Whatman 903 filter paper

The study population included 4,144 unrelated subjects living on Bioko Island These subjects aged from 1 to 75 years and received the screening for G6PD deficiency and hemoglobinopathies in Malabo Regional Hospital during April 2012 and May 2014. Questionnaires about nationality, gender, age, native status or not and written consent forms were available in Spanish (the most-common language) to ensure comprehensive understanding of the study objectives. This study was approved by the ethics committees of Malabo Regional Hospital and the First Affiliated Hospital of Shantou University Medical College. All subjects or their parents gave their informed consent by signature or thumbprint.
Blood samples were collected with EDTA-K₂ anti-coagulated tubes from each subject according to the standard procedures described previously [18 (link)], and 200uL of blood was adsorbed on Whatman 903 filter paper. These filter papers were air dried and stored individually in Ziplock bags containing silica desiccant beads and refrigerated (-20°C). Parasite detection was screened by ICT malaria Plasmodium falciparum cassette test (ICT Diagnostics, South Africa) and positive samples were treated with artesunate-amodiaquine (Camoquin plus).

Genomic DNA was extracted from dry blood spots on Whatman 903 filter paper (Whatman plc, UK) using a QIAamp DNA Mini Kit (Qiagen, USA) following the manufacturer’s instructions. The Plasmodium species were identified by amplifying the Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene using a modified protocol described by Nundu et al. [48 (link)]. In this study, of the 438 falciparum-positive samples, 248 asymptomatic infections (162 P. falciparum and 86 P. falciparum + non-falciparum) and 190 symptomatic infections (144 P. falciparum and 46 P. falciparum + non-falciparum) were used for PCR analysis [48 (link)].

Newborn blood spot screening is offered to the parents/guardians of all infants registered with the Alberta government. Newborn screening is voluntary, and consent is verbal [18 (link)]. Collection of the blood specimens is recommended to occur between 24 and 72 h of age, ideally closer to 24 h. If a newborn requires an RBC transfusion (RBCT) within the first 24 h of life, our recommendation is to collect a sample before the transfusion, followed by a second, post-transfused collection at the age equal or greater than 24 h. Dried blood spot (DBS) specimens are collected as previously described [18 (link)]. Briefly, capillary blood spots obtained by heel-stick are collected on Whatman 903 filter paper attached to the newborn screening requisition. Samples are then air-dried for a minimum of three hours and transported at ambient temperature in a sealed newborn screening envelope to the provincial screening laboratory in Edmonton. Since some conditions may be missed in low birth weight (BW) infants when screened early after birth, all infants with BW less than 2000 g are recommended to have blood spots recollected at 21–28 days of age, even if the initial screen result was normal.

The Philippine government launched a dengue mass immunization in 2016 in high-risk regions using the first licensed dengue vaccine, CYD-TDV (Dengvaxia, Sanofi Pasteur, Lyon, France). We initiated a prospective cohort study just before the mass dengue vaccination in Cebu Province.^{7 (link)} We invited healthy children residing in Bogo and Balamban, semi-urban areas in Cebu, who would be 9–14 years of age at the time of the dengue mass vaccination to participate in the study. From each participant, we collected approximately 5 mL of blood in anticoagulant-free vacutainer tubes, which was processed and aliquoted. In the initial consecutive subset of participants, 60–70 µL of blood was blotted at the center of each collection card (Whatman 903 filter paper, Whatman plc, Maidstone, Kent, UK) immediately after the blood draw, using a disposable transfer pipette. The blood on the filter paper was allowed to air-dry for at least 4 hours on a rack. After completion of the drying process, the DBS were packed individually in single, gas-permeable zipper bags with three to five desiccant sachets and a humidity indicator, and kept at ambient temperature at the field sites. The frozen sera and DBS were shipped to the University of the Philippines Manila-NIH in dry ice then stored in −80°C before testing.

The calibrators and quality controls samples were prepared in leftover blood from healthy controls after adjusting the haematocrit to 50% similar to that of a newborn. Stock solutions of PA, d9-PA (1000 μg/mL), and AASA/P6C (321 μg/mL) were prepared in water. Working stocks were prepared in 50:50%v/v acetonitrile: water and spiked into aliquots of haematocrit adjusted blood. 40μl of the spiked blood was pipetted onto Whatman 903 filter paper to prepare calibrators and quality controls (LLQC, LQC, MQC, and HQC) as per Table 1.