PLOS ONE RESEARCH ARTICLE Defining an optimal cut-off point for reticulocyte hemoglobin as a marker for iron deficiency anemia: An ROC analysis Marah Alzu’bi ID1, Hisham Bawa’neh2, Alaa Alshorman2,3, Jawad Alrawabdeh ID1, Nada Odeh1, Yazan Hamadneh ID1, Mai AlAdwan ID1, Mahmoud Odeh1, Abdalla Awidi ID1,2,4* 1 Medical School, University of Jordan, Amman, Jordan, 2 Al-Basheer Hospital, Ministry of Health, Amman, Jordan, 3 Jordan University Hospital, Amman, Jordan, 4 Cell Therapy Center, University of Jordan, Amman, Jordan * aabbadi@ju.edu.jo, Abdalla.awidi@gmail.com a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Alzu’bi M, Bawa’neh H, Alshorman A, Alrawabdeh J, Odeh N, Hamadneh Y, et al. (2023) Defining an optimal cut-off point for reticulocyte hemoglobin as a marker for iron deficiency anemia: An ROC analysis. PLoS ONE 18(7): e0288505. https://doi.org/10.1371/journal.pone.0288505 Editor: Jeffrey Chalmers, The Ohio State University, UNITED STATES Received: December 6, 2022 Abstract Reticulocyte hemoglobin (CHr) is a measure of the amount of hemoglobin in reticulocytes and a marker of cell hemoglobinization. In this study, we aimed to find the optimal cut-off point for reticulocyte hemoglobin to diagnose iron deficiency anemia using multiple methods. A total of 309 patients were included. The median age at diagnosis was 54 years. Most were females (71.2%). 68% had iron deficiency anemia. Patients with IDA had significantly lower levels of CHr compared to those who had non-IDA (p < 0.0001). The optimal cut-off value of CHr for detecting IDA, determined using various methods, was 30.15 pg. This cutoff point had a sensitivity of 87.8% and a specificity of 77.7%. CHr showed a significant positive correlation with hemoglobin, mean corpuscular volume, serum iron, serum ferritin, and transferrin saturation and a significant negative correlation with total iron-binding capacity. CHr levels correlate with most established laboratory tests for IDA. It reliably detects IDA. Our results indicate the importance of CHr in diagnosing IDA, and that CHr should be used more widely in suspected cases of IDA since it is a cheap, fast, and reliable test. Accepted: June 28, 2023 Published: July 13, 2023 Copyright: © 2023 Alzu’bi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Introduction Iron deficiency anemia (IDA) is the most prevalent anemia globally, affecting more than two billion people worldwide. IDA may be caused by poor nutrition with insufficient dietary intake of iron or may be caused by iron loss due mostly to bleeding, or may be caused by increased demands or malabsorption. It has a spectrum of symptoms ranging from fatigue, nausea, headaches, pallor, pagophagia, geophagia, restless leg syndrome to disability. Due to the high prevalence of IDA, early screening can reduce the economic, social, and human costs of IDA [1]. Ferritin is a globular protein responsible for the intracellular storage and transport of iron [2]. Additionally, ferritin acts as a buffer against iron deficiency and iron overload [3]. Accordingly, ferritin is considered the best indicator for iron deficiency in adults [4], and serum ferritin combined with blood hemoglobin (Hb) level, soluble transferrin receptor, and transferrin saturation are the most used tests to detect IDA and iron deficiency[5]. PLOS ONE | https://doi.org/10.1371/journal.pone.0288505 July 13, 2023 1/7 PLOS ONE Optimal cut-off point for reticulocyte hemoglobin as IDA marker However, using ferritin as an indicator has some drawbacks; ferritin is an acute-phase reactant, serum ferritin levels increase drastically in the presence of inflammation, giving falsely high levels even when the patient has IDA. Because ferritin acts as a buffer of iron levels in the body, low levels of ferritin only occur after the depletion of iron stores in the body. A reliable, simple and cheap test is needed to detect early IDA [6]. Reticulocytes are immature red blood cells that do not have a nucleus, however, unlike RBCs, reticulocytes still contain the remnants of their molecular machinery, a mesh-like network of ribosomal RNA [7]. Reticulocytes are the youngest erythrocytes released from the bone marrow into circulating blood, they circulate in the blood for 1–2 days before they differentiate into mature RBCs [8, 9]. Reticulocytes provide a window into the health and function of the bone marrow. The Hb content of reticulocytes (CHr) is an attractive indicator to be used as an indicator of IDA [10], and has been investigated in previous studies. In a 1999 study by Brugnara et al., CHr was shown to be the best diagnostic marker for iron deficiency and IDA in children [11]. In another study, CHr was a useful marker in diagnosing IDA in end-stage renal disease (ESRD) adult patients [12]. In another study, CHr was found to be an effective method to identify IDA and quickly measure the responsiveness of intravenous iron treatment in IDA patients [13]. A limiting factor for CHR is the lack of a standardized cut off point among researchers affecting the accuracy in diagnosing IDA [14]. The aim of this study is to find the optimal cut off point for CHr to diagnose iron deficiency anemia in a clinical setting using multiple methods. Results Characteristics of the sample Of the 306 who were included, 209 (68.3%) had iron deficiency anemia, and 97 (31.7%) had anemia due to causes other than iron deficiency. There were 218 (71.2%) females. The median age at diagnosis was 54 with an IQR of 34. The median values for ferritin, hemoglobin, and CHr were 18.2, 9.2, and 27.35, respectively (Table 1). Patients diagnosed with iron deficiency anemia had significantly lower CHr levels compared to those diagnosed with other types of anemia (p < 0.001, mean difference = 8.41 pg). In addition, those with iron deficiency anemia were more likely to be females compared to those with other types of anemia (p < 0.001). Correlation between CHr and other lab markers Pearson’s correlation was used to check for correlations between CHr levels and other lab markers in the combined sample, among the IDA group and non-IDA group. In the combined sample, CHr was significantly associated with serum iron, TIBC, and TFS (r = .456, -.588, and .424, respectively). Amongst those with IDA alone, CHr was still found to be significantly correlated with serum iron and TIBC (r = .322, -.404, respectively). Meanwhile when testing those with non-IDA alone, the said correlation was insignificant. Table 2 shows all the different correlations between CHr and other lab markers. The diagnostic value of CHr in diagnosing iron deficiency anemia A ROC curve was used to determine the diagnostic value of CHr in diagnosing iron deficiency anemia as previously defined in the methods section. CHr showed high accuracy in diagnosing IDA (AUC = 0.891, p < 0.0001). The ROC curve is shown in Fig 1. PLOS ONE | https://doi.org/10.1371/journal.pone.0288505 July 13, 2023 2/7 PLOS ONE Optimal cut-off point for reticulocyte hemoglobin as IDA marker Table 1. Demographic and laboratory data. Characteristic Frequency Percentage Gender Male 88 28.8 Female 218 71.2 Iron Deficiency Anemia 209 31.7 Anemia (Other) 97 Diagnosis 68.3 Median) IQR( Age 54 (34) Hemoglobin 9.3 (2.50) Ferritin 15 (210.23) CHr 27.30 (10.2) MCV 77.55 (29.0) WBC 6.72 (3.48) RDW 18.35 (4.0) PLT 273.00 (157.5) Reticulocyte Percentage 1.58(1.05) TIBC 360(179) Iron 35.85(42.32) TFS 10.099(21.74) B12 359.00(268.00) Folate 8.10(6.6) LDH 387.00(179.00) https://doi.org/10.1371/journal.pone.0288505.t001 Determining the optimal cut-off point for diagnosing iron deficiency anemia The ROC curve analysis generates a table with multiple cut-off points, each with a certain specificity and sensitivity value. Using 4 different methods being: Youden index, the closest to (0,1) criteria, the concordance probability method, and the index of union. An optimal cut-off point Table 2. CHr correlation with other parameters using Pearson correlation. Lab Marker Correlation (Entire Sample) P-value Correlation (IDA Only) P-value Correlation (Non-IDA) P-Value Age .241 < .001 .174 .036 -.026 0.807 Hemoglobin .227 < .001 .482 < .001 -.305 .003 WBC .075 .203 -.006 .935 -0.45 .666 MCV .343 < .001 .796 < .001 .339 <0.001 RDW -.005 .928 -.021 .765 .391 <0.001 .003 PLT -.288 < .001 -.080 .267 -.302 Retics Percentage .254 < .001 .281 < .001 .236 .022 Ferritin .283 < .001 .241 < .001 .007 .949 Iron .456 < .001 .322 < .001 .109 .298 TIBC -.588 < .001 -.404 < .001 -.023 .825 TFS .424 < .001 .133 .063 .177 .088 B12 .124 .043 .033 .653 -.155 .162 Folate .165 .011 .240 .003 -0.94 .404 LDH .349 < .001 .213 .016 .450 <0.001 https://doi.org/10.1371/journal.pone.0288505.t002 PLOS ONE | https://doi.org/10.1371/journal.pone.0288505 July 13, 2023 3/7 PLOS ONE Optimal cut-off point for reticulocyte hemoglobin as IDA marker Fig 1. Roc curve showing the diagnostic efficacy of CHr in diagnosing IDA. https://doi.org/10.1371/journal.pone.0288505.g001 was determined using each of these methods and compared to the others. The optimal cut-off point was 30.15 pg with a sensitivity of 0.878 and a specificity of 0.777. The point was found to be the same using any of the different methods. Discussion This study looked at the value of CHr and the cutoff point to be used for IDA in adolescents and adults. Most were adults and this is reflected in the population’s median age which was 54 years with predominance of females constituting 71.2%. The female predominance is in line with medical literature [15]. CHr was found to be a useful parameter that can be confidently utilized for the diagnosis of IDA [16].Our study analyzed the CHr in IDA and non-IDA populations. We showed that patients diagnosed with IDA had significantly lower CHr levels compared to those diagnosed with non-IDA. These results are consistent with previous published work [17]. The optimal cut-off point for CHr to diagnose IDA is hotly debated. A 2022 meta-analysis found the cutoff value of CHr to be 28.2 pg with a 84% sensitivity and a 91% specificity. It is important to note that this meta-analysis has combined studies conducted with different PLOS ONE | https://doi.org/10.1371/journal.pone.0288505 July 13, 2023 4/7 PLOS ONE Optimal cut-off point for reticulocyte hemoglobin as IDA marker methods. In addition, no formal methods or statistical techniques were used to define the optimal cut off point in the studies included in the meta-analysis. This leaves the question of the optimal cut off point unanswered. The aforementioned meta-analysis showed that CHr is a more effective marker in determining IDA compared to both SF and MCV levels along with many of the other more commonly used parameters [18]. Our work indicates that CHr cutoff value of 30.15 pg can identify IDA with a 87.8% sensitivity and 77.7% specificity. The aforesaid cut-off value is similar to the 30.7 pg cut-off value reported by Auerbach et al. (2021) in a cohort of 556 patients [13]. Additionally, a number of recent studies reported similar cut-off values ranging from 30 pg to 30.9 pg [19, 20]. Nevertheless, none of these studies had a sensitivity higher than that of our study. The uniqueness of our study lies in the fact that it defines an optimal cut-off point using multiple different statistical methods. This -to our knowledge- has not been done previously in literature. Previous studies reported wide ranges of cut-off points, and the aforementioned meta-analysis didn’t help specify any value as it included heterogenous studies that didn’t utilize any statistical methods for determination of cut-off points. We believe that our study will help fill the gap created by the wide range of cut-off points and help pave the way for future endeavors. In addition, no studies have been published on the Jordanian population specifically. Our study is the first to test this in our population. In the present study CHr had a strong association with serum iron, ferritin, TFS, Hb, and MCV as well as it inverse relationship with TIBC. However, CHr showed no association with either WBC or RDW. This is in line with the results of a 2020 South Asian study [21]. There are two possible limitations of this study. First, the low count of males in the registry as it would have been better if the it included more males. Second, the study may not be representative of the whole population in Jordan since it was limited to one center The size of our sample is acceptable, but larger study with more patients is warranted. It would be useful to extend the study by including patients with other comorbidities as well. Despite of these limitations, we believe that our cutoff value is satisfyingly sensitive since it remained consistent in all of the various methods we used to determine the cutoff value. It is to be noted that according to what is already published on the role of CHr in the detection of IDA, CHr is not adopted into clinical practice yet due to the lack of a universal approval on the most specific and sensitive CHr cutoff point for IDA diagnosis and this is the problem we aimed to solve in our study. Conclusion CHr is a simple, cheap, fast, and reliable test for the diagnosis of IDA. The cutoff value of 30.5pg seems to be the best value that separates IDA from the rest of non-Iron deficiency anemias. Materials and methods Study population and design A hospital-based registry of IDA in adolescents and adults with either IDA or non-IDA was analyzed retrospectively in a cross-sectional study. Data in the registry was collected from 306 patients in a university hospital. The data include age, gender, diagnosis (IDA or anemia without ID), hemoglobin, ferritin, CHr, MCV, WBC, RDW, Platelet count, reticulocytes count, serum iron, total iron binding capacity (TIBC), transferrin saturation (TFS), B12, folate and LDH. Iron deficiency was diagnosed based on either a low serum ferritin (<30 ng/mL), and/or a low TSAT (< 20%). PLOS ONE | https://doi.org/10.1371/journal.pone.0288505 July 13, 2023 5/7 PLOS ONE Optimal cut-off point for reticulocyte hemoglobin as IDA marker IRB approval and informed consent This study was approved by the IRB at the Jordan University hospital. No informed consent was required for this study because sample and data were de-identified and no additional blood was drawn. Data analysis SPSS version 26 was used for analysis. All continuous data are presented as median with interquartile range (IQR), all categorical data are described as frequency and percentage. Quantitative variables were analyzed using Student’s t-test. Pearson’s correlation coefficient was used to assess the correlation between parameters. Receiver operator characteristics (ROC) curve analysis was used to determine the value of CHr in identifying IDA, with results presented as the area under the curve (AUC) with corresponding 95% CI and P-value. The optimal cut-off point for CHr was determined using Youden’s index (J) and validated using closest to (0,1) criteria (ER), concordance probability method (CZ) and Index of Union (IU) [22]. Supporting information S1 Data. (CSV) Author Contributions Conceptualization: Abdalla Awidi. Data curation: Marah Alzu’bi, Hisham Bawa’neh, Alaa Alshorman, Jawad Alrawabdeh, Nada Odeh, Yazan Hamadneh, Mai AlAdwan, Mahmoud Odeh. Formal analysis: Marah Alzu’bi, Hisham Bawa’neh, Alaa Alshorman, Jawad Alrawabdeh, Nada Odeh, Yazan Hamadneh, Mai AlAdwan, Mahmoud Odeh, Abdalla Awidi. Investigation: Marah Alzu’bi, Abdalla Awidi. Methodology: Marah Alzu’bi, Hisham Bawa’neh, Alaa Alshorman, Jawad Alrawabdeh, Nada Odeh, Yazan Hamadneh, Mahmoud Odeh, Abdalla Awidi. Resources: Abdalla Awidi. Supervision: Abdalla Awidi. Writing – original draft: Marah Alzu’bi, Hisham Bawa’neh, Alaa Alshorman, Jawad Alrawabdeh, Nada Odeh, Yazan Hamadneh, Mai AlAdwan, Mahmoud Odeh. Writing – review & editing: Abdalla Awidi. References 1. Camaschella C. Iron-Deficiency Anemia. N Engl J Med 372, 1832–1843 (2015). https://doi.org/10. 1056/NEJMra1401038 PMID: 25946282 2. Tang Z., Wu H., Zhang Y., Li Z. & Lin Y. Enzyme-mimic activity of ferric nano-core residing in ferritin and its biosensing applications. Anal Chem 83, 8611–8616 (2011). https://doi.org/10.1021/ac202049q PMID: 21910434 3. Arosio P., Elia L. & Poli M. Ferritin, cellular iron storage and regulation. IUBMB Life 69, 414–422 (2017). https://doi.org/10.1002/iub.1621 PMID: 28349628 4. Bouri S. & Martin J. Investigation of iron deficiency anaemia. Clin Med (Lond) 18, 242–244 (2018). PLOS ONE | https://doi.org/10.1371/journal.pone.0288505 July 13, 2023 6/7 PLOS ONE Optimal cut-off point for reticulocyte hemoglobin as IDA marker 5. Daru J. et al. Serum ferritin as an indicator of iron status: what do we need to know? Am J Clin Nutr 106, 1634S–1639S (2017). https://doi.org/10.3945/ajcn.117.155960 PMID: 29070560 6. Dignass A., Farrag K. & Stein J. Limitations of Serum Ferritin in Diagnosing Iron Deficiency in Inflammatory Conditions. Int J Chronic Dis 2018, 9394060 (2018). https://doi.org/10.1155/2018/9394060 PMID: 29744352 7. Lee E. et al. The RNA in reticulocytes is not just debris: it is necessary for the final stages of erythrocyte formation. Blood Cells Mol Dis 53, 1–10 (2014). https://doi.org/10.1016/j.bcmd.2014.02.009 PMID: 24594313 8. Coulombel L., Tchernia G. & Mohandas N. Human reticulocyte maturation and its relevance to erythropoietic stress. J Lab Clin Med 94, 467–474 (1979). PMID: 572850 9. Koepke J. F. & Koepke J. A. Reticulocytes. Clinical & Laboratory Haematology 8, 169–179 (1986). https://doi.org/10.1111/j.1365-2257.1986.tb00093.x PMID: 3530617 10. Ogawa C., Tsuchiya K. & Maeda K. Reticulocyte hemoglobin content. Clin Chim Acta 504, 138–145 (2020). https://doi.org/10.1016/j.cca.2020.01.032 PMID: 32014518 11. Brugnara C., Zurakowski D., DiCanzio J., Boyd T. & Platt O. Reticulocyte hemoglobin content to diagnose iron deficiency in children. JAMA 281, 2225–2230 (1999). https://doi.org/10.1001/jama.281.23. 2225 PMID: 10376576 12. Dinh N. H., Cheanh Beaupha S. M. & Tran L. T. A. The validity of reticulocyte hemoglobin content and percentage of hypochromic red blood cells for screening iron-deficiency anemia among patients with end-stage renal disease: a retrospective analysis. BMC Nephrol 21, 142 (2020). https://doi.org/10. 1186/s12882-020-01796-8 PMID: 32321449 13. Auerbach M., Staffa S. J. & Brugnara C. Using Reticulocyte Hemoglobin Equivalent as a Marker for Iron Deficiency and Responsiveness to Iron Therapy. Mayo Clin Proc 96, 1510–1519 (2021). https://doi. org/10.1016/j.mayocp.2020.10.042 PMID: 33952394 14. Gelaw Y., Woldu B. & Melku M. The Role of Reticulocyte Hemoglobin Content for Diagnosis of Iron Deficiency and Iron Deficiency Anemia, and Monitoring of Iron Therapy: a Literature Review. Clin Lab 65, (2019). https://doi.org/10.7754/Clin.Lab.2019.190315 PMID: 31850722 15. Akbarpour E. et al. Anemia prevalence, severity, types, and correlates among adult women and men in a multiethnic Iranian population: the Khuzestan Comprehensive Health Study (KCHS). BMC Public Health 22, 168 (2022). https://doi.org/10.1186/s12889-022-12512-6 PMID: 35073904 16. Karagülle M., Gündüz E., Şahin Mutlu F. & Olga Akay M. Clinical Significance of Reticulocyte Hemoglobin Content in the Diagnosis of Iron Deficiency Anemia. Turk J Haematol 30, 153–156 (2013). https:// doi.org/10.4274/Tjh.2012.0107 PMID: 24385778 17. Cai J. et al. Evaluation of the Efficiency of the Reticulocyte Hemoglobin Content on Diagnosis for Iron Deficiency Anemia in Chinese Adults. Nutrients 9, 450 (2017). https://doi.org/10.3390/nu9050450 PMID: 28468320 18. Kılıç M., Özpınar A., Serteser M., Kilercik M. & Serdar M. The effect of reticulocyte hemoglobin content on the diagnosis of iron deficiency anemia: A meta-analysis study. J Med Biochem 41, 1–13 (2022). https://doi.org/10.5937/jomb0-31435 PMID: 35291499 19. Chinudomwong P., Binyasing A., Trongsakul R. & Paisooksantivatana K. Diagnostic performance of reticulocyte hemoglobin equivalent in assessing the iron status. J Clin Lab Anal 34, e23225 (2020). https://doi.org/10.1002/jcla.23225 PMID: 32043622 20. Toki Y. et al. Reticulocyte hemoglobin equivalent as a potential marker for diagnosis of iron deficiency. Int J Hematol 106, 116–125 (2017). https://doi.org/10.1007/s12185-017-2212-6 PMID: 28299633 21. Kariyawasan C. C. et al. Evaluation of Reticulated Haemoglobin (CHr) as a diagnostic parameter in Iron Deficiency Anemia. European Journal of Medical and Health Sciences 2, (2020). 22. Unal I. Defining an Optimal Cut-Point Value in ROC Analysis: An Alternative Approach. Computational and Mathematical Methods in Medicine 2017, e3762651 (2017). https://doi.org/10.1155/2017/3762651 PMID: 28642804 PLOS ONE | https://doi.org/10.1371/journal.pone.0288505 July 13, 2023 7/7