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Original Article

Amer Wahan, Tejashwini K. Huchannavar, Shruthi H P 

Department of Pathology, Padmashree Institute of Medical Laboratory Technology, Bengaluru, Karnataka-560072.

Corresponding author:

Dr. Shruthi H P, Padmashree Institute of Medical Laboratory Technology, Bengaluru, Karnataka-560072 E-mail: 1111.shruthi@gmail.com. Affiliated to Rajiv Gandhi University of Health Sciences, Bengaluru, Karnataka.

Received date: May 21, 2021; Accepted date: July 8, 2021; Published date: July 31, 2021

Received Date: 2021-05-21,
Accepted Date: 2021-07-08,
Published Date: 2021-07-31
Year: 2021, Volume: 1, Issue: 2, Page no. 28-32, DOI: 10.26463/rjahs.1_2_6
Views: 2721, Downloads: 98
Licensing Information:
CC BY NC 4.0 ICON
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0.
Abstract

Background: Iron Deficiency Anemia (IDA) and beta-thalassemia trait (β-TT) are the two most prevalent causes of microcytic hypochromic anemia. The current study aimed to assess the accuracy of the various indices to distinguish these two conditions.

Material & Methods: A total of 40 patients were studied retrospectively by calculating six discrimination indices. The patients were assessed according to the Mentzer, Shine & Lal, England & Fraser, Green & King, Red Cell Distribution Width Index (RDWI) and Srivastava indices. The study included 19 β-TT cases based on low Hb and mean cell volume (MCV) levels, normal serum iron and raised levels of HbA2 (>3.5%) and 21 IDA cases with low hemoglobin, low serum iron, low ferritin, high total iron-binding capacity, and normal electrophoresis. The accurately diagnosed cases were identified and their sensitivity, specificity, positive and negative predictive value and Youden’s index of the indices were calculated.

Results: None of the indices showed a sensitivity and specificity of 100%. The percentage correctly diagnosed was highest for RDWI (97.5%), followed by Green and King (95%). Youden’s index was also highest for RDWI (95) followed by Green and King (90) and Mentzer (84.7).

Conclusion: RDWI, Green & King and Mentzer are readily available and the most stable indices in differentiating between IDA and β-TT.

<p><strong>Background:</strong> Iron Deficiency Anemia (IDA) and beta-thalassemia trait (&beta;-TT) are the two most prevalent causes of microcytic hypochromic anemia. The current study aimed to assess the accuracy of the various indices to distinguish these two conditions.</p> <p><strong>Material &amp; Methods: </strong>A total of 40 patients were studied retrospectively by calculating six discrimination indices. The patients were assessed according to the Mentzer, Shine &amp; Lal, England &amp; Fraser, Green &amp; King, Red Cell Distribution Width Index (RDWI) and Srivastava indices. The study included 19 &beta;-TT cases based on low Hb and mean cell volume (MCV) levels, normal serum iron and raised levels of HbA2 (&gt;3.5%) and 21 IDA cases with low hemoglobin, low serum iron, low ferritin, high total iron-binding capacity, and normal electrophoresis. The accurately diagnosed cases were identified and their sensitivity, specificity, positive and negative predictive value and Youden&rsquo;s index of the indices were calculated.</p> <p><strong>Results: </strong>None of the indices showed a sensitivity and specificity of 100%. The percentage correctly diagnosed was highest for RDWI (97.5%), followed by Green and King (95%). Youden&rsquo;s index was also highest for RDWI (95) followed by Green and King (90) and Mentzer (84.7).</p> <p><strong>Conclusion:</strong> RDWI, Green &amp; King and Mentzer are readily available and the most stable indices in differentiating between IDA and &beta;-TT.</p>
Keywords
Iron Deficiency Anemia, Beta-thalassemia trait, Red cell distribution width index, Microcytic hypochromic anemia
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Introduction

Microcytic anemia is most common due to iron deficiency anemia (IDA) or Thalassemia. The World Health Organization (WHO) has recently emphasized its concern about anemia, which has affected an estimated two billion people; half of these cases caused by iron deficiency.1 Anemia due to lack of adequate iron to synthesize hemoglobin is the most widespread hematological disease. It has been estimated that 30% of the global population suffers from IDA, and most of those affected live in developing countries.2 Microcytic anemia in thalassemia is due to the disrupted globin chain synthesis and reduced hemoglobin (Hb) synthesis, resulting in microcytosis and hypochromia. The incidence of beta-thalassemia mutations ranges across various regions of the world with the towering prevalence in the Mediterranean, the Middle East, and Southeast and Central Asia. Approximately 68000 children are diagnosed with beta-thalassemia at birth. Its prevalence rate is 80-90 million carriers, around 1.5% of the global population.3 It is estimated that about 10% of the total world’s thalassemic are born in India every year.4 Beta thalassemia trait (β-TT) is the most frequent form of hemoglobinopathies transmitted through heredity. Individuals with β-TT are usually asymptomatic and may be unaware of their carrier status until they are diagnosed by testing. A characteristic laboratory feature of β-thalassemia trait is an elevation of HbA2 >3.5%.5

The differentiation of IDA from thalassemia minor is important as continued iron therapy in the latter condition can cause iron overload. A definitive distinction between thalassemia minor and IDA is based on the data of the electrophoresis of hemoglobin, serum iron and ferritin levels.

Hemoglobin electrophoresis is a technique used to separate and identify different hemoglobin variants by their migration within an electric field. Hemoglobin variants break apart at a different rate due to divergence in their surface electrical charge as decided by their amino acid structure. In microcytic hypochromic anemia, Hb electrophoresis is used for detection of thalassemia from other conditions.

This study aimed to determine the diagnostic value of different red blood cell indices to distinguish β-TT from IDA by estimating their sensitivity, specificity, and Youden’s index.

Materials and Methods

Retrospective analysis of complete blood count (CBC), Hb electrophoresis and serum iron profile of 40 patients with a microcytic anemia who were sent to the hematology laboratory of diagnostic center, Bangalore, India during the period from 1st of January 2018 to 31st of May 2020 were considered for the study. This sample included 21 patients with IDA and 19 cases with β-TT.

Samples were collected during routine examination in EDTA anticoagulant tubes. Complete blood counts were performed with an automated analyzer Mindary BC-6800. Serum iron (SI), total iron-binding capacity (TIBC), and serum ferritin were determined by Cobas 6000 (Roche, Germany) for all the cases. Serum levels of HbA2 were determined using Bio-rad D 10 HPLC Analyzer (Bio-Rad Laboratories, CA, USA).

Inclusion criteria

The cases with low hemoglobin, low serum iron, low ferritin, high TIBC, and normal electrophoresis were classified as IDA. Cases with normal serum iron, serum ferritin, TIBC, and high HbA2 were considered as thalassemia trait. In IDA, the cut-off range for males was hemoglobin less than 13 g/dL, serum iron less than 60 μg/dL and serum ferritin less than 30 ng/mL. The cutoff values in females were hemoglobin less than 12 g/ dL, serum iron less than 50 μg/dL and serum ferritin less than 13 ng/mL. In β-TT cases, the hemoglobin A2 cutoff was more than 3.5%.

Exclusion criteria

Newborn infants and children below the age of six months, mixed cases of IDA and β-TT were excluded from the study. Microcytic hypochromic anemia cases with normal Hb electrophoresis and normal iron profile were eliminated from the study.

Statistical evaluation

Discrimination indices used in the evaluation were calculated. Table 1 summarizes these mathematical formulas. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Youden’s Index were calculated as follows: Sensitivity = [True Positive/ (True Positive + False-Negative)] × 100 Specificity = [True Negative/ (True Negative + False-Positive)] × 100 PPV=true positive/(true positive + false positive) ×100 NPV=true negative/(truenegative + false negative) ×100 Youden’s Index = (Sensitivity + Specificity) – 100 Data was analyzed with a computerized statistical analysis software SPSS version 20.

Youden’s index was suggested by W J Youden, which is a way of summarizing the performance of a diagnostic test. It was estimated by subtracting 100 from the total performance of a diagnostic test and was calculated by subtracting 100 from the addition of sensitivity and specificity.6

Results

This study included 40 patients, 19 β-TT cases (7 women and 12 men) with a mean ± SD age of 30.05±21.62 years, and 21 IDA cases (5 men and 16 women) aged 38.81±15.02 years.

The differential value for each index was the differentiation between β-TT and IDA and the number and proportion of correctly diagnosed patients (true positive) determined using the indices as shown in Table 2.

Table 2 shows that the proportion of correctly identified patients is highest with the RDWI index (Red Cell Distribution Width Index) (97.5%), closely followed by the G&K Index (95%). Thus, according to percentage correctly diagnosed criteria, RDWI, G&K and Mentzer can be regarded as the best discrimination indices.

The sensitivity, specificity, PPV, NPV, and Youden’s Index of each distinction Index in differentiating between β-TT and IDA are listed in Table 3. None of the indices used for distinguishing between β-TT and IDA were completely sensitive or specific. Youden›s index displayed the following ranking with respect to the indices› ability to comprehend between β-TT and IDA: RDWI > Green and King >Mentzer >England and Fraser >Srivastava > Shine and Lal. The RDWI and G&K indices were, therefore, the best indicators in both the comparisons and the S&L index was the worst.

Discussion

The most widely reported diseases with mild microcytic anemia are β-TT and IDA. Distinguishing β-TT from IDA has important clinical significance because each disorder has a different cause, treatment and prognosis. The diagnosis of β-TT is determined by the presence of RBC microcytosis and increased HbA2 levels.13 Decreased levels of SI, ferritin and TS with increased levels of TIBC are the main diagnostic features for IDA.14

All the investigated cases included in the study were confirmed with the set criteria for each anemia. IDA cases were diagnosed by low Hb, RBC, MCV, serum iron, transferring saturation, serum ferritin and high TIBC. β-TT cases were confirmed with low Hb and MCV, high HbA2 levels, and normal serum iron.

Various formulas based on Hb, RBC count, MCV, MCH and RDW have been devised in an attempt to separate IDA from β-TT, as both cause microcytic hypochromic anemia. However, no study has established 100% specificity or sensitivity for any of these indices.

The present study calculated each discrimination index in the patient groups with β-TT and IDA. In addition to calculating the sensitivity, specificity, PPV and NPV of each discrimination index, we also calculated Youden›s index in their distinction between IDA and β-TT.

The study applied all six formulas, and found percentage correctly diagnosed to be highest for RDWI (97.5%), followed by Green & King (95%), Mentzer index (90.5%), England & Fraser (90%), Srivastava (90%), and Shine & Lal (55%) in decreasing order. Similar observations were made by Demir for the RDWI index (92%), Green & King Index (86%), Mentzer Index (76%), England & Fraser (78%), Srivastava (67%) and Shine & Lal (59%).15

Youden’s Index has been used previously to compare validity of differentiating indices in distinguishing between β-thalassemia minor and IDA.15 It was reported that RDWI is the most accurate discrimination index in differentiating between IDA and β-TT. This observation is in accordance with the present study. RDWI was found to be the most valid discrimination index to correctly differentiate between IDA and β-thalassemia trait (Youden’s Index 95), the Green & King came second with Youden’s Index of 90.

In 2007, Ntaios et al like red blood cell (RBC examined the diagnostic accuracy of six differentiating indices in distinguishing between IDA and β-TT and reported that the Green and King index was the most dependable index, as it had the highest sensitivity (75.06%), specificity (80.12%), and Youden’s index (70.86%) for β-TT detection. 16 In comparison, the data obtained showed that the Youden’s indices in decreasing order were as follows: RDWI > G&K > MI > E&F > SI > S&L. The RDWI was the most reliable index, as it had the sensitivity (95%), specificity (97.5%), and Youden’s index (95%), followed by Green and King index with Youden’s index of 90%.

Our data proved that the S&L index had the lowest Youden’s index, with a value of 14%, in correctly distinguishing between IDA and β-thalassemia trait, indicating that this formula is the least effective in microcytic anemia discrimination studies. The ineffectiveness of the S & L formula has been reported by Suad et al in 2007.17,18 In their study, it was observed that the E&F index had the highest Youden’s index value (98.2%), where it correctly distinguished between IDA and β-TT, while the S&L index was established to be ineffective in distinguishing microcytic anemias.

In 2009, Ehsani et al showed that the Mentzer index was accurate in providing diagnosis (94.7%).18 Even in the present study, Mentzer index could correctly diagnose (92.5%).

In an investigation conducted by Ferrara et al (2010), RDWI was established as the index with the highest sensitivity (78.9%), England and the Fraser index showed the highest specificity and Youden’s index of 99% and 64% respectively.19 Okan V et al. reported S&L and G&K indices to be best at differentiating IDA from β-TT patients, and the RDW index was found to be the worst.20 The S&L index had the highest Youden’s index value in discriminating β-TT cases from those with IDA. However, in the present study, we observed that RDWI and G&K indices to be the best at discriminating IDA from β-TT patients, and the S&L index was found to be the worst. Further investigations with larger sample size are needed.

Conclusion

In conclusion, although none of these indices showed absolute sensitivity and specificity (100%) in differentiating IDA and β-TT, the present study established RDWI to be the most reliable discriminate function in differentiating between IDA and β-thalassemia minor, as it had the highest correctly diagnosed, and Youden’s index. This was followed by the Green and King index and Mentzer. Conversely, the S&L formula was found to be ineffective.

Conflict of interests 

All the authors have contributed equally. 

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References
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