Collection and storage of Dried Blood Spots

Overview: DBS are a convenient way to collect blood samples especially in remote areas with no electricity or problems to keep blood samples frozen. After drying, the DBS can be send in a standard letter to the lab. Since there are sensitive methods available to measure in a 20 ul whole dried blood spot all 4 proteins it is very easy to get enough blood from a small nearly painless finger prick. Unfortunately there are also some disadvantages of the DBS technique. The standard laboratory approaches for quantification have to be adjusted and the accuracy of the measurement is a little bit less than in plasma or serum samples. Therefore it is always useful to check first if it is possible to collect plasma/serum samples (venous or from finger prick) and to transport them frozen to the lab. At www.nutrisurvey.de/dbs/capillary_blood.htm some information is available for collecting capillary blood samples.
RBP and AGP are very robust proteins and can resist bad collection and storage conditions but sTfR and CRP are more sensitive. Therefore it is necessary to follow some standardized procedures which are described in the following section.

Collection: Currently the recommended filter paper for collecting blood is the 903 specimen collection paper from Whatman (formerly Schleicher&Schuell) which is available in different sizes and forms. Since the paper is always the same, it is possible to cut the paper into smaller pieces to save material and reduce transport volume. The only limiting factor is sufficient space for the blood drops and clear labeling. For the collection of blood any finger prick device which is used by diabetes patients or other purposes can be used. The blood flow depends on the depth of the prick and the width of the blade and can be increased by applying a little bit vaseline on the puncture site or warming the finger (e.g. with a soft cloth moistened with warm water up to 41°C). This can be very useful in more colder environments and when for pain reduction thinner lancets are used. Some information about lancets can be found at Beckton Dickinson. Usually the middle finger is the first choice but the ring finger or the little finger can also be used and the edge of the extremity of the finger is the best puncture site. For cleaning and disinfection it is useful to to use 70% alcohol (before the finger prick the alcohol should be fully wiped away with a sterile gauze pad or just evaporated). The first drop of blood is usually removed by a sterile gauze pad (this is not absolutely mandatory) and then two or three drops of blood are soaked into the filter paper. Since the ELISA technique for the measurement of RBP, sTfR, CRP and AGP is very sensitive very small amounts of blood are sufficient to do the measurements. Therefore it is not necessary to fill the circles on the standard DBS cards (shown below in picture 1). The full circle corresponds to approx. 75 ul whole blood but as can be seen in Picture 2 already 20 ul are sufficient to do several hole punches. The most important is that the blood is soaking through the paper and that it is possible to punch out 2 or 3 holes with a diameter of 1.5 mm on the back side of the filter paper (if the size of the spot is enough 3 mm hole punches are a little bit better). To enable repeated measurements it is good to have a sufficient large spot but too large spots are more difficult to dry which could also be a problem. Picture 1 shows common problems of poorly collected DBS.

Storage: After applying the blood on the filter paper the cards should be dried horizontally with free air flow on both sides (or desiccant on one side) for at least 3 hours preferably in an environment with low humidity. After drying the DBS can be stored in a Ziploc bag containing a sachet with desiccant (ideally with a color indicator or together with a humidity indicator card, these things are available from most lab suppliers, Picture 4 shows some examples of desiccants). Since sTfR and CRP are not fully stable at room temperature it is useful to put the Ziploc bags into a refrigerator or freezer especially for long term storage. To prevent that any condensing water is going into the paper the bags should have room temperature before the DBS are taken out. For sending the DBS to the lab some days at higher temperatures can be tolerated.

Procedure for collecting DBS:

Material for collecting the DBS: finger prick device, filter paper, drying box, disinfectant, tissue (the tubes on this picture for collecting capillary blood are not necessary for the DBS)

For disinfection the finger is sprayed and wiped off with tissue containing disinfectant (70% ethanol or isopropanol)

To increase the blood flow a little bit vaseline can be put on the place where the finger prick is done (it inhibits the coagulation of blood on the finger).

The finger prick can be done with a nearly painless device which diabetic patients are using. If a higher amount of blood is necessary other more efficient lancets can be used.

Before the blood is put on the filter paper a drop of blood has to be collected.

The easiest procedure to collect the blood is to soak the blood into the paper without touching the finger. With this procedure it is also easy to check that the blood is going fully through the paper. It is very important that the filter paper is dry e.g. by storing it together with some desiccant.

In places with difficult drying conditions (humid or foggy) it is necessary to put the DBS into a simple box with desiccant. The desiccant in this box contains a color indicator and can easily be reactivated by putting it into an oven at 100°C for some hours. After 3 hours drying (or overnight) the DBS can be put into a bag which contains some desiccant (when the DBS are dry it is no problem to store them together in one bag). If the humidity is above a certain level microorganisms can grow and destroy the proteins in the filter paper

Picture 1 (taken from the Whatman website):

Picture 2: 2 spots with 20 ul blood, 1st spot with 1.5 mm hole punches and 2nd spot with a 3 mm hole punch, since the concentration of the proteins are higher in the edge the hole punches shouldn't be taken from there

Picture 4: Various desiccants and humidity indicator card

Picture 5: I can not remember where I found this picture but it is a very good example on how a simple collection method can be sufficient to dry DBS, it is much better than putting DBS into boxes in which humidity can build up.

Picture 6: This is an example for probably the most common problem when samples from tropical countries are coming to the lab. Since there is a separation of erythrocytes and plasma on the filter paper it is obvious that the values which are measured in the spot area are lower than in good DBS. It is very difficult to produce such spots in the lab but the easiest way to do it, is to make the filter paper wet. To be sure that this doesn't happen the filter paper should be stored in a bag with dessiccant and in a humid enviroment it is also better not to put too much blood on the filter paper. Smaller spots are easier to dry than bigger spots.

Picture 7: The new protein saver cards from Whatman with a card protection from both sides look very good but actually carries a high risk when they are closed immediately after collecting the DBS. It can be seen here by the blood staining on the paper card. To prevent this it would be much better to cut out the filter paper and to use a drying box with desiccant or to put them as in the example above between bamboo sticks.