a homozygous defect was demonstrated (Blanch et al., 2006) as well. The diagnosis of HAE is made based on the clinical picture in combination with a laboratory diagnosis of markedly reduced C1-Inh levels and/or function (fC1-Inh). Based on the combination of total protein and functional C1-Inh levels HAE can be divided in two types. In type I (~ 85%) low levels of C1-Inh protein are found, whereas in type II (~ 15%) C1-Inh protein levels are normal, but functional levels are low. Although heterozygosity would suggest C1-Inh levels of around 50%, typical fC1-Inh levels in untreated patients are between 5 and 30%. This may be due to C1-Inh consumption upon C1 autoactivation (Davis, 1988, Quastel et al., 1983) and/or downregulation of C1-Inh mRNA (Pappalardo et al., 2004). The recently described type III HAE yields normal functional C1-Inh levels and therefore lies outside the scope of this paper (Bork et al., 2006).

A European Union funded working group of HAE experts has been established focusing on Novel Methods for Predicting, Preventing, and Treating Attacks in Patients with HAE (preHAEAT) and aims to reach a more rapid diagnosis and uniform treatment. In the last few years excellent reviews have described consensus algorithms for diagnosis of HAE (Agostoni et al., 2004, Bowen et al., 2004, Gompels et al., 2005). The laboratory diagnosis of HAE comprises several parameters, the most important being considerably reduced (< 50% of normal) levels of C1-Inh function (Rother, 1998). In addition, levels of antigenic C1-Inh, C4 and C1q are often determined, of which measurement of C4 is important (Agostoni et al., 2004, Bowen et al., 2004) to exclude artificial C1-Inh inactivation.

Two types of tests are used to determine functional C1-Inh levels (Rother, 1998). The first assay is a chromogenic assay, which measures the inhibition of activity of the target protease C1s, by C1-Inh in the plasma sample to be tested. The other type of test is an ELISA-type assay, which detects complexes formed between C1Inh and C1r or C1s following activation of C1. In this paper we describe a survey among laboratories in Denmark, France, Germany, Hungary, Italy, Netherlands, Norway, Poland, Spain, Sweden, Switzerland, United Kingdom and Canada regarding use and performance of assays for fC1-Inh. All cooperating laboratories are nationally recognised reference centres for HAE diagnostics and treatment in their countries. The aim of the survey was to investigate the assays used and the uniformity of the results of fC1-Inh measurements used for HAE diagnosis. To this end, standardised, double-blinded plasma samples from healthy controls and HAE patients were tested. This paper describes the results and recommendations of this survey.

Section snippets

Collection of samples

Citrated plasma samples from 6 patients with HAE and 4 healthy volunteers were obtained by centrifugation of freshly collected blood at 1800 g for 10 min. Fresh serum from the same patients and healthy volunteers was obtained by centrifugation of clotted blood. The diagnosis HAE was confirmed by genetic analysis of the patients and of one of their affected family members. All samples were aliquoted and stored at − 80 °C before being sent out. The plasma/serum samples were randomised,

Assays

The chromogenic assay was used in 13/15 laboratories. Chromogenic assay kits were obtained from Technoclone GmbH (Vienna, Austria), Baxter (Deerfield, USA) or Dade Behring Holding GmbH (Liederbach, Germany). The assay supplied by Dade Behring was the most frequently used. One laboratory used a previously evaluated in-house test (Drouet et al., 1988) and one laboratory combined the protease from Dade Behring with a substrate from Bachem (Bubendorf, Switzerland). All laboratories performed the

Discussion

Based on the results of this survey, it is concluded that the diagnosis fC1-Inh deficiency is made correctly in most cases. Interestingly, in our survey most of the mainly European laboratories that are in specialised in HAE diagnostics used the chromogenic assay (13 out of 15). Although this hampers a clear comparison between the chromogenic assay and the complex ELISA, this small sample size of the latter is a clear reflection of the status quo in Europe. The observed positive predictive

Recommendations

Several recommendations may be made for fC1-Inh testing in a diagnostic laboratory:

  • The chromogenic assay can be recommended as an assay in making a correct diagnosis based on the high sensitivity, clear distinction, and low variability.

  • To avoid the occurrence of an occasional false-positive HAE diagnosis, additional determinations of for example C4 levels are recommended in case of low fC1-Inh.

  • We recommend special care of storage of samples. Storage conditions below − 20 °C are also critical,

Acknowledgements

We thank all technicians from the cooperating laboratories for their excellent technical support.

References (19)

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