This method can help answer key questions in translational research such as the chance to study biomarkers in patients from developing countries. The main advantage of this technique is the possibility to store blood and serum samples for at least two weeks without freezing and provide high quality material for downstream analysis such as qPCR and ELISA. The implications of this technique extend toward diagnosis of diseases such as cancer and infectious diseases.
This method can also be applied toward the diagnostic or research fields like the study of prenatal diseases or the analysis of infectious pathology such as HIV. We first had the idea for this method when we started comparing biomarkers between a Caucasian and an African case series for which frozen samples were not available. Visual demonstration of this method is critical as the blood spotting steps are difficult to learn.
In this procedure, three milliliters of a blood sample is collected in a three milliliter tube with 5.4 milligrams of EDTA. Write the patient code number on the bottom right-hand corner of the saver card. Carefully re-suspend the blood with a five milliliter pipette.
Next, pipette and transfer one spot of blood onto the saver card. Using a new tip for each blood aliquot, repeat the spotting to obtain a total of three to five blood spots. Leave the dried blood spots, or DBS, to dry overnight at room temperature with the cover of the saver card open in a horizontal position over an open, non-absorbent surface and avoiding direct sunlight.
On the following day, store the card at room temperature in a plastic bag with desiccant until use. DNA will be extracted from three dried blood spots from the saver card. Begin by cutting each of the DBS from the card and separating them from the card.
Cut the separated DBS into small pieces of about one millimeter in diameter. Place the small pieces from one DBS into a 1.5 milliliter centrifuge tube. To digest the samples, add to the tube 180 microliters of lysis buffer one from a commercially available DNA extraction kit, then add 20 microliters of proteinase K.Mix by vortexing.
Place the tube in a thermo-incubator and incubate with shaking at 56 degrees celsius for 60 minutes. At the end of the incubation, briefly centrifuge the tube to remove drops from the lid. Start the wash by adding 200 microliters of lysis buffer two and vortexing for 10 seconds.
Place the tube in a thermo-mixer and incubate at 70 degrees celsius for 10 minutes. Place the elusion column in a new two milliliter collection tube and discard the old one. Add 500 microliters of wash buffer and centrifuge at 6000 times gravity for one minute.
Place the column in a new two milliliter collection tube and discard the old one. Add 500 microliters of wash buffer two and centrifuge at 6000 times gravity for one minute. Place the column in a new two milliliter collection tube and discard the old one.
Centrifuge at 20, 000 times gravity for three minutes to dry the membrane. To elute the DNA, place the column in a new 1.5 milliliter centrifuge tube and discard the collection tube. Place 50 microliters of distilled water onto the center of the membrane and incubate at room temperature for 10 minutes.
Centrifuge the tube at 20, 000 times gravity for one minute. Collection the elute and place again on the center of the membrane. Centrifuge the tube again at 20, 000 times gravity for one minute.
Discard the membrane. After measuring the DNA concentration using a spectrophotometer, store the DNA at minus 20 degrees celsius until use. For this procedure, seven milliliters of peripheral blood sample is first collected in a seven milliliter tube without EDTA.
Leave the blood sample to coagulate at room temperature for 30 minutes. Write the patient code number on the bottom right-hand corner of the saver card. Centrifuge the blood sample at 1100 times gravity at room temperature for 15 minutes.
Remove the supernatant from the tube and transfer it to a new 15 milliliter tube. Carefully re-suspend the blood with a five milliliter pipette. Pipette and transfer one spot of serum onto the saver card.
Repeat the spotting to have a total of three to five blood spots. Leave the dried sero spots to dry overnight at room temperature with the cover of the saver card open. On the following day, store the card at room temperature in a plastic bag with desiccant until use.
If storage is longer than 14 days, store at minus 20 degrees celsius. Prepare for the ELISA assay by thawing the DSS for each sample if necessary. To elute the protein from the DSS, cut one DSS into small pieces of about one millimeter in diameter and put the pieces in a 1.5 milliliter tube.
Then, add four microliters of PBS. Shake the samples at low speed at four degrees celsius overnight. On the following day, use an appropriate dilution of the protein elute as a serum sample for secreted protein or growth factor analysis using a commercially available kit.
In this protocol, blood and serum samples were stored as dried spots on filter paper at room temperature. A representative protein saver card is shown before and after blood sampling. In order to confirm that the storage on filter paper and the procedure to elute blood do not interfere with the sample quality, ELISA assays for vitamin D binding protein, or DBP levels, were performed on eight samples of serum collected in a two milliliter tube and stored immediately at minus 80 degrees celsius or collected as dried serum spots.
The coefficient of variation, or CV, between matched samples ranged from 2%to 24%and the mean CV was 9.6%Once mastered, this technique can be done in 10, 15 minutes if it is performed properly. Following this procedure, downstream methods of molecular biology or protein analysis can be performed. After its development, this technique enables research in the field of translational research and biomarker analysis to collaborate with institutes from all over the world that do not have the lab equipment to store biological samples in standard conditions.
After watching this video, you should have a good understanding of how to correctly store blood samples in dried blood and serum spots. Don't forget that working with blood and chemical reagent can be extremely hazardous and precautions such as wearing gloves and a lab coat should always be taken while performing this procedure.
