Allergy. 2015 Sep;70(9):1103-11.
C1 inhibitor function using contact-phase proteases as target: evaluation of an innovative assay.
Ghannam A1,2, Sellier P1,2, Defendi F1,3, Favier B1, Charignon D1,3, López-Lera A4,5, López-Trascasa M4,5, Ponard D3, Drouet C1,3
1 Université Joseph Fourier, GREPI/AGIM CNRS FRE 3405, Grenoble, France
2 KininX SAS, Grenoble, France
3 Centre de Référence des Angioedèmes CREAK, CHU Grenoble, France
4 Unidad de Inmunología, Hospital Universitario La Paz/IdiPAZ, Madrid, Spain
5 Centro de Investigación Biomédica en Red (CIBERER U-754), Madrid, Spain
Background: Controlling prekallikrein activation by C1 inhibitor (C1Inh) represents the most essential mechanism for angioedema patient protection. C1Inh function in the plasma is usually measured based on the residual activity of the C1s protease not involved in the pathological process. We have hereby proposed an alternative enzymatic measurement of C1Inh function based on contact-phase activation and correlation with angioedema diagnostic requirements.
Methods: The contact phase was reconstituted using the purified components, with C1Inh standard or plasma sample. The kinetics of the amidase activity were monitored using Pro-Phe-Arg-pNA, independently of alpha2-macroglobulin. We prevented any interference from a possible high plasma kininogenase activity by preincubating the samples with protease inhibitor. Receiver operating characteristics (ROC) were used to calculate the assay’s diagnostic performance.
Results: The calibration curve was built using C1Inh standard (threshold limit 0.10 × 10(-3) U, i.e., 0.2 pmol), and C1Inh function was quantified in the sample, with a reference interval established based on healthy individuals (n = 281; men: 0.61-1.10 U/ml, median: 0.85 U/ml; women: 0.42-1.08 U/ml, median: 0.74 U/ml). The median values of female donors were lower than those of the others due to estrogen, yet C1Inh function remained within the reference interval. The ROC curve calculation provided the following optimum diagnostic cutoff values: women 0.36 U/ml (area under curve [AUC]: 0.99; sensitivity: 93.48%; specificity: 99.37%); and men 0.61 U/ml (AUC: 1; sensitivity: 100.0%; specificity: 100.0%).
Conclusion: The performance outcome provided features suitable for angioedema diagnostic or follow-up. Established by means of the kinin formation process, this assay should be preferred over the method based on a C1s protease target
Bradykinin-mediated angioedema represents an underestimated clinical problem. When involving airways it is a life-threatening condition, which is the cause of significant morbidity, emergency room visits, and hospitalisation. Appropriate evaluation of biological phenotype for diagnosis of patients is essential for successful management. The measurement of C1 Inhibitor (C1Inh) is the core laboratory testing for angioedema diagnosis. Patient identification requires the determination of C1Inh function, this function is currently measured using C1 protease as target. However, the primary mediator of swelling in angioedema patients is bradykinin, whose production is depending on plasma KK (Figure 1).
Figure 1. C1Inhibitor is the major control of contact phase. C1Inh, C1 Inhibitor; FXII, Factor Hageman or factor XII; HK, High Molecular Weight Kininogen; KK, plasma Kallikrein; pKK, prekallikrein.
We developed a laboratory assay for C1Inh function using contact-phase proteins, the target involved in the pathological process, without interference of α2M or plasma protease. Taking advantage of the high Kass of C1Inh for KK (table I), this assay is particularly designed to decipher pathological angioedema conditions.
The assay performance is excellent with very high sensitivity and specificity. The assay is the first test for the evaluation of C1Inh function with distinction capacity between male and female individuals. It gives the advantage of a very good sensitivity in female angioedema conditions with estrogen-dependence trigger.
An additional characteristic is its particularity to distinguish between the different SERPING1 mutants causative for the disease. As illustrative examples, the mutation products 271T and R378C cannot control contact phase whereas C1s protease is partly kept under control; the inverse situation is observed with the L107R C1Inh mutant. These features are closely associated with the circulating C1Inh molecular species.
The clinical application of the assay is suitable for angioedema diagnostic or patient follow-up: The threshold limit value to protect patient from attack is retained as 0.38 U/ml (1); this value is close to the low reference value, defined as 0.42 U/ml for women.
Table I. Control of contact phase, fibrinolysis, and complement proteases by C1 Inhibitor and α2Macroglobulin.
- Hack CE, Relan A, van Amersfoort ES, Cicardi M. Target levels of functional C1-inhibitor in hereditary angioedema. Allergy 2012 67:123-30
Arije GHANNAM MD, Ph.D
CSO KininX SAS
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