Specificity 79.0 % (95 % CI 71.4.-85.4 %) at the threshold value 9.6 Microcytic Anemia factor (MAf) in the study of iron metabolism Eloísa Urrechaga 1, Iratxe Ajuria 1, Edurne Bereciartúa 1, Luís Borque 2, Jesús F. Escanero 2 1. Hematology Laboratory. Hospital Galdakao – Usansolo. Galdakao, Vizcaya. SPAIN Department of Pharmacology and Physiology. Faculty of Medicine. University of Zaragoza. Zaragoza. SPAIN Disturbances of iron metabolism occur in many patients who are not clinically anemic as defined by the classic criteria. Certain groups, including children, adolescents and women of child-bearing age, are at particular risk of such iron metabolism disturbances. If they are not identified and treated appropriately these patients could go on to develop anemia an other related disorders. The challenge is therefore to detect these patients as early as possible, a task which is not easy because at this time only minimal analytical changes may be observed. Careful interpretation of the standard results is therefore required, together with the use of a selection of additional parameters in suspected cases. The diagnosis of iron deficiency or functional iron deficiency is particularly challenging in patients with acute or chronic inflammatory conditions because most of the biochemical markers for iron metabolism are affected by acute phase reaction. For these reasons, an iron deficient erythropoiesis may occur despite normal serum ferritin and transferrin saturation values and interest have been generated in the use of erythrocyte and reticulocyte parameters. Efforts have been made to evaluate some readily available and relatively inexpensive laboratory parameters as indirect markers of iron restricted erythropoiesis and iron availability in a clinical context influenced by inflammation and acute phase reaction. One of these parameters is Microcytic Anemic factor (MAf,) derived from the Hemoglobin (Hb)  and the mean cell volume (MCV) MAf = (MCV * Hb) / 100 The objectives of this study were to assess the values of MAf in healthy subjects and diverse types of anemia and the potential clinical utility in the study of the iron metabolism in the presence of inflammation and acute phase response. Histogram, MAf values in healthy subjects. Box & Whiskers, MAf values in the groups of patients. ROC curve, MAf and CHr diagnostic performances Materials and methods Samples from 90 healthy individuals, 72 iron deficiency anemia (IDA), 60 IDA with acute phase response (IDA APR) and 71 chronic kidney disease (CKD) were analysed on both LH 750 (Beckman Coulter Inc. Miami Fl, USA) and Advia 2120 (Siemens Medical Solutions Diagnostics, Tarrytown NY, USA) analysers, within 6 hours of collection. Ferropenic patients were analysed before iron treatment. CKD patients were managed according to the recommendations of the NKF-K/DOQI guidelines and were treated with a variety of erythropoietin doses, the majority of them were treated with a maintenance dose of intravenous iron weekly, in order to maintain Hb at the recommended level 11-0-12.0 g/dL.. The Kolmogorov-Smirnoff test was applied to verify the normal distribution of MAf values in the healthy group. Independent samples t test was applied to detect statistical deviations between the averages of the groups; values p< 0.05 are considered statistically significant. Receiver operating characteristic (ROC) analysis was used to illustrate the diagnostic performance MAf in the study of the iron metabolism. CHr = 28 pg was considered the goal standard in the assessment of erythropoietic activity. Results The values obtained in the healthy group were normally distributed (Kolmogorov-Smirnoff p = 0.882) and within the theoretician reference range, calculated employing the reference ranges of Hb and MCV, 9.6 – 16.6. The values obtained in CKD patients were intermediate between those from the healthy subjects and those from the ferropenic patients without treatment. The mean values in the groups were significantly different (p<0.001), the exception was IDA and IDA/APR, p=0.2137 ROC analysis results for MAf in the diagnosis of iron deficient erythropoiesis  defined by CHr < 28 pg AUC 0.938 ( 95 % CI 0.902-0.969) Sensitivity  92.5%  (95 % CI 84.2-97.1 %) Specificity  79.0 % (95 % CI 71.4.-85.4 %) at the threshold value 9.6 n Range Mean SD Healthy 90 12.0 – 15.6 14.0 0.9 IDA 72 3.8 – 9.9 7.5 1.7 IDA / APR 60 3.8 – 9.8 6.5 1.5 CKD 71 8.8 – 13.8 11.2 1.2 Conclusions Microcytic anemia factor (MAf) is a valuable research parameter for examining abnornal red cells modalities, since its calculation accounts for both cell size and hemoglobin content. MAf may aid in the classification of anemias characterized by microcytic red cells. Good diagnostic performance in the detection of iron deficient erythropoiesis was stated. MAf is not affected by acute phase reaction and reflects the state of the erythropoiesis; therefore it can be a good alternative to the biochemical parameters in presence acute phase response.

Box & Whiskers plot sTfR Box & Whiskers plot sTfR index The Beckman-Coulter Access® Soluble Transferrin Receptor (sTfR) in the differential diagnosis of Iron Deficiency Anemia and Anemia of Chronic Disease Eloísa Urrechaga 1, María Unceta 1, Iratxe Ajuria 1, Edurne Bereciartúa 1, Luís Borque 2, Jesús F. Escanero 2 1. Hematology Laboratory. Hospital Galdakao – Usansolo. Galdakao, Vizcaya. SPAIN Department of Pharmacology and Physiology. Faculty of Medicine. University of Zaragoza. Zaragoza. SPAIN Anemia of chronic disease (ACD) and Iron deficiency anemia (IDA) are common causes of anemia. Differential diagnosis is primary based on estimates of iron status. A highly accurate automated laboratory test detecting a biomarker that is not affected by inflammation could be a reliable method for assessing the body’s iron status in the presence of ACD. One such assay would measure serum soluble transferrin receptor (sTfR) concentration. Transferrin receptors are present on nearly every cell type, with the largest numbers found in the erythron, placenta and liver. When a cell needs iron transferrin receptor production increases to augment receptor expression on the cell surface,facilitating iron uptake. sTfR results from the proteolysis of TfR at a specific site in the extracellular domain, producing fragments that circulate in the blood complexed to ferritin. The amount of total cellular TfR is directly proportional to the concentration of sTfR in plasma or serum, so sTfR in the plasma accurately reflects the total TfR. Because most TfRs are located on erythroid progenitors, the concentration of sTfR is believed to reflect the erythroid turnover, and is determined by the erythroid proliferation rate and iron demand. sTfR increases in hemolytic anemia, reflecting the raised number of erythroid cells and in IDA due to an increased iron demand for erythropoiesis. A reduced level of sTfR is typically seen in aplasic anemia and renal failure as erythroid cell mass is reduced. The inclusion of a sTfR assay when assessing a patient with suspected anemia can provide a clearer picture of both the cause and the most appropriate treatment. The sTfR / log ferritin index (sTfR index) has an even higher specificity and sensitivity for the differential diagnosis of DA and ACD than sTfR alone. The present study assessed the clinical utility of sTfR assay and sTfR/log ferritin index, as automatically provided by Access immunochemical analyser (Beckman Coulter Inc. Miami, Fl, USA), and the best cut offs for the differential diagnosis between IDA and ACD. 2 Methods The Access sTfR assay is a paramagnetic particle chemiluminescent immunoassay for the quantitative determination of sTfR levels in human serum or heparinised plasma employing the Access family of immunoassay systems. The sTfR assay consists of a two step, sandwich assay with anti sTfR monoclonal capture antibody immobilized to solid phase and sTfR monoclonal alkaline-phosphatase conjugate detection antibody. The two step, sandwich immunoassay method combines 25 µL of serum or heparinised plasma with an anti sTfR monoclonal antibody linked to paramagnetic particles; following a 10 minutes incubation monoclonal anti sTfR conjugated to alkaline-phosphatase is added. After another 10 minutes incubation a wash cycle occurs, followed by substrate addition. sTfR concentration is determined from a stored multi point calibration curve ; results are obtained within 35 minutes. The calculation of sTfR/log ferritin index can be carried out in the “derived result function” on the Access systems. Serum iron, Transferrin and Ferritin were assayed with a chemical analyzer (Cobas c 711 (Roche Diagnostics, Manhein, Germany). Serum iron was determined spectrophotometrically with a FerroZine based method (IRON 2 Cobas, Roche Diagnostics, Manhein). Transferrin was measured with a inmunoturbidimetric method (TRSF 2 Cobas, Roche Diagnostics, Manhein). Ferritin was measured with a inmunoturbidimetric method (FERR 3 Cobas, Roche Diagnostics, Manhein). Patients Samples from 80 IDA and 90 ACD consecutive patients were analysed on the Access analyser. sTfR and ferritin assays (Beckman-Coulter) were performed. Independent samples t test was applied in order to detect statistical deviations between the groups of patients; p values less than 0.05 were considered to be statistically significant. Receiver operating characteristic (ROC) curve analysis was utilized to illustrate the diagnostic performance of sTfR and sTfR index. Results Subjects with IDA had significantly higher sTfR and sTfR index than ACD patients (p<0.0001). In the IDA group the mean sTfR concentration was 36.2 nmol/L. The mean concentration in the ACD group was 17.9 nmol/L. In the IDA group the mean sTfR index value was 31.6. The mean value in the ACD group was 8.0 . Box & Whiskers plots illustrate these data. ROC curves analysis results are summarized on table 1 and figure 2. AUC Cut off Sensitivity Specificity Ferritin, μg/L .83 < 50 80.0 81.1 Transferrin Sat, % .80 < 10 78.0 82.1 sTfR, nmol/L .905 > 21 93.1 70.3 sTfR index .958 > 11.5 92.5 86.1 Box & Whiskers plot sTfR Box & Whiskers plot sTfR index Conclusions Anemia of chronic disease (ACD) and Iron deficiency anemia (IDA) are the most prevalent forms of anemia. Accurate assessment of iron status is essential for appropriate treatment of the patients with ACD. sTfR assay and sTfR index improve detection of iron deficiency in situations where routine markers, affected by inflammation and acute phase response, become unreliable. sTrR as measured with Access is a sensitive assay in the study of patients with anemia in the presence of inflammation. Only the Access system offers automated on-board calculation of the sTfR index from the sTfR and ferritin results, giving the index value quickly and accurately, and removing the chance of clerical error.