Research paperFunctional C1-Inhibitor diagnostics in hereditary angioedema: Assay evaluation and recommendations
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:
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The chromogenic assay can be recommended as an assay in making a correct diagnosis based on the high sensitivity, clear distinction, and low variability.
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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.
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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.
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