Children 5 years of age or under who presented to six hospital study sites throughout northern and central Nigeria were prospectively screened from September 2011 through May 2015, and enrolled if they had a fever >38.5°C associated with difficulty breathing or altered consciousness. In addition, a subgroup of healthy neonatal controls were also enrolled. A detailed questionnaire was administered by a physician to obtain information on clinical history and physical examination findings. After informed consent was obtained, the skin was cleaned with alcohol swabs and 1–3 mL of blood was directly collected and inoculated into an aerobic Bactec (Becton Dickinson, Temse, Belgium) culture bottle and delivered to the regional microbiology laboratory within 4 hours of collection for processing. In addition, the finger was cleaned with an alcohol swab before being pricked, and blood was spotted onto either five spots (diameter 1.3 cm) on Whatman 903, or one spot (diameter 2.5 cm) on Whatman FTA. Whatman 903 was available for the majority of the study period at the study sites, however in the later period these were replaced by Whatman FTA. DBS were stored in a -80°C freezer until they were transported to the University of Minnesota for further processing.
Whatman 903
Manufactured by Cytiva
Sourced in United Kingdom, United States
Whatman 903 is a type of laboratory filter paper designed for collecting and storing dried blood samples. It is a cellulose-based material commonly used in clinical and diagnostic applications.
Automatically generated - may contain errors
10 protocols using whatman 903
1
Fever and Respiratory Illness in Nigerian Children
2
Dried Blood Spot PCR Analysis
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In both schools and health facilities, three capillary blood drops from finger-pricks were collected and spotted onto of Whatman 903™ filter paper (Whatman plc, UK) for PCR analysis. Whatman 903™ filter papers containing blood samples were dried and stored in individual plastic bags containing desiccant and stored at − 20 °C before transportation to Nagasaki University for PCR analysis. All DNA templates were extracted from three punched discs (6 mm in diameter) of blood spots using the QIAamp DNA Mini Kit using the QIAamp DNA Mini Kit® (Qiagen, USA) according to the manufacturer’s instructions. DNA was eluted in 50 μL of the provided buffer.
3
Dried Plasma Spot Sample Preparation
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Preparation of the DPS was done according to protocol as described by Drabe et al.47 (link). Plasma samples were thawed and spotted in 2 × 25 ul volume on Whatman 903 filter paper (Whatman, USA) per sample and allowed to air dry for 3–4 hours at ambient temperature. Filter paper samples were stored in zip-lock plastic bags with a desiccant and sent by postal service from Oslo University Hospital, Norway, to Statens Serum Instititute, Denmark.
4
Dried Blood Spot Collection for Newborn Screening
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Dried blood spots (DBS) were collected on Whatman 903 filter paper cards (Whatman Inc., USA). Whole blood was collected in the NuPED study by venous blood collection into an EDTA‐coated vacutainer (Becton Dickinson, Woodmead, South Africa) and in the STRIPE‐SA study by capillary blood collection via finger prick. Blood samples were spotted onto filter paper cards. Each filter paper card had six circles (spotting areas), and 50 μl of whole blood was spotted on each circle. The filter paper cards were allowed to dry at room temperature for 24 h, placed in zip lock bags with a desiccant and stored at −20°C for a maximum of 7 days before transportation on dry ice to CEN laboratories for storage at –80°C before shipment on dry ice to the Swiss Newborn Screening Laboratory, University Children's Hospital in Zurich, Switzerland for analysis. In the NuPED study, a serum sample was prepared from venous blood collected into a serum separator vacutainer tube (Becton Dickinson, Woodmead, South Africa) to obtain a serum sample, which was also stored on‐site at –20°C for a maximum of 7 days before transportation on dry ice to CEN laboratories for storage at –80°C before analysis.
5
Blood Sample Collection and Storage
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In total, 400 µl of blood sample was obtained via finger prick and was transferred onto a Whatman® 903 protein saver card (Whatman, Maidstone, Kent, UK). After drying at room temperature for at least 4 hours, the samples were placed individually in a sealed plastic bag, stored at 4°C, and transported to the laboratory at Juntendo University within two weeks.
6
Extracting DNA from Dried Blood Spots
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A generic single hole 6mm punch plier was used to cut the blood spots from the Whatman 903 paper card. A punch of diameter 6mm represents approximately 8.7±1.9µl of blood spotted
10 (link). 1 – 4 blood spots of size 6 mm punch was added to an eppendorf tube and incubated with 200µl PBS (readymade PBS buffer used with pH 7.4 supplied by Gibco with Ref No. 10010-023) overnight at room temperature. Our major aim was to extract the maximum amount of gDNA, therefore we have used 6mm × 1 spot to 6mm × 4 spots for extracting gDNA from DBS (
Table 1 ). We have used phosphate buffer saline (PBS) for extraction of completely dry blood matrix on Whatman 903 for easy gDNA extraction, because once the surface of blood spot becomes wet, it is easy to extract DNA from the adsorbed blood.
10 (link). 1 – 4 blood spots of size 6 mm punch was added to an eppendorf tube and incubated with 200µl PBS (readymade PBS buffer used with pH 7.4 supplied by Gibco with Ref No. 10010-023) overnight at room temperature. Our major aim was to extract the maximum amount of gDNA, therefore we have used 6mm × 1 spot to 6mm × 4 spots for extracting gDNA from DBS (
7
Comparison of POPs in DBS and Blood
8
Serum Biomarker Sampling Protocol
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Blood collection was done between 8: 00 and 8: 30 a.m. after an overnight fasting of at least 12 h. Venous blood was collected into Vacutainer serum tubes (Becton & Dickinson, Milan, Italy) and centrifuged within 4 h. All serum samples were stored at −80°C until the preparation of spots and shipment to the analytical laboratory. To prepare spot serum from patients, 20 μL of each serum were spotted on filter paper (Whatman 903; Whatman GmbH, Dassel, Germany), dried, and sent by courier to the laboratory at room temperature. The technicians of the analytical laboratory were blinded to the sample identification codes.
9
Dried Blood Spot Preparation
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DBS were prepared by spotting 5 times 70 μL (Roche and Abbott) or 50 μL (bioMérieux) of EDTA venous whole blood on Whatman 903 filter paper cards (Whatman, Maidstone, UK). The blood was left to dry overnight, packed in separate zip-lock bags with desiccant sachets and a humidity indicator card, and stored at −20°C until testing.
10
Newborn Metabolic Disorder Screening
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Dried blood spots (DBS, Whatman 903) were used (GE Healthcare, Buckinghamshire, UK) to screen 92519 newborns of Greek origin for metabolic disorders of amino acids and acylcarnitines. The blood was collected after 2-3 days of feeding with a heel prick. This procedure was approved by the Institutional Review Board of IASO hospital in agreement with the current revision of the Helsinki Declaration.
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