Take-home messages c Differentiation between functional iron deficiency and quantitative deficiency of iron stores is difficult, especially in areas of high infection pressure. c A new method of grading of iron content of fragments, macrophages and erythroblasts in the bone marrow is able to distinguish between functional and quantitative iron deficiency in anaemic children. c Severely anaemic Malawian children have less quantitative iron deficiency than controls without severe anaemia. pink background colour, which makes visualisation of cell types difficult, and hence may affect erythroblast iron assessment. The use of haematoxylin, or May–Gru¨nwald–Giemsa, for counter-staining smears has been recommended as this provides improved cellular detail.18 Tham and Macon23 demonstrated that use of a silver stain to visualise erythroblast iron was more sensitive than Perls’ stain. However, the precise chemical basis for the silver staining is still unclear. Some researchers have described erythroblast iron assessment in comparison to simply counting erythroblasts with iron. Baumgartner-Staubli and Beck11 developed a ‘‘sideroblast score’’ which assigned an arbitrary value, ranging from 1 to 4 depending on the amount and morphology of iron granules, to each erythroblast with iron. This gave a poor correlation between the sideroblast score and the marrow iron stores assessed using Gale’s grading method. The Malawi Ministry of Health does not have guidelines on whether to give iron in the management of children with severe anaemia. In practice, most children are prescribed iron, however it is often unavailable and the compliance is poor. This study has shown that approximately 30% of severely anaemic children had deficiency of iron stores (table 3) requiring iron treatment. Methods to identify these children are required in poor-resource settings that are based on simple, less invasive procedures than detailed marrow examination. Additionally this study observes a lower prevalence of deficiency of iron stores among severely anaemic children than controls. This phenomenon is not fully understood, but may support the hypothesis that iron deficiency is associated with decreased risk of infection.24 This study of a large sample of bone marrow aspirates demonstrates that using an enhanced bone marrow slide assessment provides more detailed information which allows a more precise iron status classification. Acknowledgements: Special thanks are extended to Barbara Bain, Imperial College School of Medicine, and to the children and their guardians who participated in this study. O r ig in a l a rt i c l e Funding: The study was supported by the Wellcome Trust, Gates Malaria Partnership, and Numico and Ter Meulen Foundation. Competing interests: None. Ethics approval: Ethics approval was obtained. REFERENCES 1. Roy CN, Andrews NC. Anemia of inflammation: the hepcidin link. Curr Opin Hematol 2005;12:107–11. 2. Trey JE, Kushner I. The acute phase response and the hematopoietic system: the role of cytokines. Crit Rev Oncol Hematol 1995;21:1–18. 3. Helliwell T, Kelly S, Walsh H, et al. Elemental analysis of femoral bone from patients with fractured neck of femur or osteoarthrosis. Bone 1996;18:151–7. 4. van Dieijen-Visser MP, Marell GJ, Coenen JL, et al. Quantitative determination of non- haem iron and ferritin iron in bone marrow using flameless atomic absorption spectrophotometry. A comparative study on the cytological and chemical determination of the bone marrow iron content. Eur J Clin Chem Clin Biochem 1991;29:381–4. 5. Burns E, Goldberg S, Lawrence C, et al. Clinical utility of serum tests for iron deficiency in hospitalized patients. Am J Clin Pathol 1990;93:240–5. 6. Intragumtornchai T, Rojnukkarin P, Swasdikul D, et al. The role of serum ferritin in the diagnosis of iron deficiency anaemia in patients with liver cirrhosis. J Intern Med 1998;243:233–41. 7. Finch CA, Huebers H, Eng M, et al. Effect of transfused reticulocytes on iron exchange. Blood 1982;59:364–9. 8. Gale E, Torrence J, Bothwell T. The quantitative estimation of total iron stores in human bone marrow. J Clin Invest 1963;42:1076–82. 9. Johansson SV, Plantin LO, Strandberg PO, et al. Estimation of iron in human bone marrow with histological, chemical and neutron activation analysis. Clin Chim Acta 1970;30:549–58. 10. Trubowitz S, Miller WL, Zamora JC. The quantitative estimation of non-heme iron in human marrow aspirates. Am J Clin Pathol 1970;54:71–7. 11. Baumgartner-Staubli R, Beck EA. Sideroblast score: a sensitive indicator of iron deficiency and hypoproliferative anemia. Acta Haematol 1977;57:24–31. 12. Hanif E, Ayyub M, Anwar M, et al. Evaluation of serum transferrin receptor concentration in diagnosing and differentiating iron deficiency anaemia from anaemia of chronic disorders. J Pakistan Med Assoc 2005;55:13–6. 13. Rodriguez MH, Jungery M. A protein on Plasmodium falciparum-infected erythrocytes functions as a transferrin receptor. Nature 1986;324:388–91. 14. Sullivan DJ. Theories on malarial pigment formation and quinoline action. Int J Parasitol 2002;32:1645–53. 15. Sazawal S, Black RE, Ramsan M, et al. Effects of routine prophylactic supplementation with iron and folic acid on admission to hospital and mortality in preschool children in a high malaria transmission setting: community-based, randomised, placebo-controlled trial. Lancet 2006;367:133–43. 16. Calis J, Phiri K, Faragher B, et al. Aetiology of severe anemia in Malawian children. N Engl J Med 2008;358:888–99. 17. Hughes DA, Stuart-Smith SE, Bain BJ. How should stainable iron in bone marrow films be assessed? J Clin Pathol 2004;57:1038–40. 18. Hastka J, Lasserre JJ, Schwarzbeck A, et al. Zinc protoporphyrin in anemia of chronic disorders. Blood 1993;81:1200–4. 19. Molyneux EM, Tembo M, Kayira K, et al. The effect of HIV infection on paediatric bacterial meningitis in Blantyre, Malawi. Arch Dis Child 2003;88:1112–8. 20. Pippard MJ, Hoffbrand AV. Iron. In: Hoffbrand AV, Lewis SM, Tuddenham EGD, eds. Postgraduate haematology, 4th edn. Oxford: Butterworth-Heinemann, 1999:23–46. 21. Beguin Y, Huebers HA, Josephson B, et al. Transferrin receptors in rat plasma. Proc Natl Acad Sci USA 1988;85:637–40. 22. Gabay C, Kushner I. Acute-phase proteins and other systemic responses to inflammation. N Engl J Med 1999;340:448–54. 23. Tham KT, Cousar JB, Macon WR. Silver stain for ringed sideroblasts. A sensitive method that differs from Perls’ reaction in mechanism and clinical application. Am J Clin Pathol 1990;94:73–6. 24. Murray MJ, Murray AB, Murray MB, et al. The adverse effect of iron repletion on the course of certain infections. BMJ 1978;2:1113–5. J Clin Pathol 2009;62:685–689. doi:10.1136/jcp.2009.064451 689