1. Sample Collection

1. Collect blood samples from individuals with proper informed consent. For each individual, draw 4 to 5 ml of blood into a P100 tube (BD Diagnostics, Franklin Lake, NY, USA). For comparison purposes, simultaneously collect blood in an EDTA tube.
2. The BD P100 tubes contain 15.8 ml spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also contain a mechanical separator. After collecting the whole blood, keep both tubes at room temperature for 60 min to overnight until processing occurs.
3. Centrifuge the BD P100 tubes at 2,500 g for fifteen minutes at room temperature. Transfer the plasma collected above the mechanical separator into micro centrifuge tubes.
4. Store the P100 tubes, containing the blood compacted below the separator, at -80 °C until you are ready to begin the DNA extraction.

2.1 From blood of P100 tube (P100_DNA)

1. The P100 tubes are stored at -80 °C after plasma extraction. Within 3 to 4 months, retrieve the P100 tube containing the compacted blood from the freezer and thaw at 37 ° in a water bath for 5 min (Figure 1A). Remove any residual plasma that sits above the separator. Add 2 ml of 17% sucrose solution (gradient solution tested in house – unpublished data) in the P100 tubes above the separator (Figure 1B). Centrifuge the tubes at room temperature for 20 min at 2,500 g; this process pushes the mechanical separator to the top of the tube and yields three layers of solution including the buffy coat (Figure 1C). Manually extract the separator using a steriled hooked metal paperclip (Figure 1D).
2. After retrieving the mechanical separator from each tube (Figure 1D), remove and discard the top sucrose layer. Transfer the middle layer, representing the buffy coat (BC) layer, into a sterile 15 ml conical tube. Add Phosphate Buffer Saline (PBS) to increase the buffy coat volume to 3 ml. Extract the DNA from the buffy coat using reagents from the Qiagen Puregene Blood Kit as per the manufacturer’s recommendation; except a centrifugation speed of 2,500 g (instead of 2,000 g).
3. To lyse and remove the red blood cells (RBC), add 9 ml of RBC lysis to 3 ml of buffy coat. Invert each tube several times and incubate at room temperature for 10 min with occasional inverting during incubation. Spin each mixture down at 2,500 g for 5 min and discard the supernatant. Treat the remaining pellet in each tube with 3 ml of cell lysis solution and vortex for 15 sec. Treat the lysate with 1 ml of protein precipitation solution and vortex for 15 sec.
4. Centrifuge at 2,500 g for 5 min and transfer the supernatant into a fresh 15 ml conical tube containing 3 ml of 100% isopropanol. Invert the new tubes 10 times and centrifuge at 2,500 g for 3 min. Discard the supernatant and add 1 ml of 70% ethanol. Invert the conical tubes a few times to dislodge and wash the DNA pellet. Centrifuge at 2,500 g for 1 min. Allow the pellet to dry for 3 min (place the tube upside down) at room temperature and then suspend the DNA with 250 ml of rehydration solution at 37 °C for 10 min and transfer to a pre-labeled 1.7 ml microtube. Store the tubes at -80 °C until use.

2.2 From Blood of EDTA Ttube (EDTA_DNA)

1. The whole blood for routine immunohematology testing is collected in plastic vaccutainer tubes spray-coated with 10.8 mg of K₂EDTA and stored at -80 °C. Because the tubes do not contain a mechanical separator, the DNA extraction does not include the steps involving the mechanical separator (2.1.1 and 2.1.2).
2. Within 3 to 4 months of blood storage, the DNA is extracted using the KingFisher Flex (Thermo Fisher Scientific, Waltham, MA, USA). It is an automation DNA extraction system based on magnetic particle technology and consists of cell lysis / DNA binding, several washing steps and DNA elution.
3. The kit used in the DNA extraction is the KingFisher Flex 24 DNA Blood kit (Qiagen, Germany).

3. DNA Quantity and Quality Assessment

The quantity, quality and integrity of the genomic DNA were assessed by a combination of method that includes picogreen, spectroscopy and electrophoresis.

1. The concentration of the genomic DNA is determined by Nanodrop and picogreen quantification.
2. The purity is determined from the 260/280 ratio measurement on the Nanodrop spectrophotometer.
3. The sample (500 ng) is also loaded on 1% agarose gel to assess the integrity (degradation) of the DNA.

4. Genotyping Assays and Quality Control

1. Five P100_DNA and their corresponding EDTA_DNA samples are randomly selected for further analysis using the custom Immunochip (Immuno DNA Analysis BeadChip). Immunochip is an Infinium genotyping chip containing 196,524 polymorphisms and is designed for immunogenetic studies⁵.
2. For each sample, 200 ng of DNA is amplified, fragmented, precipitated and resuspended in the appropriate hybridization buffer.
3. The denatured samples are hybridized on Immunochips at 48 °C for a minimum of 16 hr.
4. After hybridization, the Immunochips are processed for single-base extension reactions and stained.
5. The immunochips are then imaged using Illumina Hiscan Bead array reader and the normalized bead intensity data is loaded into the Illumina GenomeStudio software and converted into genotypes. Genotypes are called using the autocalling algorithm of GenomeStudio. The quality control involves exclusion of single nucleotide polymorphisms (SNPs) with a call rate <95%.
6. The SNP call rate is used as a measure of the immunochip assay efficiency. It is calculated as the percentage of the total number of assays on the chip (196,524 SNPs encoded on each chip) that achieve sufficient signal intensity and quality to receive an automated genotype assignment. High quality DNA (260/280 ratio of 1.8 or higher and absence of degradation) is expected to achieve at least the same call rate as the HapMap control DNA included in the immunochip assay. Quality control involves exclusion of SNPs with a call rate <95% in each data set.