Evaluating functional C1INH with multiple laboratory methods across Hereditary Angioedema types

Abstract

Introduction: Hereditary Angioedema (HAE) is a rare genetic disease characterized by recurrent episodes of edema and classified into HAE with C1 inhibitor deficiency (HAE-C1INH types 1 and 2) and HAE with normal C1INH (HAE-nC1INH). This study evaluates the function of C1 inhibitor (fC1INH) in patients with suspected HAE using several laboratory methods: dried blood spot (DBS), chromogenic assay, and ELISA with FXIIa and PKa (plasma kallikrein). The comparative approach aims to improve early detection and understanding of C1INH dysfunction in all HAE subtypes to reflect real-world diagnostic scenarios.

Methods: We assessed the diagnostic performance of four fC1INH assays in a cohort of 148 HAE patients: 84 with HAE-C1INH (72 type 1 and 12 type 2) and 64 with HAE-nC1INH (53 HAE-FXII and 11 HAE-UNK). The gold-standard chromogenic assay and the two substrate-specific ELISAs (PKa and FXIIa) were compared to a novel DBS-based LC-MS/MS assay using endogenous C1s activity. For all fC1INH assays, values >50% were considered within the normal range.

Results: In HAE-C1INH, the DBS assay showed the highest specificity (type 1: 98.6%, type 2: 100%) and 100% sensitivity for both subtypes. ELISA-FXIIa also performed well (specificity: 97.2% and 91.7%). In contrast, ELISA-PKa and the chromogenic assay showed reduced specificity in type 2 (25% and 66.7%, respectively). Among patients with HAE-FXII, fC1INH levels were reduced by 36.5% by ELISA-FXIIa (19/52), 19.1% by DBS (9/47), and 3.8% by ELISA-PKa (2/52), and no alterations were detected by the chromogenic assay. Some of the changes seen in other tests may be partly related to pregnancy in a few patients. In the HAE-UNK group, all 11 patients had fC1INH >50% in all methods.

Conclusion: DBS-based LC-MS/MS and ELISA-FXIIa offer promising accuracy and broader applicability for early diagnosis of HAE types 1 and 2. The use of novel substrates and the inclusion of a clinically realistic cohort may enhance the translational relevance of these findings.

Keywords: C1 inhibitor; biomarker; complement C4; diagnosis; factor XII; hereditary angioedema.

Figure 1

C4 levels according to types of hereditary angioedema. Box-and-whisker plots represent serum C4 concentrations (mg/dL) in patients with hereditary angioedema (HAE) due to C1INH deficiency type 1 (HAE-1) and HAE due to C1INH deficiency type 2 (HAE-2), as well as HAE with normal C1 inhibitor levels, subdivided into HAE with factor XII mutation (HAE-FXII) and HAE of unknown mutation (HAE-UNK). The normal reference interval for serum C4 is indicated by dashed lines. Boxes represent the interquartile range (IQR); the horizontal line within each box indicates the median, and whiskers extend from the minimum to the maximum values. Individual data points are overlaid. Statistical analysis was performed using the Kruskal-Wallis test followed by Dunn's multiple comparisons test. (****p <0.0001).

Figure 2

C1INHq levels according to HAE diagnosis. Box-and-whisker plots represent C1 inhibitor level concentrations (CIINIIq, mg/dL) in patients with hereditary angioedema (HAE) due to CIINH deficiency type 1 (HAE-1) and HAE due to C1INH deficiency type 2 (HAE-2), as well as HAE with normal C1 inhibitor levels, subdivided into HAE with factor XII mutation (HAE-FXII) and HAE of unknown mutation (HAE-UNK). The normal reference interval for CIINHq levels is indicated by dashed lines. Boxes represent the interquartile range (IQR); the horizontal line within each box indicates the median, and whiskers extend from the minimum to the maximum values. Individual data points are overlaid. Statistical analysis was performed using the Kruskal-Wallis test followed by Dunn's multiple comparisons test. (****p<0.0001).

Figure 3

Comparison of fC1INH % values by different methods in HAE-C1INH-Type1. Box-and-whisker plots represent functional C1 inhibitor (fC1INH%) levels in patients with hereditary angioedema due to C1INH deficiency type 1 (HAE-C1INH-Type1), assessed using four different methods: dried blood spot (DBS), chromogenic assay, and ELISAs employing kallikrein (PKa) or activated factor XII (FXIIa) as substrates. The dashed line indicates the lower reference limit for normal fC1INH activity (50%). Boxes represent the interquartile range (IQR); the horizontal line within each box indicates the median, and whiskers extend from minimum to maximum values. Individual data points are overlaid. Statistical analysis was performed using the Kruskal-Wallis test followed by Dunn's multiple comparisons test. (****p < 0.0001). Sensitivity was calculated by identifying true positive results defined as fC1INH activity below 50% in patients with confirmed HAE-1. Specificity was assessed using samples from individuals without C1INH deficiency, where true negatives were defined as fC1INH activity ≥50%.

Figure 4

Comparison of fC1INH % values by different methods in HAE-C1INH-Type2. Box-and-whisker plots represent functional C1 inhibitor (fC1INH%) levels in patients with hereditary angioedema due to C1INH deficiency type 2 (HAE-C1INH-Type2), assessed using four different methods: dried blood spot (DBS), chromogenic assay, and ELISAs employing kallikrein (PKa) or activated factor XII (FXIIa) as substrates. The dashed line indicates the lower reference limit for normal fC1INH activity (50%). Boxes represent the interquartile range (IQR); the horizontal line within each box indicates the median, and whiskers extend from minimum to maximum values. Individual data points are overlaid. Statistical analysis was performed using the Kruskal-Wallis test followed by Dunn's multiple comparisons test (**p < 0.01; ***p < 0.001; ****p < 0.0001); Sensitivity was calculated by identifying true positive results-defined as fC1INH activity below 50% in patients with confirmed HAE-1. Specificity was assessed using samples from individuals without C1INH deficiency, where true negatives were defined as fC1INH activity ≥50%.

Figure 5

Comparison of fC1INH % values by different methods in HAE-FXII. Box-and-whisker plots depict functional C1 inhibitor (fC1INH%) levels in patients with HAE with normal CIINH due to a Factor XII variant (HAE-FXII), assessed using four different methods: dried blood spot (DBS), chromogenic assay, and ELISAs employing kallikrein (PKa) or activated factor XII (FXIIa) as substrates. The dashed line represents the lower reference limit for normal fC1INH activity (50%). Boxes indicate the interquartile range (IQR); the horizontal line within each box shows the median, and whiskers extend from the minimum to maximum values. Individual data points are overlaid. Statistical analysis was performed using the Kruskal-Wallis test followed by Dunn's multiple comparisons test (*p < 0.05; ****p < 0.0001).

Figure 6

Comparison of fC1INH % values by different methods in HAE-UNK. Box-and-whisker plots depict functional C1 inhibitor (fC1INH%) levels in patients with HAE with normal C1INH with unknown mutation (HAE-UNK), assessed using four different methods: dried blood spot (DBS), chromogenic assay, and ELISAs employing kallikrein (PKa) or activated factor XII (FXIIa) as substrates. The dashed line represents the lower reference limit for normal fC1INH activity (50%). Boxes indicate the interquartile range (IQR); the horizontal line within each box shows the median, and whiskers extend from the minimum to maximum values. Individual data points are overlaid. Statistical analysis was performed using the Kruskal-Wallis test followed by Dunn's multiple comparisons test (**p < 0.01).