Original article Improved method for assessing iron stores in the bone marrow K S Phiri,1 J C J Calis,1,2 D Kachala,1 E Borgstein,3 J Waluza,3 I Bates,4 B Brabin,4 M Boele van Hensbroek1,2,4 1 Malawi–Liverpool–Wellcome Trust Clinical Research Programme, College of Medicine, Blantyre, Malawi; 2 Emma Children’s Hospital AMC, University of Amsterdam, Netherlands; 3 Department of Surgery, College of Medicine, Blantyre, Malawi; 4 Liverpool School of Tropical Medicine, Liverpool, UK Correspondence to: Dr K Phiri, Malawi–Liverpool– Wellcome Trust Clinical Research Programme, PO Box 30096, Blantyre 3, Malawi; kamijaphiri@gmail.com Accepted 19 February 2009 This paper is freely available online under the BMJ Journals unlocked scheme, see http:// jcp.bmj.com/info/unlocked.dtl J Clin Pathol 2009;62:685–689. doi:10.1136/jcp.2009.064451 ABSTRACT Background: Bone marrow iron microscopy has been the ‘‘gold standard’’ method of assessing iron deficiency. However, the commonly used method of grading marrow iron remains highly subjective. Aim: To improve the bone marrow grading method by developing a detailed protocol that assesses iron in fragments, in macrophages around fragments and in erythroblasts. Methods: A descriptive study of marrow aspirates of 303 children (aged 6–60 months) with severe anaemia and 22 controls (children undergoing elective surgery) was conducted at hospitals in southern Malawi (2002–04). Results: Using an intensive marrow iron grading method, 22% and 39% of cases and controls had deficient iron stores, and 40% and 46% had functional iron deficiency, respectively. Further evaluation of the iron status classification by the intensive method showed that functional iron deficiency was associated with signifi- cantly increased C-reactive protein concentrations (126.7 (85.6) mg/l), and iron stores deficiency with significantly increased soluble transferrin receptor concentrations (21.7 (12.5) mg/ml). Conclusions: Iron assessment can be greatly improved by a more intense marrow examination. This provides a useful iron status classification which is of particular importance in areas where there is a high rate of inflammatory conditions. Functional iron deficiency develops when normal physiological systems for transporting iron to target tissues are impaired in the presence of satisfactory iron stores.1 This is commonly caused by cytokines released during an acute phase response to infection, leading to impaired erythro- poiesis and later anaemia, usually termed ‘‘anaemia of inflammation’’.2 Although mass spectrometry has been recently used to give a definitive determination of iron in tissue,3 4 microscopic examination of Prussian blue- stained bone marrow aspirate has been considered the practical ‘‘gold standard’’ for determining iron depleted states.5 6 Conventionally, iron has been primarily assessed in marrow fragments which represent iron stores in the form of hemosiderin.7 8 Although some studies have shown a reasonable correlation between histological iron grading and chemical iron concentration in bone marrow,8 9 others have not, raising questions about the validity of the histological grading.10 Iron visualised in marrow fragments is from a meshwork of reticular cells which are usually undistinguishable. However, single ‘‘loose’’ macro- phages may be inspected for iron and it is hypothesised to be particularly important when iron in fragments is absent and may signify the lowest level of iron stores depletion. In areas where there is a high prevalence of inflammatory conditions, functional iron defi- ciency commonly occurs. Erythroblast iron may be indicative of cellular iron utilisation and decreased in functional iron deficiency11; however there has been little research on the use of erythroblast iron as a marker of cellular iron availability.12 Furthermore, in malaria endemic areas, interpretation of iron status may be con- founded by the presence of hemozoin.13 14 Recent studies have suggested that it may be detrimental to mass treat children with iron,15 so it is important to be able to identify children with iron stores deficiency. The aim of the present study was to determine if a more intensive bone marrow classification can distinguish iron deficiency states in severely anaemic children and their controls living in a malaria endemic area. METHODS This was a descriptive study which was part of a large case–control study investigating the aetiology of severe anaemia in Malawi. Children were recruited between July 2002 and July 2004 at Queen Elizabeth Central hospital in Blantyre and Chikwawa District hospital.16 All children who presented to hospital with severe anaemia (haemoglobin of less than 5.0 g/dl and no history of being transfused in the preceding four weeks), aged 6–60 months, were eligible for recruitment as cases. A group of children under- going elective operations with no obvious signs of infection and within the same age range as cases were recruited as ‘‘normal’’ controls. Samples of venous blood and bone marrow aspirate were collected from cases and controls under anaesthesia from either the anterior or posterior iliac crest. The first few drops of a bone marrow aspirate were collected in an EDTA tube for smear preparation.16 Written informed consent was obtained from the guardians of the children and the study was approved by the ethics committees of the University of Malawi and the Liverpool School of Tropical Medicine, UK. Bone marrow smear preparation Bone marrow smears were air-dried before being fixed in methanol for 5 minutes. Iron staining was done at the Wellcome Trust laboratories, Blantyre, using a commercial kit and according to methods recommended by the manufacturer (HematoGnost 685