Dysregulated Neutrophil Iron Homeostasis in β-Thalassemia Impairs Phagocytosis and Reactive Oxygen Species Production

Introduction Beta thalassemia is an inherited disorder characterised by ineffective erythropoiesis leading to anemia and secondary iron overload. Neutrophils are the first line of innate immune defence against infection is dysfunctional in thalassemia patients. Iron is required for the oxidative response of neutrophils to allow the production of reactive oxygen species (ROS). However, the role of iron contributing to the neutrophil dysfunction is unclear. This is the first study to characterise the neutrophil iron metabolism in β-thalassemia and its association with oxidative burst capacity, phagocytosis, and systemic iron homeostasis. Method Sixteen thalassemia patients and fourteen healthy individuals were recruited in the department of Haematology, Christian Medical College, Vellore, India. Neutrophils were purified from human whole blood collected in EDTA tube, using neutrophil magnetic isolation kit (Miltenyi Biotec). Purified population (>95%) was confirmed by the presence of the surface marker CD62L evaluated using flow cytometry. Haematological parameters were analysed according to standard methods. Serum ferritin, iron, soluble transferrin receptor were measured using immunoassay. Serum hepcidin was measured using ELISA. Neutrophil RNA was isolated using trizol method and was reverse transcribed into complementary DNA using QIAGEN kit. The relative quantification of iron related genes were measured using real-time PCR. In oxidative burst assay, neutrophils were incubated with dihydrorhodamine 123 (DHR) and stimulated with Phorbol 12-Myristate 13-Acetate (PMA). Respiratory burst of the cell was analysed by flow cytometry. Phagocytosis and acidification capacity of human neutrophils were quantified using the pHrodo Green Staphylococcus aureus BioParticles kit (Thermo Fisher). Acquisition was performed using the Beckman Coulter(Navios) flow cytometer and analysed using kaluza software. Statistical analysis was performed using SPSS software. Results We investigated a cohort of β thalassemia Major (n=5), intermedia (n=6) and sickle beta thalassemia (n=5) patients who were on regular iron chelation therapy. The demographic and biochemical parameters are tabulated in Table 1. Serum iron, ferritin levels and transferrin saturation were significantly increased in thalassemia cohort as compared to healthy donors (Fig1a). There was no significant association between ferritin and hepcidin levels. The percentage of neutrophils in thalassemia was significantly reduced incomparision to healthy donors (p=0.032). Oxidative burst capacity of neutrophils from thalassemia major and intermedia patients were significantly decreased (p=0.002) compared to healthy donors upon stimulation with PMA (Fig1b). Neutrophil phagocytosis capacity indicated by the relative amounts of phagocytized fluorescein S.aureus particles were significantly lower in thalassemia patients compared with controls, after 15min/30min incubation (Fig1c). The recognition capacity of neutrophils towards bioparticles significantly decreased at 45-minute incubation in patients (p=0.017) (Fig1d). Serum iron overload and transferrin saturation had negative association with neutrophil phagocytosis capacity (r=-0.714; p=0.045 & r=-0.857; p=0.014), respectively. Neutrophil iron related gene expression was analysed and found significantly lower expression of FPN 1A (FPN1 containing iron regulatory element (IRE)), DMT1B without IRE region and IRP2 respectively (p=0.029, p=0.029 & p=0.016) (Fig1e). FPN1B without IRE region was upregulated in thalassemia patients (p=0.016). Serum ferritin had positive correlation with FPN1B (r=0.786; p=0.036). Soluble transferrin receptor had negative association with DMT1A (containing IRE region) and IRP2 respectively (r=-0.900; p=0.037 and r=-0.750; p=0.052). Aberrant neutrophil function was found in all thalassemia patients. Although oxidative burst activity was decreased in thalassemia major and intermedia patients, sickle β-thalassemia patients had normal burst activity. Systemic iron status had inverse correlation with phagocytosis capacity of neutrophils. Dysregulation of iron transporters in neutrophils was indicated by decreased expression of DMT1 and augmented FPN1B expression, despite systemic iron overload. These findings have to be explored in a larger cohort to elucidate the clinical significance. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

SOLUBLE TRANSFERRIN RECEPTOR IS A PROMISING MARKER of IRON DEFICIENCY ANEMIA IN PREVALENT HEMODIALYSIS PATIENTS

Background Diagnosis of iron deficiency is traditionally based on ferritin and other iron parameters becomes difficult in end stage renal disease patients due to the inflammatory condition which affects these markers and masks the iron deficiency. Serum soluble transferrin receptor (sTfR) is able to be a reliable indicator for assessing iron status, as it is not affected by inflammatory procedures. Aim To evaluate the usefulness of serum soluble transferrin receptors in iron deficiency anemia detection in comparison to the classic markers of iron status in prevalent hemodialysis patients. Methods This case-control study assessed sTfR in 80 prevalent ESRD patients on regular hemodialysis in 2 groups. Group A (N = 40): CRP >10 and group B (N = 40):CRP <10 and apparently healthy 8 control subjects. Results The cut of value of STFRs in hemodialysis patients was 12.5 mg\l. The prevalence of STFRs in patients with CRP<10 was 85%, while in patients with CRP>10 was 92.5% (P-value 0.288). STFRs have high sensitivity 88.75, specificity 100, PPV100% and NPV 47.1%. The hemodialysis patients who have elevated STFRs have risk 1.22 times to have iron deficiency anemia if CRP <10 (odds ratio: 1.22) and 3.14 times if CRP>10 (odds ratio: 3.14). There was significant difference on comparing patients with CRP<10, CRP>10 and control as regard Hb and STFR with P-value 0.0001 and 0.0001 respectively. Post Hoc analysis showed significant difference in both between the patients with CRP<10 and control also in patients with CRP>10 and control (p value <0.0001). while on comparing patients with CRP<10 with patients with CRP>10 there was significant difference in STFRs p value 0.0001 despite no significant difference in hemoglobin (p value 0.642) and classic marker of iron deficiency (s.iron, TIBC, TSAT) with p value 0.701,0.192,0.382 respectively. Serum STFRs was negatively correlated with s.iron and Kt\v (r -0.372, P-value 0.018) and (r-0.416, p value 0.008) respectively in patients with CRP <10. Conclusion Serum soluble transferrin receptor is highly sensitive and specific marker for iron deficiency in hemodialysis patients especially in patients with high CRP level.

Determinants of Anaemia Among Women of Reproductive Age In South Africa: A Healthy Life Trajectories Initiative (HeLTI)

Background – Anaemia continues to be a major public health problem among women of reproductive age (WRA). A thorough understanding of anaemia risk factors is necessary to design better interventions. This paper examines the determinants of anaemia among WRA in South Africa.Methods- We included baseline data from 480 women participating in the pilot-phase of a randomized controlled trial (HeLTI). We measured haemoglobin (Hb) status using the Hemocue. Plasma iron status markers (ferritin and soluble transferrin receptor (sTfR)), markers of inflammation (C-reactive protein (CRP)) and alpha-1-acid glycoprotein (AGP)) and retinol binding protein (RBP) were assessed using the multiplex method. We used multivariate logistic regression to describe associations with anaemia and structural equation modelling (SEM) to characterise direct and indirect pathways influencing haemoglobin concentrations.Results- The prevalence of anaemia, iron deficiency (ID), and iron deficiency anaemia (IDA) was 39.4%, 38.1% and 21.6% respectively. The multiple logistic regression showed that ID (OR: 2.62, 95% CI: 1.72, 3.98), iron deficiency erythropoiesis (IDE) (OR: 1.62, 95% CI: 1.07, 2.46), and elevated CRP (OR: 1.69, 95% CI: 1.04, 2.76), increased the odds of being anaemic. SEM analysis revealed Hb was directly and positively associated with adjusted ferritin (0.0031 per mg/dl; p≤0.001), and CRP (0.015 per mg/dl; p≤0.05), and directly and negatively associated with soluble transferrin receptor sTfR (-0.042 per mg/dl; p≤0.001). While contraception use had both a direct (0.34; p≤0.05) and indirect (0.11; p≤0.01) positive association with Hb. Additionally, chicken and beef consumption had a positive indirect association with Hb concentrations (0.15; p≤0.05) through adjusted ferritin.Conclusion-A key driver of anaemia in our setting is ID, however the presence of inflammation also increases the risk of anaemia. To address anaemia, interventions should aim to improve the diet quality of women, in particular access to iron rich foods. We recommend the use of multi-micronutrient supplements with a lower dose of iron and other micronutrients which would ensure that women receive the same benefits as with iron folic acid, while alleviating anaemia of inflammation.

Continuous Veno-Venous Hemofiltration (CVVH) Improves Iron Metabolism Disorders in Patients with Sepsis：A Single-Center Prospective Cohort Study

Background: Iron metabolism disorder is commonly seen in patients with sepsis. This study aimed to evaluate whether continuous veno-venous hemofiltration (CVVH) improved the iron metabolism disorders in sepsis. Methods: In a single-center, retrospective cohort study, totally 89 sepsis patients were prospectively enrolled and divided into the CVVH group (n=39) and the control group (n=50). Clinical and laboratory data were collected and compared between the groups on days 1, 3 and 7 of ICU admission. Plasma interleukin (IL)-6, hepcidin, erythropoietin (EPO), ferritin and soluble transferrin receptor (sTfR) were determined by enzyme linked immunosorbent assay (ELISA). Sequential organ failure scores (SOFA) on days 1 and 7, and 28-day survival between groups were compared. Results: Plasma IL-6, hepcidin, ferritin and RDW on days 3 and 7 were significantly reduced in the CVVH group compared with those in the control group (all P<0.05). The CVVH group had a significantly lower SOFA score on day 7 compared with the control group (P<0.05). Hemoglobin and EPO were gradually decreased within the first week of ICU admission in both groups although no significant differences between the groups were observed. There was no significant difference in sTfR between the two groups along with the time (all P > 0.05). In addition, there were no significant differences in 28-day survival rate and median survival time between the two groups. Conclusions: CVVH improves iron metabolism disorders and the disease severity in sepsis. However, it does not alleviate anemia and fails to improve the survival.

Effect of Fortification with Multiple Micronutrient Powder on the Prevention and Treatment of Iron Deficiency and Anaemia in Brazilian Children: A Randomized Clinical Trial

Fortification with multiple micronutrient powder has been proposed as a public health intervention able to reduce micronutrient deficiencies in children. Our objective was to compare the effectiveness of fortification with multiple micronutrient powder with drug supplementation in the prevention and treatment of iron deficiency and anaemia. This was a cluster trial with anemic and non-anaemic children between six and 42 months old, in randomization data. Non anaemic children received fortification with multiple micronutrient powder or standard drug supplementation of ferrous sulfate associated with folic acid in a prevention dose. Anaemic children who were randomized to receive multiple micronutrient powder also received the recommended iron complementation for anaemia treatment. A total of 162 children were evaluated. The prevalence of anaemia decreased from 13.58 to 1.85%. Iron deficiency decreased from 21.74% to 7.89% (by serum ferritin) and iron deficiency decreased from 66.81 to 38.27% (by soluble transferrin receptor). No difference was identified between interventions for hemoglobin (p = 0.142), serum ferritin (p = 0.288), and soluble transferrin receptor (p = 0.156). Fortification with multiple micronutrient powder was effective in preventing iron deficiency and anaemia in children aged six to 48 months. In anaemic children; it was necessary to supplement the dose of multiple micronutrient powder with ferrous sulfate.

Soluble Transferrin Receptor and Soluble Transferrin Receptor/Log Ferritin Ratio are Correlated with Iron Status in Regular Hemodialysis Patients

BACKGROUND: Monitoring of iron status in chronic kidney disease patients is important, however inflammation may hinder its monitoring. Soluble transferrin receptor (sTfR) is an alternative parameter to overcome this issue, whereas ferritin play a part in the inflammation process. Hence, the correlation between the sTfR ratio and the sTfR/log ferritin ratio with conventional iron status parameters in regular hemodialysis patients is necessary to be evaluated.METHODS: A cross-sectional was conducted in the current study. As many as 5 mL of blood (2 mL for sTfR and 3 mL for serum iron and ferritin levels) was collected. sTfR level was the blood-soluble transferrin receptor level measured by the enzyme-linked immunosorbent assay (ELISA). The amount of ferritin and serum iron was determined using the immunochemiluminescent process. To evaluate the correlation, the Pearson correlation test was used.RESULTS: A total of 80 subjects was included in this study. The mean of hemoglobin was 10.25±1.66 g/dL, serum iron was 58.19±26.56 g/dL, and the median ferritin was 520.4 (49.9-3606) ng/mL. The sTfR was significantly associated only with serum iron levels with a correlation coefficient of r=-0.242; p=0.031. The sTfR/log ferritin was significantly associated with serum iron l evels (InSI)(r=-0.255, p=0.022); and transferrin saturation (r=-0.295; p=0.008).CONCLUSION: sTfR/log ferritin has a negative and significant correlation with serum iron levels and transferrin saturation, while sTfR negatively correlated with serum iron levels. sTfR and sTfR/log ferritin may be considered as an alternative iron marker in inflammation setting such as CKD.KEYWORDS: sTfR/log ferritin, iron status, serum iron, ferritin, chronic kidney disease, hemodialysis

Serum soluble transferrin receptor levels are independently associated with homeostasis model assessment of insulin resistance in adolescent girls

IntroductionMarkers of iron homeostasis are related to insulin resistance (IR) in adults. However, studies in children and adolescents are scarce and show contradictory results. The aim of this study was to evaluate the potential relationship between iron status markers and IR. Additionally, no previous study has explored the simultaneous effect of biomarkers of iron homeostasis and inflammation [i.e. high sensitivity C-reactive protein, (hsCRP)], and adipokines [i.e. retinol-binding protein 4 (RBP4)] on IR in the cohort of late adolescent girls.Material and methodsA total of 60 girls age between 16-19 years encompassed the study. Serum levels of ferritin, transferrin, soluble transferrin receptor (sTfR), hsCRP, and RBP4 were measured by immunonephelometry. Homeostasis model assessment of insulin resistance (HOMA-IR) and iron homeostasis indexes were calculated. Univariate and multivariate binary logistic regression analysis were used to investigate the possible independent associations of the examined biomarkers. Principal component analysis was used to examine its mutual effect on HOMA-IR in studied girls.ResultsFerritin, sTfR, hsCRP and RBP4 were significant predictors for higher HOMA-IR in univariate analysis (p=0.020, p=0.009, p=0.007, p=0.003, respectively). Multivariate regression analysis after adjustment for waist circumference (WC) showed that serum sTfR levels remained positively associated with higher HOMA-IR (p=0.044). Factorial analysis revealed that Obesity-Inflammation related factor (i.e., WC and hsCRP) and Adipokine-Acute phase proteins related factor (i.e., RBP4 and ferritin) showed significant difference between HOMA-IR <2.5 and HOMA-IR ≥2.5.ConclusionsSerum sTfR levels are independently associated with HOMA-IR, whereas higher serum ferritin levels together with higher RBP4 are related to higher HOMA-IR in adolescent girls.

MO789SERUM SOLUBLE TRANSFERRIN RECEPTOR IS A PROMISING MARKER OF IRON DEFICIENCY ANEMIA IN PREVALENT HEMODIALYSIS PATIENTS

Background and Aims End stage renal disease (ESRD) is chronic inflammatory condition which affects iron parameters. Serum soluble transferrin receptor (sTfR) is a reliable indicator for assessing iron status in inflammatory conditions. This study evaluates the usefulness of serum sTfR in iron deficiency anemia detection in prevalent hemodialysis patients. Method This case-control study included 40 ESRD patients on conventional hemodialysis with CRP&gt;10, 40 ESRD patients with CRP&lt;10 and 8 apparently healthy controls. Serum sTfR was measured for all patients and controls. Results STFRs predicts iron deficiency anemia in prevalent hemodialysis patients at cut off value 12.5 mg/l with area under curve 0.949, sensitivity 88.75, specificity 100, PPV 100% and NPV 47.1%. The prevalence of STFRs in patients with CRP&lt;10 was 85%, while in patients with CRP&gt;10 was 92.5% (P-value 0.288). Patients who have elevated STFRs have risk 1.22 times to have iron deficiency anemia if CRP &lt;10 (odds ratio: 1.22) and 3.14 times if CRP&gt;10 (odds ratio: 3.14). There was significant difference on comparing patients with CRP&lt;10, CRP&gt;10 and control as regard haemoglobin and STFR with P-value 0.0001 and 0.0001 respectively. Post Hoc analysis showed significant difference between the patients with CRP&lt;10 and control also in patients with CRP&gt;10 and control as regard haemoglobin and STFR (p value &lt;0.0001). on comparing patients with CRP&lt;10 with patients with CRP&gt;10 there was significant difference in STFRs p value 0.0001 despite no significant difference in haemoglobin (p value 0.642) and classic iron markers (s.iron, TIBC, TSAT) with p value 0.701, 0.192, 0.382 respectively. Serum STFRs was negatively correlated with s.iron and Kt\v in patients with CRP &lt;10 (r -0.372, P-value 0.018) and (r-0.416, p value 0.008) respectively. Conclusion Serum soluble transferrin receptor is a highly sensitive and specific marker of iron deficiency anemia in hemodialysis patients especially with high CRP level.

Hepcidin regulation in Kenyan children with severe malaria and non-typhoidal Salmonella bacteremia

Background: Severe malaria and invasive non-typhoidal Salmonella (NTS) are life-threatening infections that often co-exist in African children. The iron-regulatory hormone hepcidin is highly upregulated during malaria and controls the availability of iron, a critical nutrient for bacterial growth, within the Salmonella-containing vacuole. Methods: We first investigated the relationship between Plasmodium falciparum malaria and NTS bacteremia in all pediatric admissions aged ≤5 years between August 1998 and October 2019 (n=75,015). We then assayed hepcidin and measures of iron status in five groups: (1) children with concomitant severe malaria anemia (SMA) and NTS (SMA+NTS, n=16); and in matched children with (2) SMA alone (n=33); (3) NTS alone (n=33); (4) cerebral malaria (CM, n=34); and (5) community-based children. Results: In hospitalized children SMA, but not other malaria phenotypes, was associated with an increased risk of NTS (adjusted OR 2.88 [95% CI 1.97, 4.23]; P<0.0001). Risk of NTS increased by 30% with each 1g/dl decrease in hemoglobin concentrations. In hospitalized children median hepcidin levels were lower in the SMA+NTS (9.3 ng/mL [interquartile range 4.7, 49.8]) and SMA (31.1 ng/mL [5.5, 61.2]) groups, compared to levels in those with CM (90.7 ng/mL [38.7, 176.1]) or NTS (105.8 ng/mL [17.3, 233.3]), despite similar ferritin and CRP levels. Soluble transferrin receptor levels were lower in the CM group compared to the other hospitalized groups. Conclusion: SMA was associated with increased risk of NTS and with reduced hepcidin levels. We hypothesized that reduced hepcidin might allow increased movement of iron into the Salmonella-containing vacuole favoring bacterial growth.