32 August / September 2016
Trace 56
Fe, 63/65 Lizzie Griffin, Michael Cox & Catherine Duckett Biomolecular Sciences Research Centre, Sheffield Hallam University, Sheffield, UK
Trace metals have important biological roles to play within the human body. This work investigated the presence of 56
Fe, 63/65 Cu and 64 Zn in blood and utilised dried blood spot sampling (DBS) technology. Blood was taken from one
female subject and extraction conditions were optimised. Extracted samples were analysed by SEC-HPLC for protein separation and SEC-HPLC-ICP-MS for metal detection. Proteins from the blood samples separated via SEC-HPLC were
found to be haemoglobin, myoglobin and transferrin; SEC-HPLC-ICPMS metal determination saw correlated peaks of 56
Fe, 63/65 Cu. However, 64 Introduction
Blood diseases are very common amongst the world’s population, with anaemia being one of the world’s most common health disorders in itself and also as a result of another illness such as gastrointestinal cancer, chronic kidney disease, and others. Anaemia is associated with the deficiency of iron. In order to be able to diagnose blood diseases such as anaemia, blood samples must be taken and subsequently analysed. Conventionally this is done via venepuncture – the process of extracting blood from the veins of a patient via the use of a needle. This procedure is invasive and can be painful, especially for young children and the elderly and must be carried out by properly trained persons. Additionally, the process carries potential dangers to the healthcare professional conducting the procedure as patient’s blood may be infectious and pose a risk to their own health (Bishop, 2009) [1].
Over the past decade, the use of dried blood spots has grown in clinical settings (Déglon et al., 2012) [2]. The sampling process involves drops of capillary blood collected onto filter paper or card via a simple finger or heel prick facilitating a minimally invasive alternative from venous sampling. This allows samples to be taken in non-clinical environments by the patient themselves at home or by less extensively trained technicians, this therefore would be a less expensive service for the NHS and other healthcare providers to use. The procedure requires only small sample volumes (40 µL was
Zn peaks were much weaker and could therefore only be tentatively identified. Figure 1. Blood spots drying
used in this investigation) compared to the volume required for venous blood samples which is approximately 5 mL although this larger volume facilitates the running of several clinical tests, not solely compound determination discussed in this research. DBS samples have increased stability, can be stored at room temperature, shipped more easily, do not require any anticoagulants and reduce risk of infected pathogens such as Hepatitis C and HIV/AIDS [3].
Materials and Methods Sample Collection
The integrity of the blood sample can be adversely affected by the sampling procedure so care must be exercised through the sampling process. The use of alcohol wipes to sanitise the local site of sampling on the patient is one common practice utilised to obtain high quality samples. The hands of a healthy, 38-year-old, female subject were sanitised before sample collection and Unistik 3 Extra lancets (Owen Mumford; Oxford, UK) were used to pierce the skin of the subject’s finger and samples were collected in 40 µL Lithium Heparin capillary tubes (Cholestech LDX; Alere North America). The collected blood samples were then spotted onto Perkin Elmer 226 Spot Saver (Perkin Elmer, Greenville, South Carolina, USA) cards via capillary plungers. Capillary tubes were used to allow the acquisition of accurate and consistent sample volumes rather than applying drops of blood sample directly to the spot saver card. Spots were left to dry for 24 hours with
a continuous ambient air flow on both the front and the back of the card as can be seen in Figure 1.
Experiments were all performed in accordance with the Bioscience Research Ethics review group at Sheffield Hallam University.
DBS Sample Preparation and Extraction
Blood spots were cut from the spot saver cards using metal scissors as no ceramic scissors were available at the time. It should be noted that commercial punches are available that facilitate automation of this process, not accessible during this project. Blood spot discs were added into universal tubes containing 2 mL of ultra-pure deionised water and placed into a 40°C water bath for 60 minutes. Optimisation of this process was undertaken to ensure that recoveries were optimum and that no denaturing occurred; several time and temperature combinations were trialled and this was found to be the most suitable (data not shown). After tubes were removed from the bath, samples were left to cool at room temperature before discs were removed using tweezers. Samples were then ready for analysis. At each step of this process, control samples were prepared using blank cards.
Protein Standards
Gel marker filtration kit (mw 12,000 – 200,000 Da, MWGF200), ferritin from equine spleen (Type 1 in saline solution), myoglobin from equine heart, human haemoglobin (lyophilised powder) and transferrin bovine were all obtained from Sigma Aldrich, UK.
Cu and 64
Zn
metal analysis in whole blood by SEC-HPLC-ICPMS
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