Original article Fe, Darmstadt, Germany). Positive controls were included in each batch of slides. Bone marrow smear iron grading Bone marrow smears were graded by the conventional Gale’s method and by a new more intensive grading method. Marrow smears were first assessed by one of the authors (KP) according to Gale’s histological grading method8 which assesses only marrow fragments (table 1). In order to reduce subjectiv- ity, predefined descriptions and sample illustrations of each iron grade were used to grade fragments of all marrow smears. Only iron smears with at least seven fragments were assessed.17 Deficiency of iron stores was defined as an iron grade of none (grade 0) or very slight (grade 1). All marrow smears were then systematically assessed using an intensive histological grading method in which iron is assessed in three sites—the fragments (as in Gale’s method), macrophages and erythroblasts. Iron assessed in the fragments and macrophages represented iron stores while iron in the erythroblast represented utilisable iron. Additionally, 20 fields around and behind the fragments were examined at high power (61000) and all macrophages in these fields were examined for the presence of iron (fig 1). At high power magnification (61000), 100 erythroblasts were examined and the percentage containing iron granules in their cytoplasm (ie, sideroblasts) were enumerated. Erythroblast iron deficiency was defined when ,30% of erythroblasts had visible iron granules.18 Results of iron smear assessment using the intensive histological grading method were interpreted as normal status (normal iron stores and normal erythroblast iron); functional iron deficiency (normal iron stores and deficient erythroblast iron); iron stores deficiency (depleted iron stores and normal erythro- blast iron); and combined functional iron and iron stores deficiency (depleted iron stores and deficient erythroblast iron; table 2). Other laboratory tests Bone marrow iron status assessment was compared to peripheral blood iron markers from samples taken at the same time as the bone marrow aspirate. Haemoglobin was measured using a Coulter counter machine (Beckman Coulter, Durban, South Africa). Ferritin, a measure of iron stores deficiency, was determined using the electrochemiluminescence immunoassay (Modular Analytics E170, Roche Diagnostics, Switzerland), and soluble transferrin receptor (sTfR) levels, a measure of cellular iron need, using ELISA (Ramco Laboratories, Texas, USA). Immunoturbidimetric assay (Modular P800, Roche Diagnostics, Switzerland) was used to determine C-reactive protein (CRP) levels (measure of inflammation) in blood. Histological grading for bone marrow iron status according to No visible iron under high power magnification (61000) Small iron particles just visible in few reticulum cells under high power magnification (61000) Small, sparsely distributed iron particles just visible under low power magnification (6100) Numerous small iron particles present in reticulum cells throughout the marrow fragment (6100) Larger iron particles throughout the fragment with tendency to aggregate into clumps (6100) Dense, large clumps of iron throughout the fragment (6100) Very large deposits of iron, both intra- and extra-cellular, obscuring cellular detail in the fragment (6100) Table 1 Gale et al8 Grade 0 None Grade 1 Very slight Grade 2 Slight Grade 3 Moderate Grade 4 Moderate heavy Grade 5 Heavy Grade 6 Very heavy 686 Figure 1 Bone marrow fragment showing iron deposits at 6400 magnification (top), and 61000 magnification showing erythroblasts, iron and malaria pigment within macrophages (bottom). Statistical analysis Data were double entered using Microsoft Access and Microsoft Excel. All data were exported to SPSS for Windows for analysis. Difference in means was evaluated using Student’s t test and difference in proportions, using the x2 test. Odds ratios (OR) and their 95% CI were used to measure association between categorical variables. Table 2 Classification of iron status using the intensive grading method Iron detected in: Fragment* Macrophage{ Erythroblast{ Iron status category Present Present Present Present – – – – Present – Present – Present – Present – Present Present – – Present Present – – Normal Functional iron deficiency Iron stores deficiency Functional and iron stores deficiency *Positive fragment iron: fragment grade >2. {Positive macrophage iron: iron present in reticular cell. {Positive erythroblast iron: iron present in .30% of erythroblasts. J Clin Pathol 2009;62:685–689. doi:10.1136/jcp.2009.064451