1. Reticulocyte Hb equivalent and hypochromic red cells in the study of erythropoiesis in pregnancy Eloísa Urrechaga 1, Elia Crespo 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 is a common problem during pregnancy, iron deficiency anemia is the predominant cause in those subjects There is a high prevalence of anemia during pregnancy worldwide. One of the main reasons for anemia during pregnancy is expanded plasma volume; this hemodilution allows better circulation for the placenta and developing fetus Another common cause of anemia during pregnancy is depletion of iron. Diagnosis and prevention of iron deficiency is important, because iron deficiency is known to be associated with maternal infections, preterm delivery, and low birth weight Due to physiological alteration of plasma volume and red cell mass in pregnancy a slightly reduced Hemoglobin values can be observed, and remains a challenge to decide between physiological hemodilution and pathological status due to iron restricted erythropoiesis The aim was to assess the reliability of the extended hemogram reporting reticulocyte Hb equivalent (RetHe) and percentage of hypochromic erythrocytes (%HypoHe), in the detection of iron deficient erythropoiesis in women in the second trimester of pregnancy PATIENTS AND METHODS Seventy women (23-40 years, mean 33 y) were included in the study; the analyses had been requested by general practitioners, during the routine control in the 20 th week of pregnancy Samples were analyzed for full blood count and reticulocytes within 6 hours of collection; serum ferritin, serum iron and transferrin saturation (Tf Sat ) were measured Independent samples t test was performed in order to detect statistical deviations between the groups of patients; p values less than 0.05 were considered to be statistically significant The diagnostic performance of the standard and extended parameters was evaluated with Receiver operating characteristic (ROC) curves ; Transferrin saturation <15% was the gold standard for iron restricted erythropoiesis detection. Laboratory parameters for the assessment of iron metabolism status. The values are reported mean (standard deviation). CONCLUSIONS Due to physiologic alteration of plasma volume and red cell mass in pregnancy, Hb level are unreliable Hb determination is neither sensitive nor specific as a screening test for iron deficiency Clinical practice needs simple, straightforward, and cost-effective methods for defining diagnoses While Hb, MCV, MCH, MCHC and RDW are nowadays included in basic blood counts, a supplement of advanced cellular indices would be easy to perform Technological advances in automated full blood count analysers a few years ago allowed the hemoglobin concentration of individual red cells to be measured by flow cytometry and so, it was possible to calculate the number of individual red cells that had deficient hemoglobinization RetHe and %HypoHe reflect iron deficient erythropoiesis, could be reliable parameters for detecting iron deficiency during pregnancy and useful indicators to identify patients who will benefit from therapy Sysmex XE 500 Analytical Methods The flow fluorescence cytometry technology incorporated enables independent measurement of the volume and hemoglobin content of individual red cells Percentages of red cells subpopulations can be calculated and the new parameters %Micro R and %Macro R obtained In the reticulocyte channel blood cells are stained by a polymethine dye, specific for RNA/DNA, and analysed by flow cytometry using a semiconductor laser. A bi-dimensional distribution of forward scattered light and fluorescence is presented as a scattergram, indicating mature red cells and reticulocytes Forward scatter correlates with erythrocyte and reticulocyte hemoglobin content Ret He Percentages of red cells subpopulations can be calculated, %Hypo-He indicates the percentage of hypochromic red cells with a Hb content < 17 pg RESULTS Laboratory results are shown on table 1 Based on Tf Sat values 20 the pregnant (28.6 %) suffered iron deficiency for erythropoiesis sFerritin, MCH, MCV, Ret He and % Hypo He values in both groups were statistically different The definition of anemia during pregnancy was based on the recommendation of the World Health Organization (Hb < 110 g ⁄ L); the cut- off points for iron status measurements were selected on the basis of the reference intervals of the American College of Obstetricians, published on www.update.com www.update.com ROC analysis is summarized on the table 2. The cut offs for Ret He and %Hypo He were those which provide the best diagnostic performances Receiver operating characteristic (ROC) curves for Hb, MCH, MCV, RetHe,%Hypo He in the diagnosis of iron deficiency, defined by Tf Sat < 15 % Table 2AUC95%CICutOff Sensitivity % Specificity % Hb0.690.59-0.79110 g/L52.680.9 MCH0.710.61-0.8130 pg65.581.1 MCV0.690.59-0.7985 fL42.580.2 RetHe0.790.70-0.8829.9 pg80.771.3 %Hypo0.750.65-0.831.5 %82.368.3 Table 1 Tf Sat < 15 % N= 20 Tf Sat > 15 % N= 50 P Hb, g/L101 (9)115 (5)<0.001 MCH, pg26.3 (3.1)28.6 (2.3)0.011 MCV, fL81.2 (3.8)84.2 (3.1)0.001 sIron, µg/dL22 (11)57 (6)<0.001 sFerritin, μg/L12 (9)24 (11)<0.001 Tf Sat, %9 (3)21 (5)<0.001 Ret He, pg28.1 (2.6)31.7 (1.8)<0.001 % Hypo He, %2.8 (1.8)1.1 (0.9)<0.001