Optimizing Dried Blood Spot Sampling for Children and Adolescents With Autism Spectrum Disorder

Supplemental Digital Content is Available in the Text.

Abstract

Background:

Dried blood spot (DBS) sampling offers several advantages for therapeutic drug monitoring, including minimal invasiveness, low blood volume requirements, and potential for home sampling. However, obtaining spots of sufficient quality for reliable analysis remains a key challenge of DBS. This study aimed to evaluate the impact of enhanced visual and textual guidance on the quality of DBS by analyzing data from a prospective multicenter study involving children and adolescents.

Methods:

In total, 108 DBS cards from 56 children were assessed for spot quality. Rejection criteria included spot diameter <6 mm, smeared or irregularly shaped spots, and overlapping spots. Approval and rejection rates, overall success rates of DBS cards, and the most common rejection reasons were compared between the groups that did and did not receive the enhanced visual and textual support (intervention).

Results:

Pre-intervention: 47.6% of the DBS cards contained at least 1 spot qualitatively acceptable for analysis, and 25.7% of all spots qualified for analysis. Post-intervention: the acceptance rates increased to 86.6% and 64.3%, respectively. In both the pre- and post-intervention groups, the most common reason for rejection was the presence of smeared or irregularly shaped spots.

Conclusions:

Continued improvements in DBS quality rely on effectively addressing challenges associated with home sampling challenges, which is critical for the seamless incorporation of DBS into clinical practice.

INTRODUCTION

Since the 1960s, dried blood spot (DBS) microsampling has been extensively utilized in neonatal screening to detect genetic and metabolic disorders.¹ Recently, its application for therapeutic drug monitoring (TDM) has gained increased attention owing to its convenience, cost-effectiveness, and minimal invasiveness.² DBS involves collecting a small droplet of blood, typically obtained through a finger prick, onto a DBS collection card. This technique is highly efficient and relatively straightforward to perform, as it enables the quantification of active substances using just 1 drop of blood.³ The combination of a minimal sample volume, ease of use, and ability to preserve sample quality make DBS ideal for home sampling, allowing patients to collect and mail samples at their convenience. Therefore, the DBS method could enhance personalized treatment by making TDM more accessible.^3,4

Conventionally, TDM relies on blood sample collection through venipuncture. However, this sampling method can be particularly challenging in some patient groups, such as children, who may have an extreme fear of needles.⁵ Notably, venipuncture is particularly difficult for children with autism spectrum disorder (ASD) as they often exhibit heightened pain responses, including increased facial activity.⁶ Moreover, behavioral comorbidities and heightened sensory sensitivities can complicate venous sampling even further.^4,7 In addition, their behavioral reaction after venipuncture tends to be more intense, requiring additional time for recovery. Collectively, these factors suggest that venipuncture has a significant impact on children with ASD.⁸ Conversely, a study by Kloosterboer et al⁴ reported that a finger prick was well tolerated in this patient population, with participants reporting minimal pain based on the Visual Analog Scale. Therefore, DBS sampling offers a promising alternative that can minimize the burden on this population, increase participation in TDM, and ultimately improve health outcomes.

Despite the advantages and convenience of DBS, its use in clinical practice remains limited. This is mainly because reliable analysis for TDM requires strict adherence to certain criteria. Unlike neonatal screening, in which qualitative and semi-quantitative detection is often sufficient, TDM relies on high-quality blood spots to ensure consistent sample volume and accurate quantification. Several factors can influence reliable DBS analysis, including hematocrit concentration, handling by the analyst during sample processing, the accuracy of the analytical equipment, and the patient's collection technique. In this study, we focus solely on sample collection.

An ideal sample consists of a single, round droplet of blood applied within the designated circle on the filter paper, sufficiently large to allow a punch for analysis.⁹ In several studies employing DBS to quantify active substances, many samples failed to meet these quality requirements. For instance, a study by Zuur et al found that 34% of spots were unsuitable for analysis owing to poor blood sampling techniques. The most common reasons for rejection were contamination of the spots by fingers and spots comprising multiple droplets.¹⁰ Boons et al¹¹ found that 20% of DBS samples were rejected, primarily owing to insufficient spot size. These studies involved DBS sampling performed by health care workers for patients with tuberculosis and by adult patients with chronic myeloid leukemia, respectively. A study by Francke et al¹² demonstrated that combined visual and textual guidance, including instructional videos, can improve home-based DBS collection and minimize common sampling errors. However, when applying this technique for our study population of children with ASD, a substantial proportion of DBS samples still did not meet quality criteria. Although home-based DBS sampling is typically preferred in this population to reduce burden, it can be particularly challenging, as the procedure is typically performed by the child or their caregiver without supervision from a health care professional.

Therefore, this study aimed to evaluate the quality of home-based DBS sampling by children and/or caregivers and assess the impact of enhanced visual and textual guidance on sample quality. By addressing the practical challenges associated with DBS, the study aims to facilitate broader implementation of DBS in clinical practice for TDM, particularly in home-based settings.

MATERIALS AND METHODS

Study Design

This explorative intervention study was performed at the Department of Hospital Pharmacy in the Erasmus University Medical Center. Data were collected from the randomized controlled trial Safety and Pharmacokinetics of Antipsychotics in Children 2 (SPACe 2: STAR).¹³ DBS cards collected from the SPACe: 2 STAR study between May 2022 and January 2025 were included in the analysis. Only DBS samples collected by children and caregivers were included; those collected by researchers were excluded. The DBS cards and their individual spots were compared, with samples collected before May 5, 2023, categorized as pre-intervention and those collected on or after May 5, 2023, as post-intervention.

Intervention

Group 1 (Pre-intervention): Children and caregivers received the standard sampling guidance, which comprised an illustrative leaflet (see Leaflet, Supplemental Digital Content 1, http://links.lww.com/TDM/A909) and a corresponding animated video with instructions on how to perform DBS sampling.

Group 2 (Post-intervention): In addition to the materials provided to Group 1, children and caregivers received an additional informative personalized letter containing both visual and textual instructions. This revised material included additional information, emphasized key points using bold text, and added illustrative examples of both suitable and unsuitable spots for analysis (see Letter, Supplemental Digital Content 2, http://links.lww.com/TDM/A910).

DBS Quality Assessment

All collected DBS samples were qualitatively assessed according to the standard operating procedure of the laboratory at the Erasmus Medical Centre. Each DBS card could contain multiple spots; if at least 1 high-quality spot was present, the card was classified as qualitatively approved. A high-quality spot was defined as a well-formed droplet that did not overlap with others, was not smeared on the DBS card or irregularly shaped, and was sufficiently large to allow the punching of a 6-mm diameter disc. Spots meeting all these criteria were approved, whereas those failing to meet 1 or more criteria were rejected.

Rejected spots were categorized as follows: (A) spot diameter <6 mm, (B) smeared or irregularly shaped spots, (C) overlapping spots, and (D) any combination of A, B, and/or C. Table 1 provides a detailed overview of these categories, along with illustrative examples.

TABLE 1.

Overview of Approval and Rejection Criteria With Illustrative Examples

Data Analysis

Qualitative approval rates for the DBS cards and individual spots were determined for the pre-intervention and post-intervention groups. In addition, the most common reasons for rejection were assessed across the 2 groups. To evaluate potential learning effects over time within individuals, improvements in sampling on an individual's second attempt were examined. In addition, trends across participants within the pre- and post-intervention groups were examined to identify potential confounding factors, such as extended verbal guidance from the researcher, which may have influenced sampling quality.

RESULTS

In total, 108 DBS cards from 56 children were included. The study population had a median age of 13.0 (IQR: 9–16) years and included 38 males, 17 females, and 1 nonbinary individual. The pre-intervention group consisted of 13 children, with a median age of 10.0 (IQR: 9–13) years, whereas the post-intervention group consisted of 43 children, with a median age of 14 (IQR: 9–16) years.

In the pre-intervention group, 47.6% (10/21) of DBS cards contained at least 1 high-quality spot and were approved for analysis, corresponding to a rejection rate of 52.4%. In contrast, 86.6% (71/82) of DBS cards in the post-intervention group met this criterion, with a significant lower rejection rate of 13.4%.

In the pre-intervention group, 74 spots from 21 DBS cards were assessed, with 25.7% (19/74) meeting the approval criteria. Conversely, in the post-intervention group, 269 spots from 82 DBS cards were assessed, with 64.3% (173/269) meeting the criteria. In both groups, the primary reason for sample rejection was smeared or irregularly shaped spots, accounting for 44.6% and 20.4% in the pre-intervention and post-intervention groups, respectively. In addition, insufficient spot size (<6 mm), overlapping spots, or a combination of these accounted for 29.8% and 15.2% of rejections in the pre-intervention and post-intervention groups, respectively. Table 2 summarizes the total number of assessed DBS cards and blood spots, along with the approval and rejection rates in both groups.

TABLE 2.

Total Number of Assessed DBS Cards and Spots With Corresponding Approval and Rejection Rates

	Pre-intervention n (%)	Post-intervention n (%)
Total DBS cards	21	82
Approved DBS cards	10 (47.6)	71 (86.6)
Rejected DBS cards	11 (52.4)	11 (13.4)
Total blood spots	74	269
Approved spots	19 (25.7)	173 (64.3)
Rejected spots	55 (74.3)	96 (35.7)

Analysis of potential learning effects showed no clear evidence of improvement. Specifically, comparisons of repeated DBS collections within individuals showed that the quality of the cards did not consistently improve upon a second attempt. Similarly, performance remained consistent over time across participants within each intervention group, indicating that no confounding factors beyond the intervention itself influenced the results.

DISCUSSION

This study aimed to evaluate the quality of DBS sampling performed by children and caregivers in a home setting and assess the impact of enhanced visual and textual guidance on sample quality. After the introduction of this intervention, a notable improvement in DBS quality was noted, with a rejection rate of 13.4% in the post-intervention group, which closely aligns with findings from previous studies involving trained health care professionals.¹¹ Furthermore, the approval rate of individual spots increased from 25.7% to 64.3% after the intervention. Notably, although it might be anticipated that children and their caregivers who performed repeated DBS sampling may demonstrate a learning effect that improves sample quality, our findings revealed no clear evidence of such improvement either within individuals or across the cohort, indicating that any such effect was negligible. These results suggest that the revised guidance provides clearer instructions for both children and caregivers, leading to a higher proportion of blood spots deemed suitable for analysis. Furthermore, these findings also demonstrate that, with adequate instruction, children and caregivers are capable of collecting DBS samples at home with a quality comparable to that of clinical sampling, further supporting the feasibility of home-based TDM. However, sampling errors remain prevalent, highlighting the inherent challenges of DBS collection and the need for ongoing efforts to improve sample quality in unsupervised settings.¹⁰

To further enhance DBS quality, it is essential to understand the challenges encountered by children and their caregivers by evaluating their experiences with the sampling method and subsequently determining the specific guidance each individual requires. Thus, incorporating a questionnaire or interview could facilitate the development and implementation of novel strategies. For instance, Linder et al¹⁴ examined the perspectives of parents on DBS home sampling to identify contributing factors for successful sampling. They found that the key factor was that both the children and parents needed to feel prepared and confident. Therefore, the combination of video instructions and practical demonstration by a nurse was essential. Moreover, they highlighted that effective communication with the children was crucial.

Building on the current sampling guidance used in the SPACe 2: STAR study, which includes written instructions and a video, supervised training could be highly beneficial. This training would focus on both the practical aspects of DBS and strategies for guiding children, particularly those with ASD, through sampling. In addition, a web-based application could be developed to enable patients to upload images of their spots for quality assessment.⁹ If a spot is deemed unsuitable, patients would have the opportunity to resample before mailing the final samples. Although implementing these DBS best practices may initially be time-consuming for health care providers, the long-term benefits may justify this investment, including improved spot quality, more reliable analysis, and ultimately, accurate and timely TDM recommendations.

However, these findings should be interpreted within the context of the study's limitations. Because this was a sequential study rather than a parallel-group design, time-related factors could have potentially influenced the results. Furthermore, although our discussion noted no clear evidence of a learning effect within children or caregivers, the influence of temporal changes cannot be fully excluded. Another limitation is the unequal distribution of children between the 2 groups. In addition, as no power calculation was performed, the findings can only be interpreted for descriptive purposes. Moreover, the retrospective nature of the study limits the quality assessment of the samples, as only the visual appearance of the dried blood spots could be evaluated. For instance, excessive pressure applied to the finger during sampling cannot be detected from the spot itself, although it may have affected the results and their clinical interpretation.

CONCLUSION

In conclusion, DBS sampling guidance requires careful attention to ensure high-quality spots for reliable analysis. This present study demonstrates that incorporating visual and textual support into the sampling guidance notably improves DBS quality. Effectively addressing the challenges associated with DBS home sampling is essential for its successful implementation in clinical practice. In addition, training that emphasizes the practical aspects of DBS and effective communication with children and caregivers may provide added value to further enhance sample quality.

Supplementary Material

tdm-48-524-s001.docx ^{(3.5MB, docx)}

tdm-48-524-s002.docx ^{(1.2MB, docx)}