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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 13  |  Issue : 1  |  Page : 28-34

Role of serum hepcidin and reticulocyte hemoglobin concentration in evaluation of anemia in ulcerative colitis patients


1 Department of Internal Medicine, Faculty of Medicine, Tanta University, Tanta, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt

Date of Submission02-Sep-2021
Date of Decision21-Nov-2021
Date of Acceptance13-Dec-2021
Date of Web Publication28-Apr-2022

Correspondence Address:
Dr. Samar Reda Ammar
Elsanta, Elgharbia, Postal Code: 31511, Tanta
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/joah.joah_127_21

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  Abstract 


CONTEXT: One of the most common extra-intestinal signs of ulcerative colitis (UC) disease is anemia, which has a significant influence on patients' quality of life.
AIM: The aim was to evaluate the role of serum hepcidin and reticulocyte hemoglobin concentration (CHr) in the study of anemia in UC patients.
SUBJECTS AND METHODS: We recruited 80 UC patients and 30 healthy individuals of matched age and sex as controls. Subjects were subdivided into three groups – Group I: 50 anemic UC patients, Group II: 30 nonanemic UC patients, and Group III: 30 healthy controls.
RESULTS: CHr showed a statistically highly significant decline in Group I than Groups II and III. Serum hepcidin showed a significant difference between Groups I, II, and III. We reported a significant negative correlation between CHr and severity of UC and extension of UC and a significant positive correlation between CHr and hemoglobin level, mean corpuscular volume (MCV), serum ferritin, and transferrin saturation. While, serum hepcidin had a significant negative correlation with severity and extension of UC and a significant positive correlation with hemoglobin level, MCV, serum ferritin, transferrin saturation, and CHr.
CONCLUSIONS: CHr had an excellent performance in prediction of iron-restricted anemia and was the test of best performance in prediction of iron-deficiency anemia ± ACD. Serum hepcidin had an excellent performance in prediction of ACD.

Keywords: Anemia, hepcidin, reticulocyte hemoglobin concentration, ulcerative colitis


How to cite this article:
Ammar SR, Ghazy MA, Mabrouk MM, Gawaly AM. Role of serum hepcidin and reticulocyte hemoglobin concentration in evaluation of anemia in ulcerative colitis patients. J Appl Hematol 2022;13:28-34

How to cite this URL:
Ammar SR, Ghazy MA, Mabrouk MM, Gawaly AM. Role of serum hepcidin and reticulocyte hemoglobin concentration in evaluation of anemia in ulcerative colitis patients. J Appl Hematol [serial online] 2022 [cited 2022 Jun 25];13:28-34. Available from: https://www.jahjournal.org/text.asp?2022/13/1/28/344255




  Introduction Top


One of the most common extra-intestinal manifestations of ulcerative colitis (UC) disease is anemia, with a significant influence on patients' quality of life.[1],[2],[3] It is a good example for iron-restricted anemia (IRA), either by true or functional iron deficiency (FID) that is associated with Anemia of chronic disease (ACD).[4] Other causes include cobalamin deficiency, folate deficiency, and drug-induced anemia.[5],[6]

Hepcidin is a small antimicrobial peptide secreted from the liver that has dual value in host defense and iron homeostasis.[7]

The reticulocyte hemoglobin concentration (CHr) represents the availability of iron for hemoglobin production in the bone marrow.[8]


  Subjects and Methods Top


We studied 80 UC patients and 30 healthy individuals of matched age and sex as controls recruited from outpatient clinic and inwards of the Internal Medicine Department of Tanta University Hospital from January 2019 to June 2020.

Studied subjects were subdivided into three groups: Group I: 50 anemic UC patients (according to the WHO, anemia was defined by hemoglobin level <13 g/dL for males and <12 g/dL for nonpregnant females,[2] Group II: 30 nonanemic UC patients, and Group III: 30 healthy controls.

The exclusion criteria were as follows: patients with malignancy, systemic infection, chronic kidney disease, chronic liver disease, and patients with serious cardiopulmonary diseases.

All patients and controls were subjected to:

  1. Full history taking
  2. Full clinical examination
  3. Laboratory investigations: Complete blood count (CBC)–iron profile (serum ferritin–transferrin saturation)–serum hepcidin level–CHr.


Blood sampling

Eight milliliters of venous blood was collected from each subject. Two centimeters was put in ethylenediaminetetraacetic acid tube for CBC and CHr and 2 cm was put in vacuum ESR tube. The rest of sample was allowed to clot for half an hour in water bath at 37°C and then it was centrifuged for 15 min at 3000 r.p.m for separation of serum by means of clean dry tubes for liver function, renal function, C-reactive protein, and hepcidin.

Hepcidin enzyme-linked immunosorbent assay

To assess the level of human hepcidin (Hepc) in samples, the kit employs a double-antibody sandwich enzyme-linked immunosorbent assay. Incubate hepcidin (Hepc) is a monoclonal antibody enzyme well that has been precoated with human hepcidin (Hepc) monoclonal antibody; then add hepcidin (Hepc) antibodies labeled with biotin and combined with streptavidin-horseradish peroxidase to form immune complex; then incubate and wash to remove uncombined enzyme. When you add chromogen solution A, B, the color of the liquid turns blue, and when you add acid, the color turns yellow. The concentration of the human substance hepcidin (Hepc) in the sample and the chroma of color were positively correlated.

Reticulocyte hemoglobin concentration

Using automated hematology analyzers produced by Siemens (Advia2120) approved by Food and Drug Administration in 1997, CHr is measured during reticulocyte analysis. CHr is calculated using light scatter measurements taken from two distinct angles after isovolumetric sphering of oxazine 750-stained reticulocytes. The amount of light dispersed at two distinct angles is used to determine the volume and hemoglobin concentration of individual reticulocytes. The cellular volume multiplied by the cellular hemoglobin concentration equals CHr. As a result, the hemoglobin concentration rises and the cell volume falls.

Statistical analysis

Analyses were done using SPSS version 21 (SPSS Inc.company, united states, chicago). Quantitative data were analyzed by mean ± standard deviation, median, range, and interquartile range and evaluated Mann–Whitney U-test (in case of two groups of nonparametric variables) or Kruskal–Wallis (in case of more than two groups of nonparametric variables). Categorical data were presented by number and percent and assessed by the Chi-square test, and when it was inappropriate, it was replaced by Monte Carlo exact test. Spearman correlation was done for the linear relation between nonparametric variables. Receiver operating characteristic (ROC) curve was done for agreement of sensitivity and specificity of the markers. The P value was considered significant at the level of ≤0.05.


  Results Top


Group I was subdivided into Group Ia –33 patients (66%) with true iron-deficiency anemia (IDA) as ferritin level <30 ng/ml, Group Ib – 5 patients (10%) with mixed true and FID (ACD/ID) that was defined as ferritin level 30–100 ng/ml, and Group Ic –11 patients (22%) with FID caused by anemia of chronic disease (ACD) that was defined as ferritin >100 ng/ml. There was one patient (2%) with megaloblastic anemia.

Comparison between all groups showed an insignificant difference in age and gender [Table 1].
Table 1: Demographic data of the studied groups

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Group I shows a statistically highly significant higher severity and more extended disease than Group II. The comparison between Groups Ia, Ib, and Ic was statistically insignificant [Table 2].
Table 2: Disease severity and extension of ulcerative colitis

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Regarding hemoglobin, there was a statistically highly significant decrease in Group I than Groups II and III and there was a statistically significant decrease in Group Ia than Groups Ib and Ic. Severe anemia in IDA patients can be explained by more severe and extended disease found in this group leading to more blood loss and anemia [Table 3].
Table 3: Complete blood count of the studied groups

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Regarding mean corpuscular volume (MCV), there was a statistically highly significant decline in Group I than Groups II and III (P1 < 0.001) and there was a statistically insignificant difference between Group Ia, Group Ib, and Group Ic (P3 = 0.13) [Table 3].

Regarding platelet count, we reported a statistically highly significant increase in Group I than Groups II and III (P1 < 0.001) and a statistically significant increase in Group Ia than Groups Ib and Ic (P3 < 0.004). Increased platelet count in IDA patients may be due to reactive thrombocytosis associated with IDA [Table 3].

Regarding total leukocyte count, there was a statistically insignificant difference between all groups (P1 = 0.09, P2 = 0.928, and P3 = 0.616) [Table 3].

Regarding serum ferritin, we noticed a statistically highly significant difference between Group I and Group II and Group III (P1 < 0.001) and there was a statistically highly significant decrease in Group Ia than Group Ib than Group Ic (P3 < 0.001) [Table 4].
Table 4: Serum ferritin level and transferrin saturation percentage of studied groups

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Regarding transferrin saturation, there was a statistically highly significant decrease in Group I than Groups II and III (P1 < 0.001) and there was a statistically highly significant decrease in Group Ia than Group Ib than Group Ic (P3 < 0.001) [Table 4].

Regarding CHr, there was a statistically highly significant decline in Group I than Groups II and III (P1 < 0.001), there was a statistically highly significant decrease in Group I than Group II (P2 < 0.001), and there was a statistically highly significant decrease in Group Ia than Group Ib than Group Ic (P3 < 0.001) [Table 5].
Table 5: Reticulocyte hemoglobin concentration and serum hepcidin level of studied groups

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Regarding serum hepcidin, there was a statistically significant difference between Groups I, II, and III (P1 < 0.023), there was a statistically significant difference between Group I and Group II (P2 < 0.004), and there was a statistically highly significant decline in Group Ia than Group Ib than Group Ic (P3 < 0.001) [Table 5].

CHr showed a highly significant negative correlation with severity and extension of UC and a highly significant positive correlation with hemoglobin level, MCV, serum ferritin, and transferrin saturation [Table 6].
Table 6: Correlation between reticulocyte hemoglobin concentration and serum hepcidin level with other parameters

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Serum hepcidin showed a significant negative correlation with severity and extension and a highly significant positive correlation with hemoglobin level, MCV, serum ferritin, transferrin saturation, and CHr [Table 6].

At cutoff value ≤27.7 pg of CHr to predict IRA, the sensitivity was 90%, specificity was 79.6%, area under the curve (AUC) was 0.914, and P value was <0.001 [Figure 1].
Figure 1: ROC curve for Serum ferritin, Transferrin saturation, CHr, Hepcidin for diagnosis ulcerative colitis with IRA

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At cutoff value ≤24.2 pg of CHr to predict pure IDA, the sensitivity was 94.7%, specificity was 90.9%, AUC was 0.989, and P value was <0.001 [Figure 2].
Figure 2: ROC curve of serum ferritin, CHr, serum hepcidin to predict IDA

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At cutoff value ≤24.85 pg of CHr to predict IDA ± ACD, the sensitivity was 100%, specificity was 94.7%, AUC was 0.999, and P value was <0.001 [Figure 3].
Figure 3: ROC curve of serum ferritin, CHr, serum hepcidin to predict IDA± ACD (IDA-ACD/ID)

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At cutoff value ≥255.05 ng/ml of serum hepcidin to predict ACD, the sensitivity was 100%, specificity was 86.7%, AUC was 0.960, and P value was <0.001 [Figure 4].
Figure 4: ROC curve of serum ferritin, CHr, serum hepcidin to predict ACD

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  Discussion Top


While treating anemia in UC patients, it is critical to determine the type of anemia and the degree of ID and inflammation in each patient, in order to decide treatment in an evidence-based manner. Traditional markers (serum iron, ferritin, transferrin saturation, and soluble transferrin receptor) have some limitations.[6]

Reticulocyte indices including CHr and hepcidin are promising markers that can help in solving the issue of evaluation of anemia in inflammatory bowel disease patients.

IDA is the most frequent type of anemia in UC (up to 90% of patients) followed by anemia of chronic illness (prevalence: 11%–42%). Other uncommon causes include Vitamin B12, folic acid deficiency, hemolysis, and drug-related anemia.[9]

We studied 50 anemic UC patients, and among them, 33 patients (66%) had true iron IDA, 5 patients (10%) had mixed true and FID (ACD/ID), 11 patients (22%) had FID (ACD), and there was only one patient (2%) with megaloblastic anemia. In line with our results, Stadnicki et al.[2] recruited 74 UC patients and 19 healthy individuals as controls. Anemia was found in 22 UC patients, which constitutes 30% of enrolled patients. Twelve patients (54.5%) had IDA, 7 patients (31.8%) had ACD, and in 3 patients (13.7%), both types of anemia coexisted (IDA/ACD).

Comparison between all groups showed an insignificant difference in age and gender, and this is also in agreement with Stadnicki et al.[2]

The risk of anemia in UC (blood loss and ACD) is related to the disease activity and extension.[2] The previous study reported that Group I showed a statistically highly significant higher severity and more extended disease than Group II. The comparison between Groups Ia, Ib, and Ic was statistically insignificant.

We reported more severe anemia in Group Ia that can be explained by more severe and extended disease found in this group leading to more blood loss and anemia. In agreement with these results, Stadnicki et al.[2] reported a statistically highly significant decline in Hb level in the IDA group than ACD group, nonanemic group, and control group (P < 0.001, 0.001, and 0.001). However, in Urrechaga et al.,[10] it was found that the median of Hb was 10.8 g/dl in the IDA group, 10.9 g/dl in the ACD group, and 11.6 g/dl in the ACD/ID group, and there was a statistically insignificant difference between groups (P = 0.128).

In the previous study, it was reported that CHr had a statistically highly significant decrease in Group I than Groups II and III (P1 < 0.001), a statistically highly significant decrease in Group I than Group II (P2 < 0.001), and a statistically highly significant decrease in Group Ia than Group Ib than Group Ic (P3 < 0.001). This is because CHr depends on the amount of iron available for erythropoiesis and Group I is associated with IRA, and IDA cases are accompanied by depleted iron store. In partial accordance with these results, Torino et al.[11] showed that the median of CHr was 25.2 pg in the IDA group, 29.7 pg in the ACD group, and 26 pg in the ACD/ID group. There was an insignificant difference between the IDA and ACD/ID groups. However, a statistically highly significant decline in the ACD/ID group than ACD group was reported (P < 0.001).

Regarding serum hepcidin, it showed a statistically significant difference between Groups I, II, and III (P1 < 0.023) with a statistically highly significant decrease in Group Ia than Group Ib than Group Ic (P3 < 0.001). This is due to effect of iron status and inflammation on hepcidin levels as IDA leads to inhibition of hepcidin production and inflammation associated with ACD leads to upregulation of hepcidin. In agreement with our results, Cheng et al.[12] reported a statistically significant level between the IDA, ACD, and ACD/ID groups (P = 0.01).

In line with our results, Karagülle et al.[13] who evaluated the clinical importance of reticulocyte hemoglobin content in the study of IDA noticed a significant positive correlation between CHr and hemoglobin (r = 0.775), mean corpuscular hemoglobin (MCH) (r = 0.883), serum iron (r = 0.648), MCV (r = 0.868), MCH concentration (r = 0.685), and transferrin saturation (r = 0.764) while there was a significant negative correlation with transferrin (r = −0.599) and total iron-binding capacity (r = −0.613).

Interestingly, the previous results were partially in agreement with Singh et al.[14] who reported a positive correlation between CHr and serum ferritin in the IDA group (P = 0.04) and in the ACD/ID group (P = 0.002). Furthermore, in the ACD group, CHr was positively correlated with SI and transferrin saturation (P = 0.02). However, CHr had a weak correlation with severity of anemia.

In agreement with our results, Mecklenburg et al.[15] reported only a significant correlation between serum ferritin levels and serum hepcidin (Spearman's rho = 0.491; P < 0.001) with insignificant correlation between hepcidin and the inflammatory markers.

Moreover, Paköz et al.[16] concluded that hepcidin levels were not correlated either with the disease activity indexes or with the inflammatory markers.

We found that serum hepcidin had an excellent performance in prediction of ACD. CHr had an excellent performance in prediction of IRA and was the test of best performance in prediction of IDA ± ACD.

In the study performed by Mast et al.,[17] it was reported that using Prussian blue staining of the bone marrow aspirate to diagnose iron deficiency, CHr of <28 pg had a sensitivity (74%) and specificity (73%).

Furthermore, Urrechaga et al.[10] reported that using ROC analysis of CHr for detecting ID at cutoff (30.3 pg), the sensitivity and specificity were 82.2%and 83.3%, respectively, while for ferritin sensitivity 75.5% and specificity 72.2% at cutoff (77 g/L).

Furthermore, Singh et al.[14] showed that CHr at the best cutoff 23.95 pg in IDA patients had a sensitivity of 96.6% and specificity of 85.7%. While, in ACD, CHr at the best cutoff value of 26.2 pg had a sensitivity of 85.7% and specificity of 81.8%.

In addition, CHr was considered the most accurate screening test for diagnosing ID with or without anemia as reported by Mateos et al.[18] who found that CHr at cutoff value of 25 pg had a sensitivity of 94% and specificity of 80%.

In agreement with these results, Chikwanda et al.[19] reported that in IDA, ferritin at ≤30 ng/mL cutoff value had the most accurate performance with 96.4% sensitivity and 73.8% specificity whereas hepcidin at ≤5 ng/mL cutoff value had the worst performance with 89.3% sensitivity and 34.2% specificity. However, in ACD, hepcidin showed the most valuable performance with 95.2% sensitivity and 63.8% specificity compared to ferritin.


  Conclusions Top


CHr and hepcidin can be considered valuable markers in evaluation of iron status in UC patients. Serum hepcidin had an excellent performance in prediction of ACD. CHr had an excellent performance in prediction of IRA and was the test of best performance in prediction of IDA ± ACD, so it can solve the problem of detection of iron depletion in the presence or absence of inflammation which is considered the main issue in evaluation of anemia in UC.

Ethical policy and Institutional Review Board statement

Approval from the Institutional Ethics Committee was taken.

Patient declaration of consent statement

written consent was taken from each participant after explaining the aim and procedures of the study.

Data availability statement

The data set used in the current study is available on request from Ammar Samar R. ([email protected]).

Reporting guidelines

The manuscript adheres to the STROBE reporting guidelines (for cross-sectional studies).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Silverberg MS, Satsangi J, Ahmad T, Arnott ID, Bernstein CN, Brant SR, et al. Toward an integrated clinical, molecular and serological classification of inflammatory bowel disease: Report of a Working Party of the 2005 Montreal World Congress of Gastroenterology. Can J Gastroenterol 2005;19 Suppl A:5A-36A.  Back to cited text no. 1
    
2.
Stadnicki A, Bojko B, Maciążek-Jurczyk M, Klimacka-Nawrot E, Korzonek-Szlacheta I. Differentiation of anemia in patients with ulcerative colitis. SciTZGastroenterol 2016;1:1002.  Back to cited text no. 2
    
3.
Gasche C, Lomer MC, Cavill I, Weiss G. Iron, anaemia, and inflammatory bowel diseases. Gut 2004;53:1190-7.  Back to cited text no. 3
    
4.
Dignass AU, Gasche C, Bettenworth D, Birgegård G, Danese S, Gisbert JP, et al. European consensus on the diagnosis and management of iron deficiency and anaemia in inflammatory bowel diseases. J Crohns Colitis 2015;9:211-22.  Back to cited text no. 4
    
5.
Weiss G, Gasche C. Pathogenesis and treatment of anemia in inflammatory bowel disease. Haematologica 2010;95:175-8.  Back to cited text no. 5
    
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Oustamanolakis P, Koutroubakis IE, Messaritakis I, Kefalogiannis G, Niniraki M, Kouroumalis EA. Measurement of reticulocyte and red blood cell indices in the evaluation of anemia in inflammatory bowel disease. J Crohns Colitis 2011;5:295-300.  Back to cited text no. 6
    
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Oustamanolakis P, Koutroubakis IE, Kouroumalis EA. Diagnosing anemia in inflammatory bowel disease: Beyond the established markers. J Crohns Colitis 2011;5:381-91.  Back to cited text no. 7
    
8.
Agarwal MB, Pai S. Reticulocyte hemoglobin content (CHr): The gold standard for diagnosing iron deficiency. J Assoc Physicians India 2017;65:11-2.  Back to cited text no. 8
    
9.
Bergamaschi G, Di Sabatino A, Albertini R, Ardizzone S, Biancheri P, Bonetti E, et al. Prevalence and pathogenesis of anemia in inflammatory bowel disease. Influence of anti-tumor necrosis factor-alpha treatment. Haematologica 2010;95:199-205.  Back to cited text no. 9
    
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Urrechaga E, Hoffmann JJ, Bernal A, Arévalo JA, Cabriada JL. Reticulocyte hemoglobin content (MCHr) in the assessment of iron deficient erythropoiesis in inflammatory bowel disease. Dig Liver Dis 2018;50:1178-82.  Back to cited text no. 10
    
11.
Torino AB, Gilberti MD, Costa ED, Lima GA, Grotto HZ. Evaluation of erythrocyte and reticulocyte parameters as indicative of iron deficiency in patients with anemia of chronic disease. Rev Bras Hematol Hemoter 2015;37:77-81.  Back to cited text no. 11
    
12.
Cheng PP, Jiao XY, Wang XH, Lin JH, Cai YM. Hepcidin expression in anemia of chronic disease and concomitant iron-deficiency anemia. Clin Exp Med 2011;11:33-42.  Back to cited text no. 12
    
13.
Karagülle M, Gündüz E, Sahin Mutlu F, Olga Akay M. Clinical significance of reticulocyte hemoglobin content in the diagnosis of iron deficiency anemia. Turk J Haematol 2013;30:153-6.  Back to cited text no. 13
    
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Singh BG, Jain N, Chaturvedi V, Patel J, Kotwal J. Evaluation of reticulocyte hemoglobin for assessment of anemia in rheumatological disorders. Int J Rheum Dis 2019;22:815-25.  Back to cited text no. 14
    
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Mecklenburg I, Reznik D, Fasler-Kan E, Drewe J, Beglinger C, Hruz P, et al. Serum hepcidin concentrations correlate with ferritin in patients with inflammatory bowel disease. J Crohns Colitis 2014;8:1392-7.  Back to cited text no. 15
    
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Paköz ZB, Çekiç C, Arabul M, Sarıtaş Yüksel E, İpek S, Vatansever S, et al. An evaluation of the correlation between hepcidin serum levels and disease activity in inflammatory bowel disease. Gastroenterol Res Pract 2015;2015:810942.  Back to cited text no. 16
    
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Mast AE, Blinder MA, Lu Q, Flax S, Dietzen DJ. Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron deficiency. Blood 2002;99:1489-91.  Back to cited text no. 17
    
18.
Mateos ME, De-la-Cruz J, López-Laso E, Valdés MD, Nogales A. Reticulocyte hemoglobin content for the diagnosis of iron deficiency. J Pediatr Hematol Oncol 2008;30:539-42.  Back to cited text no. 18
    
19.
Chikwanda E, Daka V, Simakando M, Kowa S, Kaile T. Evaluation of hepcidin as a biomarker for the differential diagnosis of iron deficiency anaemia and anaemia of chronic disease. Asian J Med Sci 2018;9:15-20.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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