Measurement of the Vitamin A and Iron Status in Blood Samples

In Nutrition Surveys for micronutrient deficiency there is often no alternative than to measure blood samples to get a clear picture of the situation. For the detection of Vitamin A and iron deficiency it is possible to measure certain proteins (RBP for Vitamin A deficiency, Ferritin and sTfR for iron deficiency) with a sensitive and inexpensive Sandwich ELISA technique (Erhardt JG et. al. 2004) which can also be easily combined with the measurement of CRP and AGP as indicators for acute and chronic infection. The infectious status can be of interest by itself but also be used to correct RBP and ferritin which are influenced by infection (click on Ferritin or Vit. A to get the publications for this procedure). The combined Sandwich ELISA technique needs some experience and there are problems with the availability of the antibodies but this method allows to measure these 5 proteins for approx. 5 USD/sample or 1 USD per protein. As material serum or plasma from venous or capillary sampling can be used and already 15 ul are sufficient to do a double measurement of these 5 proteins. Therefore a finger or heel prick is usually sufficient to get enough blood for doing these measurements. To increase the blood volume warming the finger, using more efficient lancets (e.g. the blue ones from BD), wiping off the blood with a tissue to initiate again the blood flow or adding vaseline/silicon spray to the puncture site can be helpful procedures. For the collection of capillary blood there are various tubes available (see pictures below) and all of them fit into a small inexpensive mini centrifuge. If no electricity is available there is also a manual centrifuge available but more expensive and not so convenient. After centrifugation the serum/plasma should be stored in 0.2 mL PCR tubes to enable the use of them directly in an automatic pipetting system to avoid the tedious and error prone manual pipetting. Serum/plasma should be stored frozen but if this is not possible storage in non frozen form for some days is possible. Extensive tests at the CDC in Atlanta have shown that proteins in plasma are stable at room temperature for one week. Since the conditions in the field can be much worse (temperature > 25°C or higher risk for bacterial growth) it's better to freeze the serum/plasma as soon as possible or at least to keep it in a cool environment. A car battery driven refrigerator or a big ice block in a good styrofoam box can be sufficient to keep the samples in good condition for several days until they can be frozen. Serum is often more accurate for pipetting (there are less particles in it) and should therefore be prefered but the yield is less than for plasma. Therefore this can be a problem when capillary samples are collected. The following pictures show some examples on how to collect, process and measure blood samples for the Vit. A and iron status.


This shows a typical collection of material for getting blood from the finger: Storage box, 0.2 mL PCR tubes, labels, blood collection tubes, lancets, transfer pipette (e.g. Sarsted No. 86.1180) and a manual centrifuge. Not shown is the standard material for blood collection (disinfectant, tissue,...).
capillary_sampling

These are examples on how to collect capillary blood from the finger. All of these tubes fit into a cheap mini centrifuge where the erythrocytes can be separated in less than 5 min. The material shown here is from Sarstedt (No. 20.1292 and 16.444 are appropriate) but there are several alternatives which can also be used and are as good as this.
sample tube

The serum or plasma can be stored in such a 0.2 mL PCR tube (Sarstedt no. 72.737.002) for which appropriate and easy to print labels from Avery/Zweckform (No. L7658) Herma (No. 4333) or Brady (LAT-29-799) are available. To keep the first label (Avery or Herma) fixed it is useful to wrap the label with the fingers around the conical part of the tube. With the smaller Brady label which can be seen in the second picture it is easier to check how much volume is in the tube and if there are any particles in it which might block the tip of the pipette.


vol tubes
These are some examples on the volumes in the 0.2 mL PCR tubes. To prevent spilling over the volume should never be more than 150 ul. The best is to fill it only half.
storage box

These are pictures of a storage box with a 14*14 grid (196 tubes per box) to store efficiently the tubes and a rack which can be directly used in an automatic pipetting system (Abgene AB-1417). Both options are a safe way to keep the samples sorted and labels protected during storage and transport. The easiest and quickest procedure to open the lids of the tubes in the red rack is to cut the lid attachment of the 0.2 mL tube and to remove the lid with tweezers.



If samples are already collected the most efficient procedure to prepare the samples is to put them directly into a 384 well plate. It's less work than putting the samples into the 0.2 mL tubes. Together with the blanks and QC's 182 samples  are fitting on one plate to make an independent double measurement in two different positions. With special plates from Ependorf the sample numbers of a template underneath the plate are easily to see and the wells for the blanks and QC's can be marked to help that these wells are not unintentionally filled with samples. With an adhesive foil the plates can be safely closed and shipped frozen to the lab.
styrofoam box

These two pictures show a high efficient styrofoam box with more than 5 cm thick walls and tightly closing lid. In the picture on the right side storage racks for approx. 4000 samples are added. There is still space for 20 kg dry ice which is sufficient for more than 7 days. Alternatively frozen cool elements (if possible at -70°) can be put in such a box and can keep samples frozen for several days (this has to be checked for the specific box) in case dry ice is not available or forbidden for the transport. 


 This is an automatic pipettor with the 0.2 mL tubes in a special rack which can be used to replace the tedious and error prone manual pipetting.



 This shows the final result on the 384 well plate (in this picture with CRP as example). The values can then be presented in the following form for the samples and the calibration/quality control.


Examples on how samples shouldn't come to the Lab:

Bad tubes

 The first two tubes have not enough volume for an automatic pipetting (the 2nd label is also not in the right position and not firmly wrapped around), the next two tubes are labeled with non sticky labels and handwritten which is very often difficult to read, the 5th tube has lost it's label and the last two tubes are totally overfilled. This makes a clean working in the field and the lab nearly impossible and there is a risk that the cap of the tube opens automatically during freezing. Already in the field HIV, hepatitis and other viruses will be spread on any surface which are touched with the fingers who have to handle such tubes.




These are two examples for tubes which were prepared with good intentions (wrapping with parafilm or adhesive tape) but which is not necessary and makes a lot of additional effort in the field and the lab. In some situations it might be necessary to use an additional fixing material. Then a thin transparent adhesive tape which is put around the lower part of the tube can be used.



This is the worst example on how a tube can come to the lab. Wrapped with heavy tape it was impossible with normal forces to open it and the analysis had to be rejected.