Development of a High-throughput Serological Testing Platform for Mass Screening Purposes of Both Pediatric and Adult Cohorts

Orthomyxoviridae, Flaviviridae, and Coronaviridae are viral families from which a pandemic/epidemic agent is expected to emerge in the coming years. In particular, highly pathogenic avian influenza viruses of the H5N1 subtype and MERS-CoV are respiratory viruses that are known to cause sporadic infections in humans in vast areas of the world where these infections are endemic in animals. These pathogens are on the high-priority list of the World Health Organization given their potential to increase their fitness in humans, their ability to reassort/recombine with other respiratory agents, and the unpredictable potential to gain human-to-human transmission abilities. Zika and other exotic zoonotic flaviviruses like Dengue, Usutu, or Mayaro viruses are not respiratory pathogens, but the rising temperatures associated with climate change are making the European region a more favorable ecological space for the introduction and maintenance of these viruses.

The development of a serological high-throughput platform for mass screening is a fundamental element in increasing preparedness for pandemic scenarios. In the case of large emergency serosurveys, innovation in the sampling techniques is an often neglected but relevant aspect to facilitate the enrolment of subjects in both adult and paediatric populations. Venipuncture is considered the gold standard for sampling blood. Nonetheless, this method includes several important drawbacks: the great volume that is collected (exceeding 1 ml), its invasiveness, the need for a sterile environment as well as specialized personnel to perform it, and the potential risk of exposure to pathogens, which demands a great effort in sample preparation and shipping conditions (affecting costs) (reviewed in Bossi et al. 2024).

As a result, starting in 1963 with dry blood spots (DBSs), blood microsampling methods, that is, the collection of small volumes (≤150 μl) of capillary blood derived usually through a finger prick, have been proposed. Capillary sampling is a standard procedure for neonatal screening testing and diabetic patients, while venipuncture is the standard approach to blood sampling and, in particular, for those oriented to the screening of antibodies against infectious diseases. The finger and heel-prick techniques have been rarely adopted for the titration of neutralizing antibodies due to the limited volume of sera available and the cumbersome laboratory process required to recover antibodies (i.e., puncturing DBSs, eluting the spots overnight, etc.).

Microsampling devices, known as volumetric tip microsampling (VTM), represent a novel approach to sampling and self-sampling. These tools serve the same purpose as the DBS (i.e., filter papers), which has been commonly used for neonatal hematology, but with the advantage of the ease of automation and the standardization of sample volumes. Further, VTM can be particularly beneficial in remote non-specialized centers and domestic settings where venipuncture is not possible and where logistical impediments challenge the storage of samples.

VTM is a method that permits a correct quantification of the sample. Hematocrit, along with other parameters, such as humidity and drying conditions, influence spot size, spot homogeneity, and extraction recovery of analytes in DBS, making it inadequate for procedures that require quantification of analytes (reviewed in Thangavelu et al. 2023). Moreover, filter paper properties, a fundamental component of DBS, can introduce further bias by influencing "the maximum loading capacity, blood spreadability, chromatographic effects, analyte stability and recovery." (reviewed in Thangavelu et al. 2023).