In determining vitamin A status the measurement of retinol in blood is still the most accepted method of assessment. Since the retinol level in blood is homeostatically controlled and the liver stores have to be depleted to see a decrease, serum retinol is not a perfect indicator for vitamin A status. Additionally, infection decreases the concentration of vitamin A in blood. Currently the most frequently used procedure for measuring vitamin A status is to take venous blood samples, to centrifuge them and, after freezing and transporting to the lab, to measure the retinol content in serum by HPLC. It is obvious that this is a tedious and expensive procedure. The aim of this SIGHT AND LIFE project was therefore to improve and evaluate the use of dried blood spots (DBS) for measuring the vitamin A status. The collection of DBS in the field is very simple. Only a small drop of blood from a finger prick has to be applied to filter paper and can be sent after drying by normal mail to the lab. Since retinol is protected by the retinol binding protein (RBP) from oxidation we have already shown (Erhardt 2002) that it is possible to measure the retinol content in this DBS by HPLC. A less expensive and more efficient way is to measure RBP. This correlates very well with the retinol content, can be measured by an inexpensive sandwich ELISA technique and can also be easily combined with the measurement of C-reactive protein (CRP) and alpha-1 glycoprotein (AGP) as indicators for acute and chronic infections. It also allows the correction of the retinol or RBP values in blood (Thurnham 2003). Methods Collection of DBS in the field: For the collection of DBS the standard finger prick procedure which every diabetic patient uses can be applied. Since the ELISA technique is very sensitive, only a small DBS, corresponding to approx. 15–20 µl whole blood, is necessary to perform all the measurements. A drop of blood is put on the filter paper, dried overnight and then put for storage in a plastic bag. Under humid conditions a desiccant should be added to fully dry the blood on the filter paper. In dry form the DBS are very stable at normal temperatures for several months. Only at high humidity and temperatures above 25 oC does degradation seem to occur. ELISA technique for measuring RBP, CRP and AGP in DBS: Figures 1and 2 show the procedure for this technique. For extraction of the proteins from the DBS, a 3-mm hole punch is taken from the DBS card and extracted overnight in the fridge in phosphate buffered saline. This extract is then put on the analysis plate of a standard sandwich ELISA technique. It uses a primary antibody (DAKO, Denmark) to capture the proteins, and a secondary antibody, which is coupled to a peroxidase, to perform a color reaction which is proportional to the amount of protein in the sample. Only different dilutions of the extract and different antibodies are used for the three proteins. The details of this procedure can be found in an article which was published recently in the Journal of Nutrition (Erhardt 2004). The only expensive part of this technique are the antibodies. A pair of antibodies cost around USD 600 but they are sufficient for more than 10,000 measurements. If a large number of samples is measured the average cost of chemicals is reduced to 50 cents per sample for all three measurements. This Use of dried blood spots (DBS) – A simple and field-friendly method of collecting blood samples for the measurement of vitamin A status Juergen Erhardt, PhD, SEAMEO-TROPMED, Regional Center for Community Nutrition, University of Indonesia, P.O. Box 3852, Jakarta 10038, Indonesia, email@example.com Figure1. Taking of blood by finger prick and collection of DBS in a storage box with reusable dessiccant. NEWSLETTER 1/2005 5 SIGHT AND LIFE does not include the cost of running a laboratory and salaries, but the cost of chemicals is usually the most critical factor in deciding whether to perform a biochemical assessment or not. With a simple multichannel pipette the measurement can also be done very efficiently. With some training, 80 samples per day can be easily measured for all three parameters.
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