Holly Nicholls (Presenter)
University of East Anglia
Bio: I studied BSc Biomedical Sciences (Sandwich Degree) at Sheffield Hallam University. I currently work at the Bioanalytical Facility at The University of East Anglia, under Professor William Fraser. My work mainly involves the analysis of vitamin D using liquid chromatography mass spectrometry. Recently I have helped to develop a method for measuring 25 dihyroxy vitamin D3 using dried blood spots and volumetric absorptive micro-sampling.
Authorship: Holly Nicholls (1), Jonathan Tang (1), John Dutton (1), James Rudge (2), Christopher Washbourne (1), Isabelle Piec (1), William Fraser (1)
(1) University of East Anglia, (2) Neoteryx
| Short Abstract The use of dried blood spots (DBS) has been extensively reported as a convenient and less invasive alternative to venepuncture, yet the haematocrit present in whole blood can adversely affect the quality of measurements. Haematocrit affects the viscosity of whole blood thus altering the distribution of the blood droplets on the cards. Mitra™ tips are volumetric absorptive micro-samplers (VAMS) which take up a fixed volume of blood via an absorptive tip. We have developed a LC-MS/MS assay for the quantitation of 25(OH)D3 in whole blood collected using DBS and Mitra™ tips. In this study we compared the two micro-sampling techniques against the plasma assay and investigated the impact of different haematocrit levels on the 25(OH)D3 concentration. |
Long Abstract
Background
The role of vitamin D in bone disorders is firmly established and its potential influence in a wide scope of other diseases, such as mental health problems and cancers, has been a hot topic of research. Liquid-chromatography mass spectrometry (LC-MS/MS) is recognised as the gold standard method for the measurement of 25-dihyroxy vitamin D3 (25(OH)D3) in serum or plasma. The use of dried blood spots (DBS) has been extensively reported as a convenient and less invasive alternative to venepuncture, yet the haematocrit present in whole blood can adversely affect the quality of measurements. Haematocrit affects the viscosity of whole blood thus altering the distribution of the blood droplets on the cards. Mitra™ tips are a form of volumetric absorptive microsampler (VAMS) which take up a fixed volume of blood via an absorptive tip. We have developed a LC-MS/MS assay for the quantitation of 25(OH)D3 in whole blood collected using DBS and Mitra tips. In this study we compared the two micro-sampling techniques against the plasma assay and we investigated the impact of different haematocrit levels on the 25(OH)D3 concentration.
Method
To create calibration standards, a pool of whole blood was created and centrifuged. The plasma layer was removed and the cells were washed using 0.9% saline. The wash step was repeated four times. Vitamin D depleted serum was added at a volume equal to that of the plasma that was discarded. The pool was separated into five and spiked with commercial certified standards to obtain calibrators at 0, 17, 25, 50 and 125 nmol/L.
Calibrators, quality controls and whole blood EDTA samples from patients (n=97) were sampled by Mitra tips and pipetted onto Whatman 903 cards. The samples were allowed to dry at room temperature overnight and then stored at -20°C until use. Blood spot samples were obtained from the cards by cutting out a whole disc around the DBS and by making 2x3mm sub-punches from a DBS using a card puncher. All samples were extracted using supported liquid extraction plates and derivatised with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) before being analysed by LC-MS/MS. Plasma 25(OH)D3 measurements were obtained from the same patient samples using a previously validated method to provide a reference value.
The LC-MS/MS method was validated against inter-assay (n=10) and intra-assay (n=6) imprecision, extraction recovery in spiked samples, the lower limit of detection (LLoD) and the lower limit of quantification (LLoQ). Sample stability was assessed by analysing samples stored at -20°C for up to 50 days. The effect of haematocrit on the measurements of 25(OH)D3 was investigated by progressively removing plasma or adding red blood cells to samples.
Results
The whole blood results, prepared with DBS or Mitra, correlated well with the plasma results (DBS whole disc, r²=0.89; DBS 2x3mm sub-punches, r²=0.92; Mitra, r²=0.87), although the 25(OH)D3 concentration was significantly lower in whole blood. Regression analyses on DBS or Mitra against the plasma reference assay gave a straight line equation for each method, which was used to calculate a plasma equivalent value from the whole blood results. This conversion equation was applied to a second set of 60 patient samples. Lin's concordance correlation coefficient showed there was a good agreement between the derived plasma values and the actual plasma values (DBS whole disc, Rc= 0.97; DBS 2x3mm sub-punches, Rc = 0.92; Mitra, Rc = 0.95). However the Bland-Altman plots showed a small positive bias in the derived plasma values. The inter-assay and intra-assay CV in DBS whole disc, DBS 2x3mm sub-punches and Mitra were <17%, <16% and <14% respectively, across the concentration range. Recovery of 25(OH)D3 in DBS whole disc, DBS 2x3mm sub-punches and Mitra was on average 92.8%, 103.3% and 103.6% , respectively. The LLoD for each sampling method was; DBS whole disc 1.4 nmol/L, DBS 2x3mm sub-punches 1.8 nmol/L, Mitra 1.2 nmol/L. The LLoQ for DBS 2x3mm sub-punches was greater (2.5 nmol/L) than for DBS whole disc and Mitra (1.6 nmol/L and 1.5 nmol/L). The 25(OH)D3 value decreased as haematocrit levels increased in the samples.
A small reduction in 25(OH)D3 concentration in stored samples was observed in 12 of the 18 samples analysed after 50 days. The average (±SD) difference between day 1 and day 50 in DBS and Mitra were 2.0 nmol/L (± 4.2 nmol/L) and 1.8 nmol/L (± 3.4 nmol/L), respectively.
Conclusion
In summary, our findings showed that whole blood 25(OH)D3 collected in Mitra tips gave more precise results across the concentration range than blood spots collected in Whatman cards. However using the whole disc DBS did give a slightly higher Rc value and smaller bias. We found 25(OH)D3 is mainly present in the plasma portion of whole blood, therefore a conversion calculation is needed to obtain a plasma equivalent value. We have demonstrated that in patient samples with normal haematocrit levels, the derived 25(OH)D3 value from dried blood spots reflects the concentration in plasma, and thus shows the potential of using Mitra micro-sampling device as an alternative to venous blood to assess an individual's vitamin D status.
References & Acknowledgements:
Spooner N, Denniff P, Michielsen L, et al. A device for dried blood microsampling in quantitative bioanalysis: overcoming the issues associated with blood hematocrit. Bioanalysis. 2015;7(6):653-659.
Heath AK, Williamson EJ, Ebeling PR, Kvaskoff D, Eyles DW, English DR. Measurements of 25-hydroxyvitamin D concentrations in archived dried blood spots are reliable and accurately reflect those in plasma. J Clin Endocrinol Metab. 2014;99(9):3319-3324.
Eyles D, Anderson C, Ko P, et al. A sensitive LC/MS/MS assay of 250H vitamin D-3 and 250H vitamin D-2 in dried blood spots. Clin Chim Acta. 2009;403(1-2):145-151.
Kvaskoff D, Ko P, Simila HA, Eyles DW. Distribution of 25-hydroxyvitamin D3 in dried blood spots and implications for its quantitation by tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2012;901:47-52.
Kvaskoff D, Heath AK, Simila HA, Ko P, English DR, Eyles DW. Minimizing Matrix Effects for the Accurate Quantification of 25-Hydroxyvitamin D Metabolites in Dried Blood Spots by LC-MS/MS. Clin Chem. 2016;62(4):639-646.
de Vries R, Barfield M, van de Merbel N, et al. The effect of hematocrit on bioanalysis of DBS: results from the EBF DBS-microsampling consortium. Bioanalysis. 2013;5(17):2147-2160.
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