Validating and utilizing dried blood spots for family screening: Screening Programme Providing Outreach for Testing Hereditary Angioedema (SPPOT-HAE)

Abstract

Background

Hereditary angioedema (HAE) is a rare genetic disorder with potentially life-threatening consequences, traditionally diagnosed by conventional laboratory methods that can be resource intensive and inconvenient. Incorporating dried blood spot (DBS) tests may be a promising alternative for diagnosing HAE and family screening.

Objective

This study aimed to validate DBS with conventional laboratory assays among confirmed C1 esterase inhibitor (C1-INH) HAE patients and assess the utility of DBS in a Screening Programme Providing Outreach for Testing Hereditary Angioedema (SPPOT-HAE).

Methods

In part 1, 16 Chinese C1-INH-HAE patients from 7 families participated in the validation of DBS for detecting C4, C1-INH, and functional C1-INH (fC1-INH). The results were compared with conventional laboratory assays. In part 2, DBS was utilized in family screening for HAE in a large Chinese family with relatives previously refusing testing.

Results

The study found strong correlation between conventional assays and DBS in measuring C4 (r = 0.870, P < .0001), C1-INH (r = 0.978, P < .0001), and fC1-INH (r = 0.756, P < .0001). There were no false-negative results from the DBS for C4, C1-INH or fC1-INH. SPPOT-HAE successfully recruited 9 additional relatives for family screening, of whom 22% were confirmed to have HAE. The use of DBS in an outreach program overcame barriers of prior family screening initiatives.

Conclusion

This is the first study to validate measurement of fC1-INH using DBS in C1-INH-HAE with conventional assays. An outreach program using DBS is a promising strategy overcoming previous barriers of family screening. Further large-scale, multicenter studies are required to establish the role of DBS, compare cost-effectiveness with prior strategies, and maximize diagnosis in resource-constrained countries.

Introduction

Timely diagnosis of hereditary angioedema (HAE) is paramount to ensure appropriate treatment and reduces unnecessary procedures, hospitalization, and even death.1, 2, 3 Prior research has highlighted the considerable impact of earlier HAE diagnosis, particularly in underserved and rural areas, such as those within the Asia-Pacific region, where there are limited diagnostic resources.4, 5, 6, 7, 8 Diagnosing HAE due to C1 esterase inhibitor (C1-INH) deficiency (type I) or dysfunction (type II), collectively referred to as C1-INH-HAE, necessitates a combination of clinical history and laboratory testing.⁹ Traditionally, conventional laboratory techniques involve turbidimetry, nephelometry, or radial immunodiffusion for quantitatively measuring C4 and C1-INH antigenic levels, and chromogenic assays or enzyme-linked immunosorbent assays (ELISA) for determining functional C1-INH (fC1-INH) levels.10, 11, 12 Despite being a reasonable screening marker, finding of normal C4 level cannot rule out C1-INH-HAE, with sensitivity as low as 56% among Chinese HAE patients.¹³ In diagnosing type II HAE, fC1-INH is particularly crucial because C1-INH levels can be normal or even elevated. However, establishing accredited laboratory facilities with expertise might not be feasible or affordable in some regions. In resource-limited areas where fC1-INH is not easily accessible, low C4 with elevated C1-INH values has been suggested to screen for type II HAE.¹⁴ Inappropriate handling of blood tests can result in in vitro protein denaturation and falsely low outcomes.¹¹ Storage temperatures can also influence fC1-INH measurements, necessitating blood sample processing within 2 hours if stored at room temperature, or functional levels may decrease, leading to a falsely positive HAE diagnosis.^10,12 If samples need to be transported for over 24 hours, blood samples must be centrifuged at the collection site, stored at a minimum of −20°C, and transported on dry ice.¹⁵ This is frequently not possible in remote locations, especially among resource-constrained countries. Furthermore, the reported sensitivity of fC1-INH has also been inconsistent—as low as 57% when using ELISA.¹⁶

The potential of dried blood spot (DBS) testing has generated significant interest, particularly in areas lacking access to laboratory facilities.^4,5 DBS also allows testing patients in remote areas or screening those who may decline to return to clinics for formal laboratory testing. Benefits over conventional laboratory methods include easier blood collection, stability of DBS samples in storage, and ease of transport. Iurașcu et al developed a DBS method for quantifying C4 and C1-INH using in-house liquid chromatography–mass spectrometry in both HAE and acquired angioedema (AAE) patients.¹⁷ The results were validated against conventional measurements of C4 using turbidimetry and C1-INH using radial immunodiffusion. However, the measurement of fC1-INH was not explored, which is vital for diagnosing type II HAE. Lai et al also developed a DBS method to quantify C4, C1-INH, C1q, and fC1-INH using liquid chromatography–mass spectrometry, demonstrating distinct levels between healthy individuals and C1-INH-HAE patients (22 type I and 1 type II HAE).^18,19 Nevertheless, the results were not validated with more conventional laboratory methods, as mentioned previously.

Our study aimed in part 1 to validate the DBS method with conventional complement assays in patients with confirmed C1-INH-HAE; and in part 2 to assess the utility of DBS in SPPOT-HAE. Levels of C4, C1-INH, and fC1-INH were measured using turbidimetry, nephelometry, and chromogenic assay, respectively. Details of laboratory tests are provided in Text E1 in the Online Repository available at www.jaci-global.org. Results were indicated either as low (defined as below the lower limit of the reference range/value) or not low (defined as within the normal reference range/value or higher). Clinical parameters, including symptomatology, age at onset and diagnosis, type of C1-INH-HAE, and treatment history, were reviewed.

Patients with confirmed C1-INH-HAE from the Angioedema Centres of Reference and Excellence (aka ACARE) site in Hong Kong, Queen Mary Hospital, were recruited. The family pedigree was drawn and updated, and confirmed HAE patients were asked to invite relatives for family screening using DBS. Relatives previously screened as not having HAE or with no risk of inheriting HAE (eg, offspring of relatives confirmed to not have HAE, or siblings of HAE patients confirmed to have a de novo mutation through family trio sequencing) were excluded. An outreach program that used trained medical nurses to perform DBS in the community setting (eg, within patient’s homes or community centers)—Screening Programme Providing Outreach for Testing for HAE (SPPOT-HAE)—was performed between June and July 2024. All patients were asked to return for confirmatory testing by conventional laboratory methods. SPSS Statistics 26 (IBM, Armonk, NY) and GraphPad Prism 8 (GraphPad Software, La Diego, Calif) were used to calculate the Pearson correlation coefficient (r) between the DBS and laboratory results.

All patients provided informed consent to participate in this study. This study was approved by the institutional review board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster.

Results and discussion

Part 1 of this study focused on evaluating and validating the DBS results in diagnosing C1-INH-HAE. A total of 16 Chinese C1-INH-HAE patients (type I, n = 11; type II, n = 5) from 7 families participated in the validation analysis of DBS for detecting C4, C1-INH, and fC1-INH. Among the participants, 8 were male and 8 were female, and 14 were symptomatic (87.5%); they had a median (range) age at symptom onset of 19 (5-44) years, at diagnosis of 44 (12-66) years, and during blood collection of 50 (9-79) years. Five (20.8%) were receiving HAE-specific prophylactic medications. A detailed comparison of conventional complement assays and the new DBS-based method is presented in Table I.

Table I.

Comparison of results between conventional and DBS laboratory testing

Patient no.	C4 level				C1-INH level				Functional C1-INH assay
	Conventional		DBS		Conventional		DBS		Conventional		DBS
	Normal: 9-35 mg/dL	Result	Normal > 89.57 μg/mL	Result	Normal: 21.4-38.7 mg/dL	Result	Normal > 126.66 μg/mL	Result	Normal: 0.7-1.3 U C1-INH/mL	Result	Normal > 62.8%	Result
1	22	NL	139.99	NL	67.8	NL	444.25	NL	0.30	L	<LLoQ	L
2	8.2	L	56.6	L	6.6	L	21.52	L	NA		<LLoQ	L
3	10	L	30.17	L	6.1	L	9.3	L	0.03	L	<LLoQ	L
4	8	L	28.89	L	57.2	NL	366.06	NL	0.07	L	<LLoQ	L
5	8	L	0	L	98.3	NL	481.54	NL	0.49	L	<LLoQ	L
6	8	L	41.52	L	68.3	NL	380.66	NL	0.07	L	<LLoQ	L
7	8	L	31.09	L	70.5	NL	423.32	NL	0.07	L	<LLoQ	L
8	8	L	33	L	3.3	L	12.01	L	0.08	L	<LLoQ	L
9	10	L	34.54	L	8.1	L	29.66	L	0.26	L	<LLoQ	L
10	4.6	L	19.49	L	3.4	L	11.48	L	0.08	L	<LLoQ	L
11	<8	L	25.39	L	4.7	L	15.95	L	NA		<LLoQ	L
12	<8	L	42.58	L	5.7	L	15.85	L	NA		<LLoQ	L
13	18	NL	103.25	NL	8.3	L	32.21	L	0.32	L	<LLoQ	L
14	3.8	L	14.17	L	4.7	L	2.95	L	0.16	L	<LLoQ	L
15	32	NL	155.64	NL	17.4	L	40.15	L	0.16	L	<LLoQ	L
16	8	L	83.63	L	20.3	L	49.63	L	0.50	L	<LLoQ	L
17	33	NL	181.04	NL	29.3	NL	181.04	NL	1.21	NL	71.3	NL
18	17	NL	98.39	NL	4.6	L	16.15	L	0.20	L	<LLoQ	L
19	<8	L	17.64	L	5.0	L	14.97	L	0.16	L	<LLoQ	L
20	21	NL	105.96	NL	35.7	NL	160.22	NL	1.41	NL	76.8	NL
21	21	NL	112.01	NL	22.0	NL	106.33	L	0.78	NL	59.3	L
22	14	NL	79.64	L	21.5	NL	94.97	L	0.82	NL	48.3	L
23	36	NL	239.61	NL	35.2	NL	173.29	NL	NA		78.7	NL
24	NA		172.11	NL	NA		148.62	NL	NA		70.1	NL
25	27	NL	168.16	NL	29.2	NL	98.97	NL	1.14	NL	68.6	NL

L, Low; LLoQ, lower limit of quantification; NA, not available; NL, not low.

Part 2 of this study examined the potential role of DBS in an outreach HAE family screening program, SPPOT-HAE. A prior comprehensive family screening program, where all contactable relatives had been invited to return for family screening, had already been implemented in Hong Kong and reported previously.¹³ In one large Chinese HAE family, the prior family screening program managed to screen 14 relatives; 7 were confirmed to have HAE, of whom 2 had exhibited symptoms before screening. After approaching relatives who had persistently declined family screening previously (the result of reluctance to travel to the clinic for blood tests), an additional 9 relatives (patients 17-25) from this family (1 male and 8 female subjects) agreed to DBS testing as part of SPPOT-HAE, with DBS performed at their homes and during a community event celebrating the annual HAE day.

DBS results indicated abnormalities (patients with low C1-INH and fC1-INH) in 4 of these 9 relatives (patients 18, 19, 21, and 22). After simultaneous and repeat testing using conventional laboratory methods, 2 of 4 patients (patients 21 and 22, with very borderline low C1-INH and fC1-INH results on DBS) had normal C1-INH and fC1-INH, without clinical history of angioedema. The remaining patients (patients 17, 20, 23, 24, and 25) who were screened as not having HAE had concordant results.

Overall, a strong correlation was demonstrated between the DBS and conventional laboratory testing for C4 (r = 0.870, P < .0001), C1-INH (r = 0.978, P < .0001), and fC1-INH (r = 0.756, P < .0001) (Fig 1). With reference to conventional laboratory testing as the standard, there were no false-negative DBS results, including C4 (9/9), C1-INH (9/9), and fC1-INH (3/3).

Fig 1.

Correlations of conventional complement assays with new DBS-based method for (A) C4 level, (B) C1-INH level, and (C) C1-INH function.

To our knowledge, this is the first study that validates the measurement of fC1-INH using DBS in C1-INH-HAE patients via conventional laboratory methods. It is also the first study to use DBS for the purpose of family screening for HAE in a small pilot outreach program. Our results showed strong correlation between conventional methodology and DBS results in measuring C4, C1-INH, and fC1-INH. Our small pilot study did not detect any false-negative results, which give confidence that DBS may be a promising tool for family screening (ie, low likelihood to miss genuine C1-INH-HAE). However, larger studies will be needed to further confirm the positive and negative predictive values of DBS in family screening.

Furthermore, the success of SPPOT-HAE presents a potential solution to overcome some barriers in diagnosis and family screening related to lack of resources and individual barriers from relatives (eg, reluctance to travel or return to clinic for blood sampling). Previous larger-scale studies have utilized other in-house DBS methodologies, with some inconsistent results.¹⁷ DBS has also been shown to be a viable method for genetic analysis and for quantifying C1q, which may be useful in aiding diagnosis of HAE with normal C1-INH or AAE.¹⁸ The use of DBS in measuring cleaved high-molecular-weight kininogen is currently being explored.²⁰ Our study has several limitations, including small sample size and lack of comparison with other methodologies like ELISA or radial immunodiffusion. Additionally, patients with AAE were not included, and larger cohorts of normal control subjects to validate our C1-INH comparisons were not available. This emphasizes the importance of larger validation studies to assess diagnostic accuracy in the future.

We propose that future research should focus on adopting strict clinical criteria for DBS screening in patients with angioedema and further validating DBS with conventional laboratory assays among different populations. We postulate that the added value of incorporating DBS into ongoing screening initiatives can help rapidly boost HAE diagnosis, especially among resource-constrained countries. Prospective studies to explore the cost-effectiveness of SPPOT-HAE across mainland China and in other Asia-Pacific countries are also in progress. It would also be imperative to compare the cost-effectiveness of SPPOT-HAE with previous clinic-based screening strategies. In conclusion, DBS in HAE is highly promising, and further large-scale, multicenter studies will further consolidate its role in the HAE diagnosis landscape.

Key messages.

• •DBS testing correlates with conventional tests of HAE.
• •Outreach programs using DBS can overcome prior barriers in family screening.
• •Further research is needed to optimize DBS utilization and assess cost-effectiveness

Disclosure statement

The dried blood spot tests were funded by Takeda (Massachusetts, United States) and Revvity Omics (Pennsylvania, United States). Takeda and Revvity did not have any input in designing the study, conducting measurements, analyzing/interpreting results, or writing the report.

Disclosure of potential conflict of interest: P. H. Li was speaker and advisor for and has received research funding from CSL Behring, KalVista, Pharvaris, and Takeda. The rest of the authors declare that they have no relevant conflicts of interest.