Reticulocyte Indices Research Articles

Automated Blood Cell Counts

The CBC count and leukocyte differential count (LDC) are among the most frequently requested clinical laboratory tests. These analyses are highly automated, and the correct interpretation of results requires extensive knowledge of the analytic performance of the instruments and the clinical significance of the results they provide. In this review, we analyze the state of the art regarding traditional and new parameters with emphasis on clinical applications and analytic quality. The problems of some traditional parameters of the CBC count, such as platelet counts, some components of the LDC such as monocyte and basophil counts, and other commonly used indices such as red cell volume distribution width and platelet indices such as mean platelet volume and platelet distribution width are considered. The new parameters, evaluated from analytic and clinical viewpoints, are the available components of the extended differential count (hematopoietic progenitor cells, immature granulocytes, and erythroblasts), the immature reticulocyte fraction, the reticulocyte indices, the fragmented RBCs, and the immature platelet fraction.

Altered Erythrocyte Membrane Protein Composition in Chronic Kidney Disease Stage 5 Patients under Haemodialysis and Recombinant Human Erythropoietin Therapy

Our aim was to evaluate red blood cell (RBC) membrane protein composition in chronic kidney disease (CKD) stage 5 patients under haemodialysis (HD) and recombinant human erythropoietin (rhEPO) therapy, and its linkage to rhEPO hyporesponsiveness. We evaluated in 63 CKD stage 5 patients (32 responders and 31 non-responders to rhEPO therapy) and in 26 healthy controls RBC count, haematocrit, haemoglobin concentration, haematimetric indices, reticulocyte count, reticulocyte production index, RBC osmotic fragility test and membrane protein analyses. CKD stage 5 patients presented significant changes in membrane protein composition, namely a reduction in spectrin, associated to altered protein 4.1/spectrin and spectrin/band 3 ratios. Non-responder CKD stage 5 patients were more anaemic, with more microcytic and anisocytic RBCs, than responders; significantly altered ankyrin/band 3 and spectrin/ankyrin ratios were also observed. CKD stage 5 patients under HD are associated with an altered protein membrane structure, which seems to the disease itself and/or to the interaction with HD membranes.

A Case Report of Refractory Warm Autoimmune Hemolytic Anemia Treated With Plasmapheresis and Rituximab

A 30-year-old male presented with severe, warm autoimmune hemolysis 17 months subsequent to a matched, unrelated peripheral hematopoietic stem cell transplant. The patient responded poorly to conventional therapy with steroids and immunoglobulin, prompting the initiation of rituximab. On account of persistent, severe hemolysis, therapeutic plasma exchange was employed as a bridge until the rituximab therapy became effective. Immediately following plasmapheresis, the patient demonstrated clinical improvement followed by attenuation of the hemolysis and improved reticulocytosis. The hemoglobin concentration and reticulocyte index demonstrated further improvement following subsequent doses of rituximab and continued following the cessation of plasmapheresis. This case suggests the utility of plasmapheresis and rituximab in severe, life-threatening cases of warm autoimmune hemolytic anemia refractory to conventional therapy.

Hubungan antara Indeks Produksi Retikulosit (IPR) dengan Red Blood Cell Distribution Width (RDW) pada Klasifikasi Anemia berdasarkan Defek Fungsional

Reticulocyte Production Index (RPI) is a shift correction for the presence of reticulocyte that can assesses the real erythropoietic response of the bone marrow. RPI can be used to classify anemia as result from functional defect, i.e. RPI 3 for hemolytic anemia. Measurement of Red Blood Cell Distribution Width (RDW) is the mathematical representation of variance within the size distribution of the red cell population. The objective of this study was to identify the variation of the red cell size within the population measured (anisocytosis) enable to determine the cell population as homogeneous (normal RDW) or heterogenous (increased or high RDW). The role of RDW to differentiate functional defect anemia with RPI 3 classified as hemolytic anemia. Means for reticulocyte count, corrected reticulocyte count, RPI, and RDW for hypoproliferative and maturation defect anemia were 2,8% and 2,3%; 1,1% and 1,1%; 0,4 and 0,5; 21,8% and 17,8% respectively, with p value by the Wilcoxon Signed Ranks Test are = 0,322; 0,52; 0,273; and 0,27 (CI = 95%). The proportion of anisocytosis and normocytosis for hypoproliferative anemia were 76,08% and 23,9% and for maturation defect anemia were 76,19% and 23,8% with Chi-Square test 0,000 and p = 0,993. No correlation between was observed RDW and RPI<2 with Spearman correlation (r = 0,0297 and p = 0,993). It is concluded that the use of RDW and RPI is not always useful to differentiate hypoproliferative and maturation defect anemia on the group of anemia categorized as RPI<2 without support from another laboratory test especially in combined anemia.

Hematological parameters in preterm infants from birth to 16 weeks of age with reference to iron balance

The objective of this study was to describe the natural kinetics of serum soluble transferrin receptor (S-TfR), ferritin and reticulocyte indices in preterm neonates, and to find out whether these analytes relate to hematocrit (Hct) level in determining the need for red cell (RBC) transfusions. During a 2-year period, 100 preterm neonates were recruited in a tertiary level neonatal intensive care unit. Inclusion criteria were gestational age < or =34 weeks or birth weight <2000 g. Biochemical markers of iron deficiency and hematological indices were serially analyzed from birth. This report focuses on the first 16 weeks after birth. The trends of the studied analytes were presented with reference ranges. RBC transfusions did not have a significant effect on reticulocyte hemoglobin content (CHr) or reticulocyte count. Reticulocytes were lowest after the first week and S-TfR at 9 weeks of age. CHr and fraction of immature reticulocytes were highest at birth and decreased thereafter. CHr and reticulocyte count were significantly different in two groups determined by Hct level (Hct < or > or =0.30). This difference was not observed in S-TfR or ferritin concentrations. In addition to reflecting the activity of erythropoiesis, S-TfR seems to reflect iron balance in preterm neonates. By using CHr and reticulocyte, it is possible to obtain more information about iron balance in relation to erythropoiesis, and it might be useful to combine this information with Hct before making a decision about a transfusion.

Acute erythroid toxicity in visceral leishmaniasis: A rare complication of antimonial therapy

The spectrum of side-effects of sodium stibogluconate is well described, however, little is known regarding the acute erythroid toxicity caused by this drug. We hereby present a case with this unusual complication of antimonial therapy. A 6-year-old male with leishmaniasis was started on parenteral sodium stibogluconate. During the course of treatment, his hemoglobin (Hb) dropped from 7.2 g/dl to 3.5 g/dl. Bone-marrow aspirate showed karyorrhexis in many erythroid precursors with several Leishmania donovanii bodies. Sodium stibogluconate was stopped and amphotericin-B was started. Four days after the cessation of the antimonials, the patient's Hb improved to 5 gm/dl with a corrected reticulocyte count of 10% indicating bone-marrow erythroid regeneration. The exact mechanism of this acute erythroid toxicity of sodium stibogluconate remains unexplored.

Elevated serum erythropoietin concentration is associated with coordinated changes in red blood cell and reticulocyte indices of pregnant women at term

The aim of this study was to evaluate the relationship between the erythropoietin (EPO) concentration and both the advanced cellular indices reflecting the haemoglobin contents of red blood cells and reticulocytes and the serum markers of iron status. The study population comprised pregnant women at term (n = 210). The serum EPO, transferrin receptor (TfR), ferritin, the percentages of hypochromic red blood cells (%HYPOm) or reticulocytes (%HYPOr) and the cellular haemoglobin in mature red blood cells (CHm) or in reticulocytes (CHr) were measured in maternal blood before delivery. The EPO concentration was elevated above the reference limit (>31.5 mIU/mL) in 16 % of all pregnant women, and appeared to correlate in a linear fashion, especially with %HYPOm (r = 0.52, p<0.001), %HYPOr (r = 0.57, p<0.001) and CHr (r = −0.45, p<0.001). The significant (p<0.05) predictors of EPO in the multivariate stepwise regression analysis were %HYPOr, Hb, %HYPOm and MCV. In general, the lower the cellular haemoglobin content, the higher the overall maternal EPO production. In conclusion, elevated %HYPOm and %HYPOr reflecting iron‐deficient erythropoiesis are associated with an increase in EPO concentration in maternal blood. This could be explained by subclinical iron deficiency being accompanied by a compensatory EPO response.

Effect of iron and zinc supplementation in the treatment of malaria in children

Background Iron and zinc administration for children withmalaria in endemic area were known to decrease parasitemia butdata on their effectiveness when given together to increasereticulocytes as erythropoiesis parameter and hemoglobin isinsufficient.Objective To determine the effect of zinc to increase ironabsorption in the treatment of Plasmodium falciparum malaria inchildren.Methods Children with positive Plasmodium falciparum on theirblood smear (n=86) examination were randomly assigned to dailysupplementation of iron 6 mg per kg body weight per day plusplacebo or iron plus zinc 10 mg per day for 30 days. Venous bloodspecimens were collected at the start and at the end of the study.Results Sixty-nine children completed the supplementations andhad both baseline and follow-up blood specimen study. After 30-day supplementation, the iron plus placebo and iron plus zincgroups showed significant difference on hemoglobin concentration(0.58 and 0.09 g/dl; P&lt;0.05). There was no significant differencein reticulocyte production index and reticulocyte count beforeand after intervention in both groups. There was only significantdifference in red blood cells concentration after supplementationof iron plus placebo and iron plus zinc (4.7 in 4.5 million/μl;P&lt;0.05).Conclusions Iron supplementation with or without zinc showssignificant increase of hemoglobin concentration. It is slightlyhigher in iron plus placebo group.

Erratum

European Journal of HaematologyVolume 80, Issue 1 p. 92-92 Free Access Erratum This article corrects the following: Use of advanced red blood cell and reticulocyte indices improves the accuracy in diagnosing iron deficiency in pregnant women at term Mari Ervasti, Sanna Kotisaari, Seppo Heinonen, Kari Punnonen, Volume 79Issue 6European Journal of Haematology pages: 539-545 First Published online: September 15, 2007 First published: 13 December 2007 https://doi.org/10.1111/j.1600-0609.2007.01002.xAboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat The article by Ervasti et al. (1) was found to contain a few errors. In the text on page 541 and in Tables 1 and 2, the correct number of pregnant women in the non-iron deficient study group (TfSat >11%) is 167 instead of 67. In Table 2, the correct percentage of gestational diabetes mellitus (GDM) in the iron deficient group (TfSat ≤11%) is 16% instead of 6%. The correct version of Table 2 is printed below. Table 2. Clinical characteristics of pregnant women with and without iron deficiency Variable TfSat ≤ 11%, n = 31Mean ± SD TfSat > 11%, n = 167Mean ± SD P-value Maternal age 28.1 ± 4.8 28.9 ± 5.5 0.54 Duration of pregnancy 40 + 2 ± 1 + 0 40 + 2 ± 1 + 0 0.96 Number of previous pregnancies 1.35 ± 1.36 1.31 ± 1.42 0.70 Prepregnancy weight (kg) 67.1 ± 14.3 67.9 ± 13.0 0.53 GDM 16% 11% 0.46 Pre-eclampsia 0% 3% 0.33 Smoking 13% 11% 0.81 P-value is the difference between the groups calculated with Mann–Whitney U test for continuing variables, or Chi-Square test for categorical variables. GDM, gestational diabetes mellitus. Reference 1 Ervasti M, Kotisaari S, Heinonen S, Punnonen K. Use of advanced red blood cell and reticulocyte indices improves the accuracy in diagnosing iron deficiency in pregnant women at term. Eur J Haematol 2007; 79: 539– 45. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume80, Issue1January 2008Pages 92-92 ReferencesRelatedInformation

Use of advanced red blood cell and reticulocyte indices improves the accuracy in diagnosing iron deficiency in pregnant women at term

Detection of iron deficiency during pregnancy with hemoglobin (Hb) and serum measurements is insignificant as the measurements may be affected by e.g. hemodilution or accelerated erythropoiesis. This study tests whether cell indices will give a more reliable measure of iron deficiency in pregnant women at term. The population was 202 pregnant women. Using the ADVIA 120 hematology system, Hb, mean cell volume (MCV), percentage of hypochromic red blood cells (%HYPOm) and reticulocytes (%HYPOr), and cellular hemoglobin in reticulocytes (CHr) were tested. Additionally, transferrin saturation (TfSat), ferritin, and transferrin receptor (TfR) were analyzed. Receiver operating characteristic (ROC) curves and area under the ROC curves (AUC) were used as statistical methods. When TfSat (< or = 11%) was used as the reference test for iron deficiency, %HYPOm and CHr had a sensitivity of 58.1% and 80.7%, while the specificities were 82.6% and 71.3%, respectively. Additionally, the AUC values were %HYPOr 0.80, CHr 0.79, ferritin 0.77, %HYPOm 0.75, TfR 0.67, MCV 0.63 and Hb 0.64. The results provided by the cell indices alone (%HYPOm or CHr) were in good agreement with the results based on the usage of a combination of three commonly used tests (Hb, MCV, ferritin). This study suggests that the most practical way to diagnose iron deficiency in pregnant women at term is to use cell indices such as CHr and %HYPOm provided by the automated hematological analyzer. Further studies are needed to determine the usefulness of the cell indices in diagnosing iron deficiency longitudinally during the course of pregnancy.

Effect of Hydroxyurea on Immature Reticulocyte Fraction in Sickle Cell Anemia

Immature reticulocyte fraction (IRF) is a good indicator of bone marrow erythropoiesis in response to hemolysis or tissue hypoxia and is markedly increased in sickle cell disease (SS). We compared IRF changes in SS patients with those who were treated with hydroxyurea (SS-HU), and those who had concurrent alpha globin gene deletion (SS-(- deletion). Forty-two patients including 16 SS, 16 SS-HU, and 10 SS-alpha-deletion patients were studied. Significant decreases (P <.01) in reticulocyte indices including IRF, the reticulocyte percentage, and absolute reticulocyte count (ARC) were observed in SS-alpha-deletion compared to SS patients. On the other hand, although the reticulocyte percentage (P <.01) and ARC (P <.01) were significantly decreased in SS-HU compared with SS patients, the IRF was persistently elevated in both groups (P = .4), suggesting continuous bone marrow stimulation in SS-HU patients in response to tissue hypoxia. The possible underlying physiological mechanisms are discussed.

Diagnostic clues to megaloblastic anaemia without macrocytosis

Masking of the macrocytic expression of megaloblastic anaemia (MA) by coexisting thalassaemia, iron deficiency and chronic illness has been widely reported. We described the haematological and clinical features of 20 Chinese patients with MA presenting with mean corpuscular volume (MCV) < or =99 fl, and analysed the steps leading to the final diagnosis of MA with concomitant thalassaemia trait (n = 11), thalassaemia trait and iron deficiency (n = 3), iron deficiency (n = 4) and chronic illness (n = 2). We also compared the haematological characteristics of this group of patients with a group of normocytic anaemic patients without vitamin B(12)/folate deficiency, and identified certain laboratory information useful for differentiating the two groups. Statistically significant parameters included the mean values of haemoglobin, MCV, red cell distribution width (RDW), reticulocyte index, platelet count and serum bilirubin. All provided clues to maturation disorders within the marrow. A decision flowchart for the diagnosis of MA without macrocytosis was proposed. In the studied population, by using the parameters of haemoglobin <10 g/dl, MCV 80-99 fl, RDW > or = 16% and reticulocyte index < or = 2% as indicators, there was a 58% chance that a patient had MA without macrocytosis if he/she had all the four indicators, and a 2.2% chance of having it if he/she did not have these indicators. We emphasized the importance of including peripheral blood smear examination in the diagnostic procedures for such patients, as well as the importance of paying attention to patients' medical history, racial background and previous MCV value.

The relationship between red blood cell and reticulocyte indices and serum markers of iron status in the cord blood of newborns

The aims of this study were to assess the relationship between red blood cell and reticulocyte indices and biochemical iron status measurements, and to define reference values of these markers in the cord blood of newborns. In cord blood samples from 199 full-term newborns, cellular indices were assessed using an ADVIA 120 hematology system and iron status was analyzed by measurement of serum iron, transferrin, transferrin saturation (TfSat), transferrin receptor (TfR) and ferritin. Cellular hemoglobin in red blood cells or reticulocytes was independent of serum iron markers such as TfSat, TfR and ferritin. The percentage of hypochromic red blood cells (%HYPOm) and reticulocytes (%HYPOr) correlated significantly with TfSat and TfR-F index (TfR/log ferritin). Importantly, %HYPOm and %HYPOr were also positively correlated with the high immature reticulocyte fraction (IRF-H) and the mean cell volume of red or reticulocytes. In newborns, accelerated erythropoiesis is a major contributor to red blood cell and reticulocyte indices, which provide conflicting results when compared with serum markers of iron status. Apparently, the serum proteins ferritin, transferrin and TfR are more appropriate tools for the diagnosis of iron status in newborns.

Hematological Characteristics of Infants and Young Children Presenting with Different Levels of Malarial Anemia at a Rural District Hospital in Western Kenya.

In areas of intense P. falciparum exposure, such as western Kenya, the most lethal complication of malaria is severe malarial anemia (SMA). The pathogenesis of SMA is multi-factorial and not fully understood. Our ongoing studies are, therefore, investigating the hematological manifestations of MA in a cohort of children presenting at a rural district hospital in western Kenya. A total of 619 children aged 3–33 mos. were categorized into 5 groups: SMA (Hb<6.0g/dL); moderate MA (MdMA; 6.0≤Hb<8.0g/dL); mild MA (MlMA; 8.0≤Hb<11.0g/dL); uncomplicated malaria (UM, Hb≥11.0g/dL), and healthy aparasitemic controls (HC). Parasite density, CBC, and reticulocyte count was obtained on all subjects (see table).HC (n=62)UM (n=36)MlMA (n=139)MdMA (n=161)SMA (n=221)P valueaParasitemia (/μL)082471±1592551711±530051513±5798)62934±64160.107Hb (g/dL)1.81±0.1111.66±0.149.27±0.07*6.86±0.05*4.78±0.06*<0.001MCV (fL)71.87±0.7173.42±1.1268.57±0.63*69.25±0.76*71.34±0.670.001Reticulocyte count (%)1.57±0.201.64±0.242.55±0.16*4.21±0.29*5.50±0.31*<0.001RPI1.83±0.331.90±0.491.44±0.091.50±0.101.15±0.07*0.001Data are presented as mean±SEM. aDifferences between the groups were compared using the Kruskal-Wallis test. *Denotes p<0.05 (Mann-Whitney U test) relative to the HC group.Peripheral blood smears (n=150) were randomly selected from all groups for examination of RBC and WBC morphology, and identification of any clinically significant abnormalities, such as presence of hemozoin (Hz). The majority of the children with malaria had inadequate bone marrow responses (77.6%), as determined by the reticulocyte production index (RPI). Relative to the UM group, the SMA group had increased microcytosis (p<0.005). Moreover, numbers of nucleated RBC (nRBC) and Hz-laden monocytes correlated with increasing degrees of anemia (p=0.08 and p<0.005, respectively). In contrast, levels of parasitemia, prevalence of high-density parasitemia (≥10,000 parasites/ml) and reactive lymphocytes did not differ among the four malaria groups. No significant increases were observed in the number of schistocytes, acanthocytes or spherocytes in any of the groups. Macrocytosis was rarely observed and sickle cells were not detected. These findings illustrate that parasitemia is not associated with the severity of anemia in this population. Conversely, the number of Hz-containing monocytes was a strong predictor of anemia severity, suggesting that SMA results from more chronic malaria infections. Finally, although nRBC were most commonly associated with SMA, the RPI was not appropriately increased for the degree of anemia, illustrating that these children have suppressed and/or ineffective erythropoiesis.

Altered Immune Response in Severe Malaria Anemia in Children.

We prospectively assessed immune markers in children <6 years with severe malarial anemia (hemoglobin <5.0 g/dL; n = 72) and uncomplicated malaria (n = 69) who presented to Macha Mission Hospital in Zambia's Southern Province. We also studied 70 children <6 years who presented to well child clinics in Harare, Zimbabwe as controls. Compared to controls, children with uncomplicated malaria had significantly higher temperatures and parasite counts, lower hemoglobin and platelet concentrations, higher plasma levels of interferon-gamma, tumor necrosis factor alpha, and interleukin 10 and lower levels of monocyte inhibitory factor (MIF). Compared to uncomplicated malaria, severe malaria anemia was associated with younger age, longer duration of fever and lower temperature on admission. Reticulocyte index and serum concentrations of bilirubin and LDH did not differ between the malaria groups, suggesting that unusually severe extra- or intra-medullary hemolysis did not explain the severe anemia. Higher white blood cell and platelet counts in the severe malaria group suggested that pan-suppression of the marrow was also not the primary cause. Of originally selected measures of inflammation, plasma levels of TNF-alpha and MIF did not differ between the malaria groups, but concentrations of both interferon-gamma and interleukin-10 were significantly lower in the severe anemia group (P <0.006). Additional testing revealed levels of interleukin-1alpha, interleukin-6, and IP-10 to be lower and levels of sFAS to be higher in the children with severe anemia versus uncomplicated malaria (P <0.0005). In a logistic regression model, severe malarial anemia was associated with younger age (P = 0.010), prior treatment with sulfadoxine/pyrimethamine or traditional medicine (P <0.32), lower levels of Interleukin-10 (P = 0.025) and higher levels of sFAS (P = 0.003) and TNFa (P = 0.013). Our results are consistent with a multifactorial cause of severe malarial anemia, possibly including infection with resistant plasmodia, over-expression of TNF-alpha in conjunction with under-expression of IL-10, and increased apoptosis.

Successful Treatment of Pure Red Cell Aplasia (PRCA) Associated to Phenitoin without Blood Transfusions in a Jehovah's Witness Patient.

A 17 year-old Jehovah's Witness girl was admitted to the neurosurgery ward with a subarachnoid hemorrhage Fisher IV, Hunt and Hess II, due to an arteriography-confirmed aneurism of the left posterior comunicating artery (LPCA). CT scan showed hemorrhages of the left lateral, 3rd and 4th ventriculi. Her blood counts were normal. She underwent surgery and during the procedure she developed neurologic deterioration due to brain edema. The procedure was stopped and she was started on prophylactic Phenitoin. Her hemoglobin levels began to drop progressively until they reached 3.6 gr/dL with 0.8% reticulocytes while her platelet and WBC counts remained within normal ranges (278 and 5.9 × 109/L respectively) with a normal differential count. No bleeding site was found, and hemorrhage was discarded as cause of the anemia. The serum iron was 218, the IBC 213 and the saturation index 102.5 (37.3%), all within normal values. The bone marrow aspirate showed a slight increase of the cellularity with normal megakariocytes and granulocytes with slight eosinophilia and a total absence of the erythroid series. The lymphocytes were also normal. This image was consistent with a PRCA. The existence of a thymoma as well as several viral infections including Parvovirus, EBV, HIV, HB and HC were discarded as cause of the PRCA. Phenitoin was stopped and treatment with steroids, cyclosporine, mesterolone and erythropoietin 8,000 U/d SC was started. Despite the evidence of very deep anemia with dispnea and prostration, which required red cell transfusions, the patient and her parents absolutely refused them for religious reasons and the supportive treatment was enhanced. Her clinical status improved after 48 hours and the Hb began to increase progressively. She was discharged one month later with a Hb of 13.1 gr/dL, Ht 39.6%, with a corrected reticulocyte count of 1.8%, 6.2 × 109/L WBC with a normal differential count and 284 × 109/L BP. Even though no tests could be done to confirm phenitoin as the triggering factor of the PRCA, the temporal association between its use and the development of the anemia very strongly suggests a causal association even though it can not be assured. The results we had with erythropoietin, high-dose iron, oxygen and parenteral feeding were very rewarding. Jehovah's Witnesses patients with severe anemia like this one have a poor prognosis and pose a strong therapeutic challenge that may discourage the medical staff to continue a struggle that seems hopeless. We feel that by accepting it and sharing experiences like this one we can help these patients who will rather die than accept blood derivatives.

Reticulocyte indices in a canine model of nutritional iron deficiency

Reticulocyte indices, especially reticulocyte hemoglobin content (CH retic), have shown promise as markers of iron deficiency (ID), but there have been no prospective investigations of reticulocyte indices in experimental models of ID. The objective of this study was to compare reticulocyte indices with conventional hematologic and biochemical indices as markers of ID in dogs. Iron deficiency was induced in 7 dogs by feeding an iron-deficient diet, and corrected by restoring dietary iron and by giving iron parenterally. Blood samples were collected at weekly intervals. Results of hematologic and biochemical tests were compared using t-tests and receiver operator characteristic (ROC) curve analysis. Comparing mean values on days 0 and 35, by which time hemoglobin concentration decreased to 90% of baseline in all dogs, % Macro retic, % Hypo retic, % Low CH retic, and % High CH retic differed by greater than 3-fold, whereas no conventional hematologic or biochemical indices differed by as much as 2-fold. Comparing conventional hematologic and reticulocyte indices by ROC curve analysis using 4 different biochemical diagnostic criteria of ID, CH retic, % Hypo retic, % Low CH retic, and % High CH retic had higher areas under the curve (AUC) than either MCV conv or MCHC conv according to all 4 criteria, and % Macro retic and MCV retic had higher AUC values according to 3 of 4 criteria (differences were not always statistically significant). Results of this study support the value of reticulocyte indices in the diagnosis and monitoring of ID in dogs. Species similarities in the pathophysiology and hematologic manifestations of ID suggest these findings also may be relevant to ID in people.

Successful Treatment of Iron Overload with Phlebotomies in Two Siblings with Congenital Dyserythropoietic Anemia – Type II (CDA-II)

Successful treatment of iron overload by phlebotomies has been reported in two splenectomized siblings with congenital dyserythropoietic anemia--type II (CDA-II). In both patients 400 ml of blood were withdrawn every month. During three years 12 200 ml of blood were removed. The serum ferritin levels decreased from 1450,4 microg/L and 1131,7 microg/L to 447 microg/L and 457 microg/l, respectively. The transferrin saturation dropped from 0,99 at the start of the therapy to 0,64 and 0,86, respectively. The values of Hb, Hct, erythrocyte counts and MCV did not change as well as did not change reticulocyte counts, reticulocyte index, and RDW. Both patients tolerated repeated phlebotomies well. The decrease of bilirubin and normal values of haptoglobin might be the concequence of diminished destruction of erythrocytes and their precursors. Our observation confirms that phlebotomies can be used with success in CDA patients with mild anemia as treatment modality of iron overload.

Immature Reticulocyte Fraction after Iron Therapy for Iron Deficiency Anemia.

Background: Automated counting of reticulocyte introduced new reticulocyte parameters such as immature reticulocyte fraction(IRF). IRF is thought to be more sensitive than absolute or corrected reticulocyte count for detecting recovery of bone marrow function. The aim of this study is to assess the role of IRF for predicting the response of therapy after treatment of iron deficiency anemia.Methods: Patients with previously untreated iron deficiency anemia (blood hemoglobin < 10.0g/dL and serum ferritin < 10ng/mL) were enrolled into this study. On the 8th day of iron therapy, Complete blood count(CBC) with reticulocyte count and IRF was assessed by RAM-1 reticulocyte autoanalyzer(Sysmex. Tokyo, Japan). IRF was defined as sum of the reticulocyte fraction of high-fluorescence intensity regions plus the fraction of middle fluorescence intensity regions. After 1 month of iron therapy, CBC was reassessed to evaluate the response of iron therapy. The correlation of the 8th day IRF and the change of hemoglobin level after 4 weeks of therapy was evaluated. The 8th day corrected reticulcyte count was also evaluated.Results: From Jan. 2001 to May 2003, 27 patients with iron deficiency anemia (3 men and 24 women) entered into this study. We evaluated 21 patients, excluding 6 patients who were lost to follow-up or refused blood sampling after 1 month of iron therapy. The range of pretreatment hemoglobin level was 3.3–9.7g/dL (median 7.5/dL). The 8th day IRF was 0.06–0.39(median 0.15). Ten patients had the low IRF (IRF ≤ 0.15). Four of them (40%) had their hemoglobin level increased by more than 3g/dL. On the other side, 11 patients had high IRF (IRF > 0.15) and 10 of them (91%) had their hemoglobin level increased by more than 3 g/dL. This difference was significant (P=0.024). However, the 8th day IRF was not correlated with the change of hemoglobin as strongly as the 8th day corrected reticulocyte count (Pearson coefficient 0.420 vs 0.693).Conclusion: For the patients with iron deficiency anemia, the 8th day of treatment IRF correlates with the response of iron treatment, but the 8th day of treatment corrected reticulocyte count has a stronger correlation.

Study of Uncoupling Protein 2 (UCP2) Function during Erythroid Differentiation and Maturation.

Mitochondrial oxidative stress is thought to play a key role in sideroblastic anemia and the myelodysplastic syndrome. Potential sources of reactive radicals reside in the heme biosynthetic pathway involving the import and production of pro-oxidant agents, such as ALA and iron and in the respiratory chain. Antioxidant mechanisms are, therefore, expected to be an integral function in erythroid differentiation and their impairment is expected to affect hemoglobinization and maturation. The mitochondrial uncoupling proteins have been shown to reduce oxidative stress through the generation of proton leak across the inner membrane of the mitochondria. They have been implicated in a wide range of physiological and pathological states, including obesity, diabetes, aging neurodegenerative, and immunological diseases.Here we report that UCP2 is induced during erythroid differentiation and that UCP2 deficient mice have a delayed recovery from anemia.We hypothesized that erythroid heme biosynthesis is accompanied by oxidative stress, which results in the induction of UCP2, and that UCP2 plays a role in erythroid maturation by preventing oxidative stress and damage.We found that UCP2 transcripts and protein are induced following the activation of GATA-1 in G1ER cells and during DMSO, butyrate and heme -induced differentiation of murine erythroleukemic (MEL) and K652 erythroid cell lines. Similarly, differentiation of primary mouse c-kit+ / Ter119− erythroid progenitors to Ter119+ is accompanied by induction of UCP2 transcripts. To test the functional significance of UCP2 in erythroid differentiation we studied a UCP2 null mouse. Peripheral blood analysis from UCP2 KO mice revealed a mild elevation of the reticulocyte index as compared to wild type (WT C57BL/6J) mice, which may be related to mild anemia. To test the role of UCP2 in recovery from anemia, we treated WT and UCP2 KO mice with phenylhydrazine for 3 days and studied erythropoiesis using FACS analysis of Ter119 and CD71 surface markers in cells isolated from bone marrow. Stimulation of erythropoiesis was more rapid in the WT mice as compared to the UCP2 KO. The delay in the mutant is more pronounced at the stage of the proerythroblast and is also reflected in the peripheral blood where a higher level of reticulocytes was transiently observed. By 9 days the UCP2 KO mice peripheral blood count was identical to the WT. Analysis of oxidative damage confirmed that UCP2 acts to reduce oxidative damage of mitochondrial proteins. The delayed reticulocytosis could not however be explained by cell death or by reduced hemoglobinization. The increased oxidative damage present in the UCP2 null cells during erythroid differentiation and maturation did not result in the stimulation of apoptosis as revealed by identical Annexin V staining profile of UCP2 KO and WT mice. Remarkably, iron incorporation and hemoglobin content assays ruled out a function of UCP2 in the process of heme biosynthesis per-se. We therefore conclude that UCP2 deficiency regulates maturation in the erythroid lineage independent of the heme biosynthetic pathway.