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Division of Gastroenterology and Nutrition and Programs in Metabolism and Integrative Biology Research Institute, The Hospital for Sick Children Toronto, Canada
Division of Gastroenterology and Nutrition and Programs in Metabolism and Integrative Biology Research Institute, The Hospital for Sick Children Toronto, Canada Division of Gastroenterology and Nutrition and Programs in Metabolism and Integrative Biology Research Institute, The Hospital for Sick Children, Departments of Paediatrics and Nutritional Sciences and The Center for International Health, University of Toronto, Toronto, Canada
Dear Editor,
We are writing in reference to the recent article by Shell-Duncan and McDade (1). We share the authors’ interest in the development and application of a field-friendly screening method for detecting iron deficiency in community settings. The authors reported a community-based survey based on the measurement of soluble transferrin receptor (sTfR) using a modified whole-blood spot method, a method which required a single finger prick blood sample placed onto a spot of filter paper. We feel it is also necessary to bring to your attention some inaccuracies and limitations of the method used.
Firstly, the authors claimed that their method for measuring sTfR on whole blood spot samples can be performed with the commercially available Ramco kit. We disagree with this important comment because the standards and sample diluents used for the assay were not part of the commercial kit, but were "home-made" in the laboratory of the authors. This would mean that even with the Ramco kit, the dilution buffer and standards would be inconsistent among laboratories with potentially different results.
The authors appropriately attempted to lower the matrix difference between standards and unknowns by diluting the purified TfR protein stock with washed erythrocytes prior to application onto the filter paper (2). However, by washing the erythrocytes, the plasma and buffy coat were lost; thus matrix difference still existed between the standards and the unknowns. Furthermore, erythrocytes are subject to biological variation and thus may not be an appropriate reference preparation to create a standard curve. An alternate strategy to decrease the impact of the varying matrices might be the use of a monoclonal anti-TfR antibody (instead of the polyclonal antibody in the kit) to coat the microwell strips, which will increase antigenic specificity.
As has been previously noted, the sTfR concentration varied when discs of whole blood were punched out at varying distances from the center of the same blood spot; the concentration of sTfR in peripheral discs was on average 12% higher compared to discs punched from the center (2,3). Unfortunately, there is no simple solution to this problem, although measurement of the blood volume prior to spotting the sample onto the filter paper and punching out the entire dried blood spot sample would likely have produced more accurate and uniform results.
The sensitivity of the method described in this manuscript is quite low. The authors report that the lowest concentration of sTfR detected with their method was 0.55 mg/L (2 SD above the zero standard). With other commercially available sTfR EIA kits sensitivity is 0.1 mg/L (3 SD above the zero standard) (Table 1). Thus, compared to currently available methods, the filter-paper spot method described is not very sensitive.
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Developing a field-friendly method to detect iron deficiency is an important goal. The authors have described an initial step in the process, yet because of in-house modifications to the commercial kit, low sensitivity, and a debatable cutoff value to detect iron deficiency, we question the current applicability of this screening tool and the generalizability of its results and implications.
LITERATURE CITED
1. Shell-Duncan, B. K. & McDade, T. W. (2004) Use of combined measures from capillary blood to assess iron deficiency in rural Kenyan children. J. Nutr. 134:384-387.
2. McDade, T. W. & Shell-Duncan, B. K. (2002) Whole blood collected on filter paper provides a minimally-invasive method for assessing human transferrin receptor level. J. Nutr. 132:3760-3763.
3. Cook, J. D., Flowers, C. H. & Skikne, B. S. (1998) An assessment of dried blood-spot technology for identifying iron deficiency. Blood 92:1807-1813.
4. Bland, J. M. & Altman, D. G. (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1:307-310.[Medline]
5. Platt, R. W., Hanley, J. A. & Yang, H. (2000) Bootstrap confidence intervals for the sensitivity of a quantitative diagnostic test. Stat. Med. 19:313-322.[Medline]
6. Metz, C. E. (1978) Basic principles of ROC analysis. Semin. Nucl. Med. 8:283-298.[Medline]
7. Campbell, G. (1994) Advances in statistical methodology for the evaluation of diagnostic and laboratory tests. Stat. Med. 13:499-508.[Medline]
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