DIAGNOSTIC ACCURACY OF SERUM FERRITIN AND SOLUBLE SERUM TRANSFERRIN RECEPTOR, TAKING BONE MARROW IRON STAIN AS A GOLD STANDARD FOR IRON DEFICIENCY ANEMIA IN HETEROGENOUS GROUP OF PATIENTS

Objective: To determine the diagnostic accuracy of serum ferritin and soluble serum transferrin receptor (sTfR), taking bone marrow iron stain as a gold standard for iron deficiency anaemia in heterogeneous group of patients. Study Design: Cross-sectional diagnostic accuracy study. Place and Duration of Study: Department of Diagnostic, Combined Military Hospital Lahore, from Mar to Aug 2020. Methodology: A total of 55 adult patients, of both genders, undergoing bone marrow examination for any reason were enrolled. Patients with known hemolytic condition (sickle cell anemia, megaloblastic anemia), taking erythropoietin/iron supplements, transfused red cell concentrate (RCC) recently or undergoing chemotherapy were excluded. Age, gender, clinical history and results of bone marrow examination, complete blood count (CBC), serum Ferritin and C-reactive protein (CRP) were recorded. Results: Serum ferritin was found to be less sensitive (28%) but more specific (100%) for reflecting reduced bone marrow iron stores as compared to sTfR (sensitivity: 60%, specificity: 96.6%). sTfR had highest likelihood ratio (15) and diagnostic accuracy (80%). On Receiver Operator Characteristic (ROC) graph Transferrin index (AUC=0.908) showed maximum accuracy, followed by Ferritin (AUC=0.884) and sTfR (AUC=0.879). Conclusion: Serum soluble transferring receptor (sTfR) and transferrin index has advantage over serum ferritin alone in predicting the bone marrow iron stores and differentiating iron deficiency anemia from anemia of chronic disease.

Increased Bone Marrow Iron at Diagnosis Is Associated with Inferior Prognosis in Patients with Myelodysplastic Syndromes

Introduction: Iron storage in patients (pts) with myelodysplastic syndromes at the time of diagnosis may vary from normal to iron overload. Even before the first blood transfusion, storage iron can be increased due to down-regulation of hepcidin and subsequent increase in duodenal iron uptake. Iron overload is known to worsen the prognosis of MDS patients, partly due to iron-related organ damage after long-term transfusion therapy, and partly due to an increased risk of infections. However, it is unclear whether increased storage iron at the time of diagnosis already has a prognostic influence. We assessed bone marrow iron stores at the time of MDS diagnosis and correlated them with clinical outcome. Methods: In a retrospective analysis of 3762 adult MDS patients from the Düsseldorf MDS Registry, Prussian blue staining of marrow smears was performed in our cytology lab to assess iron stores according to the following categories: normal or decreased iron stores versus increased iron stores versus iron overload. Patients were followed up for survival and AML evolution until June 2021. Median time of follow-up was 20 months. 67.4% of the patients died during the course of the disease. Results: The study included 3.762 adult patients who received their initial diagnosis of MDS between 1970 and 2021. 58% were diagnosed as non-blastic MDS ( MDS SLD (RS) (n=240), MDS MLD (RS) (n=350), MDSdel(5q) (n=107), and MDS-U (n=25). Iron stores were decreased in 8% of the patients, normal in 44%, increased in 41%, and strongly increased in 7% (massive iron overload). In 282 cases, histologic assessment of storage iron was available. When comparing cytologic and histologic assessment, we found a strong correlation (p<0.0005), since 87% of the patients with increased iron on cytomorphology also showed increased iron as assessed by histopathology. However, 37% of the patients who cytologically showed normal iron stores, were reported to have slightly increased iron as assessed by histopathology. Median and mean serum ferritin values of patients with normal or decreased iron stores were 295 and 629 µg/l, respectively, as compared to 548 and 902 µg/l, respectively, in patients with increased iron stores. The cumulative risk of AML evolution was not associated with the results of iron staining. Regarding survival, we found that patients with decreased or normal storage iron had a median survival of 31 months, whereas those with increased iron had a median survival of 28 months (p=0.007). Focusing on patients with non-blastic MDS, the difference was not significant (46 vs 44 ms). However, patients who presented as EB I (n=435), EBII (n=510), AML MRC (n=264), CMML I (n=254), or CMML II (n=77), showed a prognostic impact of storage iron; patients with increased iron had a median survival of 11 months, as compared to 16 months in patients with normal or decreased iron (p<0.0005). Conclusion: Increased tissue iron in the bone marrow at the time of diagnosis is associated with inferior survival in patients with MDS, primarily in patients with higher risk MDS. At diagnosis, patients are not yet transfusion-dependent. This suggests that increased iron reflects a prolonged period of increased duodenal iron uptake as a consequence of ineffective erythropoiesis. Therefore, increased marrow iron at the time of MDS diagnosis seems to be a surrogate parameter of hematopoietic insufficiency, which is the real cause of inferior prognosis. Disclosures Nachtkamp: Jazz: Honoraria; Bsh medical: Honoraria; Celgene: Other: Travel Support. Gattermann: Novartis: Honoraria; Takeda: Research Funding; Celgene: Honoraria. Germing: Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding; Janssen: Honoraria; Novartis: Honoraria, Research Funding.

FC 075POST-MORTEM HEPATIC AND BONE MARROW IRON CONTENT IN HEMODIALYSIS PATIENTS: A PROSPECTIVE COHORT STUDY

Background and Aims Studies using T2 MRI liver scans among Hemodialysis (HD) patients raised concern about the presence of iron overload in this population, regularly treated with intravenous (IV) iron. Histological evidence of tissue iron overload is scarce, since the majority of studies were performed in pre-erythropoiesis- stimulating agents (ESA’s) era, when blood transfusions were common. Primary objective: to quantify iron in the liver and bone marrow by biochemical and histological analysis, in adult CKD stage 5-HD. Secondary objectives: To explore association of clinical, laboratorial parameters, IV iron therapy and iron stores. Method After approval of local Ethical committee and informed consent from families, liver biopsy and bone marrow aspirate were obtained in the first 24h post-mortem from 21 chronic HD patients with anemia or under anemia treatment who died in Hospital Fernando Fonseca. Exclusion criteria: blood transfusion in the previous 2 weeks, acute or chronic liver disease, HIV infection, known hematologic or oncologic disease. Clinical, laboratorial and anemia therapy data were retrieved from hospital registry and outpatient HD centers. Biochemical liver iron content (LIC) was quantified by atomic absorption spectrophotometry. Histological semi-quantitative grading of iron storage was made in the liver and bone marrow using Scheuer’s and Gale’s criteria of grading Perls’ stain, respectively. Results Of 21 patients included, 10 (47,6%) were male, median (IQR) age 76.0 (67.5-85.5) years old, 18 (85.7%) white, 3 (14.3%) black, dialysis vintage was 47.0 (12.5-104.0) months. Charlson Comorbidity index was 10.0 (7.5-11.0), 7 (33%) patients had diabetes, and 11 (52.4%) used an arteriovenous fistula as vascular access. The cause of death was infection (n=9, 42.9%), cardiovascular (N=6, 28.6%), HD withdrawal (n=2, 9.5%) and unknown =3 (14.3%). Median (IQR) hemoglobin was 9.8 (8.5-11.4) g/dl and 11 (52.3%) patients had hemoglobin <10 g/dl. Ferritin was 494.0 (136.0-850.5) ng/ml and TSAT 19.9 % (13.3-26.0). 19 (90.5%) patients were receiving IV iron therapy. Median (IQR) IV iron administered in the previous 6 and 12 months before death was 800 (300-1250) mg and 1500 (650-2175) mg, respectively. All patients were on ESA therapy, median (IQR) dose 5000 (3000-9000) UI/week and erythropoietin resistance index was 9.6 (4.2-16.6). Median (IQR) liver iron content determined by atomic absorption was 42.5 (22.9-69.7) µmol/g. 9 patients (42.9%) had normal LIC (<36 μmol/g), while the remainder had mild to moderate overload. Median (IQR) Scheuer grade was 2 (1-3) and 13 (62%) of liver biopsies had increased (Scheuer grade > 1) iron deposition at histology. Median (IQR) grade of Perls staining in the bone marrow was 3 (3-4) and 9 (45%) had increased (Gale’s grade >3) iron content in the bone marrow. Iron semi-quantitative scores in liver and bone marrow had strong positive correlation (r=0.71, p<0.001). There was a strong positive correlation between LIC and ferritin (r=0.86, p < 0.001) and also TSAT (r=0.56, n=16, p=0.02). Hemoglobin was negatively associated with LIC (r= -0.46, p=0.04), and with iron content in the bone marrow (p=0.04). LIC did not associate with ESA dose, C-reactive protein, dialysis vintage or other clinical parameters. There was no statistically significant association between the dose of IV iron administered in the previous 6 and 12 months with LIC, ferritin,TSAT or iron scores in bone marrow and liver. Conclusion In these HD patients, there was biochemical and histological evidence of iron accumulation in liver and bone marrow. Ferritin and TSAT showed strong correlation with iron deposits, but none was found with the dose of IV iron administered. In this study, anemia severity was associated with higher degree of iron storage both in the liver and bone marrow, suggesting a multilevel blocking mechanism of iron’s utilization.

P0861THE RELATIONSHIP BETWEEN HEPCIDIN-25 AND BONE MARROW IRON IN ANEMIC, NON-DIALYSIS, CHRONIC KIDNEY DISEASE PATIENTS

Background and Aims Hepcidin-25 (Hep25) is a key known regulator of iron metabolism and its interactions with inflammation, iron stores and erythropoietic activity were involved in the pathogenesis of chronic kidney disease (CKD)-associated anemia. Therefore, our aim was to assess the determinants of serum Hep25 level in non-dialysis CKD patients. Method In this cross-sectional, single-center study, 52 subjects (56% men, 65±13 years) with CKD [estimated glomerular filtration rate, eGFR 14.5 (95%CI 16 to 25) mL/min] and anemia [hemoglobin, Hb 9.8 (95%CI 9.2 to 9.9) g/dL], not treated with erythropoiesis-stimulating agents (ESA) or iron in the previous 6 months, were enrolled. Patients with anemia of other causes than CKD, active infectious and inflammatory diseases, malignancy, severe hyperparathyroidism, transfusions during the last 3 months, and immunosuppressive therapy were excluded. The iron status was evaluated using both peripheral and central parameters. The iron stores were assessed by serum ferritin (Fer) and iron content in bone marrow macrophages (iMf, measured quantitively on a scale from 0 to 6). The iron available for erythropoiesis was assessed by transferrin saturation (TSAT) and the percentage of sideroblasts (%Sb). Anemia was further evaluated by a peripheral blood smear, erythrocytes indices and reticulocyte index. Serum Hep25 and erythropoietin (Epo) were assessed by ELISA (Bachem®, and Abcam® 119522, respectively). C-reactive protein (CRP), albumin, and parameters of kidney disease (eGFR, proteinuria) were also measured. Mann-Whitney, Kruskal-Wallis, Chi2 tests, Spearman bivariate correlation and multiple linear regression were used for statistical analysis. Results The median serum Hep25 of the whole cohort was 82.1 (95%CI 68.7 to 92.1) ng/mL. According to median Hep25, subjects were clustered in Group 1 (below median - G1) and Group 2 (above median - G2). %Sb and reticulocyte index had higher levels in G2 than in G1 [9 (95%CI 5 to 14) vs. 5 (95%CI 4 to 7) %, p=0.003 and 0.55 (95%CI 0.39 to 0.77) vs. 0.41 (95%CI 0.32 to 0.58), p=0.05, respectively], while the proportions of subjects with iMf suggestive for iron deficiency or iron overload were similar in G2 and G1 (38% vs. 50%, p=0.40, and 26% vs. 23%, p= 0.75, respectively). Peripheral blood smear, peripheral iron indices and all the other studied parameters were also alike. In bivariate analysis, Hep25 was positively associated both with indices of iron stores, i.e. Fer (rs = 0.30, p=0.03) and iMf (rs = 0.34, p=0.01) and indices of iron available for erythropoiesis, i.e. %Sb (rs = 0.55, p<0.001) and (marginally) with TSAT (rs = 0.26, p=0.06). Meanwhile, Hep25 was not related to serum Epo, CKD parameters or inflammation markers. In a multivariate linear regression model that explained 28% of Hep25 variation, the percentage of bone marrow sideroblasts, i.e. the tissue iron available for erythropoiesis, was the only independent determinant of Hep25: Variables entered in the first step: reticulocyte index, percentage of medullary sideroblasts (%Sb), iron content in the bone marrow macrophages (iMf), serum ferritin, and transferrin saturation Conclusion In stable patients with advanced CKD, not treated with ESA or iron, with low to moderate inflammation, serum hepcidin was related only to bone marrow iron available for erythropoiesis, suggesting that in this clinical setting the need of iron for erythropoiesis prevails over inflammation in regulation of hepcidin synthesis.

Disbalanced Erythroid Ferroportin Expression Contributes to Ineffective Erythroid Output in Anemia of Chronic Disease

Iron is essential for proper red blood cell development. During erythroid maturation in the bone marrow iron dependency starts at the basophilic stage, and lasts until the reticulocyte stage. Thereby the majority of iron is delivered in a Tf-TfR dependent manner and is incorporated in hemoglobin of developing red cells. Just recently, it has been discovered that erythroblasts and mature red blood cells not only incorporate iron but also express the sole known iron exporter, ferroportin (Fpn), throughout their development, and thus can also effectively export iron that is not used for heme synthesis. Fpn surface expression is regulated via hepcidin, a hepatocyte-derived peptide, which binds directly to surface-Fpn, inducing its internalization and consequent degradation in lysosomes. Among patients suffering from anemia of chronic disease (ACD), hepcidin is constantly induced due to long-lasting inflammation, leading to impaired iron export capacity. While splenic tissue iron overload, which is associated with the restricted capacity of red pulp macrophages to export recycled iron form degraded erythrocytes is well established, the effect of high hepcidin levels in ACD and its consequences on developing erythroblasts in the bone marrow has not been investigated. First we induced chronic kidney disease (CKD) in C57BL/6 mice via an Adenine - diet. Alongside microcytic anemia, reduced Tf-saturation, increased hepcidin levels and splenic tissue iron overload, we found a ~38% increase in tissue iron content in bone marrows of CKD mice compared to control mice. In parallel, Western blot analysis revealed massively reduced Fpn protein levels in the bone marrow. Moreover, the individual iron-dependent erythroblast precursor populations (i.e. basophilic, polychromatic, orthochromatic erythroblasts and reticulocytes) showed higher levels of intracellular iron as measured by Calcein fluorescence via flow cytometry. We could further corroborate these results by Western blot analysis and flow cytometric work-up of reticulocytes and mature red blood cells in the blood stream, both revealing highly reduced Fpn protein levels on these cells in mice suffering from CKD. Based on these findings we performed additional experiments to investigate the detrimental effect of iron overload on erythroid development and to exclude the possibility of a direct inflammation-regulated process in the CKD model. Therefor we established an iron-overload mouse model via repeated parenteral iron dextran applications. Despite significantly increased Tf-saturation levels as well as hepatic hepcidin levels (>4-fold) and reduced bone marrow Fpn protein levels among iron-treated mice, iron overload led to higher stress levels among erythroid precursor populations. In detail, we could demonstrate via flow cytometry that higher intracellular iron pools (measured by Calcein), correlated with higher levels of mitochondrial stress and higher levels of lipid peroxidation (determined by mean fluorescence intensity of MitoSOX and Bodipy 581/59, respectively). These data indicate that iron is a critical regulator of stress during erythroid development and can be regulated via the hepcidin-Fpn axis. In conclusion, we clearly show that Fpn expression on erythroid precursor cells is inversely regulated to systemic hepcidin levels and affects the erythropoietic bone marrow iron content. Moreover our results reveal a novel model for ineffective erythroid output in patients suffering from ACD due to hepcidin-triggered Fpn internalistation. Based on our data, anti-hepcidin treatment strategies are promising to overcome restricted erythropoiesis, which will be evaluated in future experiments. Disclosures Theurl: Kymab Ltd: Consultancy, Equity Ownership.

Hepcidin and conventional markers to detect iron deficiency in severely anaemic HIV-infected patients in Malawi

IntroductionIron deficiency is a treatable cause of severe anaemia in low-and-middle-income-countries (LMIC). Diagnosing it remains challenging as peripheral blood markers poorly reflect bone-marrow iron deficiency (BM-ID), especially in the context of HIV-infection.MethodsSevere anaemic (haemoglobin ≤70g/l) HIV-infected adults were recruited at Queen Elizabeth Central Hospital, Blantyre, Malawi. BM-ID was evaluated. Accuracy of blood markers including hepcidin alongside mean corpuscular volume, mean cellular haemoglobin concentration, serum iron, serum ferritin, soluble transferrin receptor (sTfR), sTfR -index, sTfR–ratio to detect BM-ID was valued by ROC area under the curve (AUCROC).ResultsSeventy-three patients were enrolled and 35 (48.0%) had BM-ID. Hepcidin and MCV performed best; AUCROCof 0.593 and 0.545. Other markers performed poorly (ROC<0.5). The AUCROCof hepcidin in males was 0.767 (sensitivity 80%, specificity 78%) and in women 0.490 (sensitivity 60%, specificity 61%).ConclusionBM-ID deficiency was common in severely anaemic HIV-infected patients and is an important and potential treatable contributor to severe anaemia. Hepcidin was the best, though still suboptimal, marker of BM-ID. Hepcidin, which is directly linked to iron absorption, is a very promising marker to guide curative iron supplementation policies in severely anaemic HIV-infected patients.

Anaemia and iron deficiency in chronic heart failure patients

Tis review focused on prevalence of anemia and iron defciency (ID) in CHF and their effect on the course and prognosis of this condition. Based on evaluation of numerous laboratory data defnitions of anemia and ID were suggested. Specifcally, a diagnostic value of measuring serum iron, serum ferritin, transferrin saturation, total iron-binding capacity, and concentration of soluble transferrin receptors was discussed. Te review highlighted the importance of measuring bone marrow iron, which is rarely used in everyday clinical practice even though this test is considered a «gold standard» of ID diagnosis. Te review provided an insight into pathogenetic mechanisms of ID in CHF including insufcient iron supply, role of inflammation, erythropoietin, RAS, and effects of some pharmacological therapies. Te authors described physiological consequences of ID and anemia, activation of hemodynamic and non-hemodynamic compensatory mechanisms, which develop in response to anemia and not infrequently aggravate CHF. Special atention was paid to current approaches to treatment of anemia and ID in CHF, including a discussion of efcacy and safety of oral and intravenous dosage forms of iron and hemopoiesis stimulators.

Perl's Stain Grade in the Bone Marrow Aspirate Correlates with Overall Survival in Low Risk Myelodysplastic Patients

Background Myelodysplastic syndromes (MDS) are heterogeneous group of acquired clonal hematopoietic stem cell disorders characterized by atypical stem cells maturation and genetic instability leading to an enhanced risk of progression to acute myeloid leukemia. Low risk MDS patients have a lower probability to evolve in leukemia but are commonly characterized by dyseritropoiesis. These patient are incline to long term accumulation of iron in the organs due mostly to red blood cell transfusion (RBC) but iron overload may also occur in MDS patients who do not receive RBC transfusions due to the ineffective erythropoiesis. It is well known the effect of oxidant-mediated tissue's injury through the formation of free toxic iron species in the liver and heart site, but recent knowledges assumes that this mechanism is effective also in the bone marrow nice, where oxidative stress seems to impaired the haematopoietic stem cells growth. At this moment microscopic examination of the stainable iron in the bone marrow is considered the gold standard for determining the iron stores. The effect of bone marrow's iron overload on overall survival in the low risk MDS has been a matter of unresolved debate. We aimed to investigate the predictive value of bone marrow iron accumulation as demonstrated by Perl's staining on outcome in such patients. Design We retrospectively analyzed all low risk,intermediate-I MDS patients who had diagnosed in our institution in the last 20 years (since 1998). Diagnosis of MDS was made according to WHO criteria. Patients were stratified based on International Prognostic Scoring System (IPSS). Patients had undergone bone marrow aspiration as part of the diagnostic work up for their MDS. Two different experienced hematologist analyzed all samples. Bone marrow aspiration slides with at least seven fragments were considered suitable. Perl's Prussian blue stain was used to stain bone marrow, assessed by modified Gale's grading (Tab. 1) and then correlated with outcome. Patients and methods Marrow staining of one hundred and fourteen consecutive MDS patients were revised and analyzed. Median age was 70 years (range 32-93). Eighty three patients were IPSS low- risk and 30 Intermediate I. All patients were evaluated for bone marrow iron stores with Perl's stain. Twenty-seven patients had grade 1 (+), 31 grade 2 (++) and 56 grade 3 (+++). Patients had never or minimally received RBC . None of these patient had received iron chelation before marrow examination. Probability of overall survival (OS) was estimated by the Kaplan-Meier method and the significance was assessed by the log-rank test. Results 20-year OS was significantly lower in patients with higher Perl's score (median = 80 ±7 months in grade 3; median = 70 ±17 months in grade 2; median = 144 ±18 months in grade 1 , P=0.011); Fig. 1 Conclusions We evaluated retrospectively the bone marrow aspirate from 114 consecutive new MDS low-risk, Intermediate-I IPSS patients with Perl's stain for iron detection. Although Perl's grading is a qualitative method, it is still the gold standard to detect iron storage in the bone marrow. Our results correlate Perl's stain at diagnosis with long term outcome in MDS patients. We show how higher grade of iron storage at diagnosis can impact on outcome in these patients. We conclude that Perl's stain, together with Ferritin and blood transfusional burden could be another marker at diagnosis of iron-related toxicity that predict overall survival. Disclosures Pilo: Novartis Italy: Honoraria. Angelucci:Jazz Pharmaceuticals Italy: Other: Local ( national) advisory board; Vertex Pharmaceuticals Incorporated (MA) and CRISPR CAS9 Therapeutics AG (CH): Other: Chair DMC; Roche Italy: Other: Local (national) advisory board; Novartis: Honoraria, Other: Chair Steering Comiittee TELESTO Protocol; Celgene: Honoraria, Other: Chair DMC.