The target population consists of all women who give birth in Norway. The study is described in detail elsewhere [5
]. Briefly, pregnant women and their partners are recruited by mail prior to their ultrasound appointment at 17th week’s gestation; 99% of pregnant women in Norway have routine ultrasounds. Direct participation contact occurs at the ultrasound appointment and at delivery. The opportunity for collection of biological material exists only on these occasions. shows the progression of the study enrolment over time; currently 50 of 52 hospitals that perform deliveries in Norway are participating.
Number of participating hospitals in the MoBa Study and number of sample sets registered and processed at the Biobank (Numbers per 07/07/06)
Biological specimens are collected from the mothers at 17–18 weeks of gestation and at the time of delivery. Blood samples from the fathers are collected at the ultrasound appointment; recruitment of fathers began in 2000. Cord blood samples are drawn at delivery or, if cord blood is unavailable, a capillary sample from the newborn child is collected at the time of phenylketonuria (PKU) screening (3–4 days after birth). Over the course of the study, the specimens collected have changed as research interests have grown. presents a timeline of the specimen collection activities.
Timeline of sample collection strategies in MoBa.
Initially, only two 7-ml EDTA tubes (Becton-Dickinson (BD), Plymouth, UK) were drawn from women at the ultrasound appointment. One of the EDTA tubes is spun following sample collection in the hospital laboratory and plasma is pulled o3 before shipping; the other is shipped in the vacutainer directly to the Biobank. In 2002, based on particular interests in environmental toxins, the sample set was extended to include an additional 7-ml anticoagulant tube for plasma analysis and one plastic 3-ml EDTA tube (BD, Plymouth, UK) of blood for trace metal analysis. Additionally, 8-ml of urine is collected in a urine cup and transferred to a urine transport tube. (Becton-Dickinson (BD), Franklin Lakes, NJ, USA). Urine was initially shipped in tubes without bacterostatic additive (BD, Plymouth, UK), but most of the urine samples showed bacterial growth upon receipt in the Biobank. We therefore tested the impact of urine preservation methods on potential environmental analyses of interest (e.g., phthalates, bisphenol A) [6
]. As a result of this evaluation we changed the protocol in 2003 to ship urine samples in tubes with chlorhexidine (UAP Vacutainers, BD, Franklin Lakes, NJ, USA). From fathers, the umbilical cord and mothers one day after delivery two 7-ml EDTA tubes are drawn.
Sample processing at the Biobank
Samples are shipped overnight from hospital laboratories to the Biobank. A majority of samples are received the day after collection. Samples are processed the day of receipt. A specially designed computer program (The Mother–Child program) supports all processing at the Biobank. This Laboratory Information Management System (LIMS) controls all steps where sample identity can be mixed-up, e.g. transfer of samples from one tube to another. Specimen tubes from hospitals are labelled with the woman’s name and national identity number; upon registration in the Biobank the sample sets get a unique Biobank ID which is linked to the MoBa participant ID for this pregnancy. At registration the LIMS records maternal ID, date of sampling, date of receipt, collection hospital, and sample status at arrival (e.g., incomplete volume, haemolysed, coagulated). Bar codes with the unique Biobank identifier are automatically printed for further sample processing. Our LIMS also stores locations for all aliquots for each Biobank ID (which freezer, rack, plate, and position on the 96 well plate). Access to the Mother-Child program is highly restricted, and a tape back-up of the database is made each night and stored in a fire-protected area.
Aliquoting of samples
Whole blood is aliquoted into two polypropylene deep-well plates (930 μl in each, ABgene, Surrey, UK). For the plasma pulled off at the hospital laboratories before shipment, a total of 1.8-ml is aliquoted onto 6 different polypropylene microtitre plates (300 μl each, ABgene, Surrey, UK) using Tecan pipetting robots (three Genesis RSP 200 and one Freedom Evo, Tecan Ltd, Männedorf, Switzerland). The plates are sealed with heat-sealing foil sheets (Easy Peel, ABgene, Surrey, UK). Maternal plasma collected for environmental analyses is spun at the Biobank and aliquoted into individually labelled vials on three Matrix™ plates (TrackMates tubes, Hudson, NH, individual nanobar code; e.g. 2D bar code on each tube, 930 μl in each). Urine is divided into 930 μl aliquots on six Matrix plates.
The 930 μl volume was selected because this is the maximum efficient volume for the Tecan machine; larger volumes require a much greater aliquoting time and thus all samples are stored in 930 μl or smaller volumes. DNA is extracted manually using the FlexiGene kit (Qiagen, Hilden, Germany). Quality control is performed on all DNA samples using a spectrophotometer (Spectramax 190, Molecular Devices, Sunnyvale, CA) where optical density (OD) is tested in triplicates. Stringent parameters must be adhered to for DNA to be accepted. These parameters include a DNA purity of 1.6–2.0 260/280 ratio, a DNA concentration in excess of 20 ng/μl, and OD at zero for the negative control included in each batch of 24 samples (e.g. extracted at the same time). All indications of quality outside the requirement are marked in our LIMS. The DNA concentrations are subsequently normalised to 100-ng/μl using a Tecan pipetting (Generis 2000, Ma¨ nnedorf, Switzerland) robot; which also tracks the amount of DNA aspirated throughout the process. DNA normalisation is based on four different computer programs that communicate with each other (Softmax Pro 3.0 for the spectrophotometer, Gemini for the Tecan machine, MCS (specially made for communication between Softmax Pro 3.0 and Gemini). DNA is then aliquoted into four to ten 1.4-ml deep-well plates (930 μl in each) dependent on the DNA yield, the volume in each well which is not totally filled is calculated and recorded, and the file from the Gemini program of the Tecan robot is imported to the Mother–Child program.
DNA is stored at −20 °C while whole blood, urine and plasma are stored at −80 °C. Plates containing aliquots from the same sample are stored in two different chest freezers. The 3-ml EDTA tube for metal analysis is stored at −20 °C. All freezers are connected to an external alarm system, and temperatures are logged routinely. An emergency electrical system is connected to all freezers to provide backup power.
We plan to retrieve specified samples using automated equipment for all samples stored in the 96-well format. Study investigators will identify subjects and samples of interest; these personal identification numbers will be linked to the encrypted Biobank IDs and pull lists will be generated. Once the samples to be retrieved are identified, the retrieval file from the Mother–Child program will be transferred to a MultiPROBE II robot (Perkin Elmer, Wellesley, MA). Up to 100 plates are loaded into the MultiPROBE II robot for automatic retrieval and transfer to a delivery plate. The retrieved volume from a certain position is tracked in the Mother–Child program.
Quality Assurance and Quality Control (QA/QC) procedures
Conduct of such a large study over such a long period of time requires a comprehensive quality assurance program to ensure the ability to track changes over time. In addition to standard operating procedures (SOPs) which are constantly updated, the Biobank is being ISO-certified (ISO 9001). As part of the QA, we keep five samples from each lot of materials used in the study; this includes vacutainers, pipettes, tips and plates. Because a majority of sample collection and processing occurs at the 50 hospitals, annually we conduct a written survey of all hospitals involved to assess collection, processing, and storage procedures in order to identify variations in protocols throughout the study period. We are conducting an evaluation of changes in different markers in plasma and urine during repeatedly freeze-thaw cycles and long-term storage. Sodium, cholesterol, triglycerides, free fatty acids, vitamin E and aspartic aminotransferase were analysed in plasma while sodium and creatinine were analysed in urine [7
]; results will be presented in a future manuscript.