European Journal of Cardiovascular Medicine

Anemia is a significant public health concern, particularly among pregnant and postpartum women in low- and lower-middle-income countries. The highest burden of anemia affects populations residing in rural areas, those with lower socioeconomic status, and individuals with limited educational attainment. Globally, it is estimated that anemia affects 37% of pregnant women and 30% of non-pregnant women aged 15 to 49 years (1). The etiology of anemia is multifactorial, with contributing factors including deficiencies in iron and vitamins, inadequate intake or absorption of nutrients, infections, chronic diseases, gynecological and obstetric conditions, and inherited red blood cell disorders.

Among pregnant women, the primary causes of anemia include iron deficiency anemia (IDA), beta-thalassemia trait (BTT), sickle cell trait, and malaria (2). India carries a substantial burden of hemoglobinopathies, with thalassemia being one of the most prevalent inherited disorders. Thalassemia is classified into minor, major, and intermedia forms, depending on the level of impairment in α- or β-globin chain synthesis (3). The prevalence of β-thalassemia varies across different regions of India, with reported rates of 6.5% in Punjab, 4.3% in southern India, and 3.5% in West Bengal. Additionally, certain ethnic groups, such as the Sindhi, Luvana, tribal populations, and Rajputs, exhibit a higher prevalence of β-thalassemia (4). In Gujarat, the incidence ranges from 10% to 15% (5). According to the National Health Mission (2016), the average prevalence of BTT in India is 4%, and the expected annual incidence of thalassemia major births is estimated at 0.5 per 1,000 live births (6).

Screening for thalassemia during pregnancy is crucial for early detection, risk assessment, and effective management to ensure maternal and fetal health. Early identification facilitates genetic counseling and enables couples to make informed decisions regarding further diagnostic procedures, such as chorionic villus sampling (CVS) or amniocentesis. Detecting thalassemia minor at an early stage helps prevent unnecessary iron supplementation, which can lead to iron overload and subsequent complications. Additionally, timely screening allows for appropriate antenatal care and planned hospital deliveries, thereby reducing maternal mortality in high-risk anemic patients.

To minimize the burden of β-thalassemia, well-integrated strategies and effective implementation at both state and national levels are essential. Screening adolescents in schools and all pregnant women is a feasible preventive strategy for reducing the prevalence of thalassemia intermedia and major. Premarital screening, followed by prenatal diagnosis and medical termination of affected pregnancies, is widely accepted within Indian communities. The success of screening programs in India hinges on increasing awareness among the general population and healthcare professionals to ensure timely testing and improved detection rates.

A considerable proportion of microcytic hypochromic anemia cases detected through peripheral blood smear examination may, in fact, be undiagnosed thalassemia trait cases masquerading as IDA. Misdiagnosis and inappropriate iron therapy can exacerbate the condition. In remote healthcare settings, confirmatory tests such as high-performance liquid chromatography (HPLC) and serum iron profiling may not be readily available, necessitating the use of cost-effective and accessible screening methods. Various hematological indices have been developed to differentiate IDA from thalassemia, particularly during pregnancy, when physiological changes can affect baseline hematological values. These indices are based on parameters such as mean corpuscular volume (MCV), red blood cell count (RBC), mean corpuscular hemoglobin (MCH), and red cell distribution width (RDW). The present study evaluates the diagnostic performance of several hematological indices, including the Shine & Lal index (MCV × MCV × MCH/100), the Mentzer index (MCV/RBC), and the Srivastava index (MCH/RBC), in identifying thalassemia minor in low-resource settings.

This study employed a retrospective descriptive design and was conducted at a tertiary care center in the eastern region of India. Retrospective data were collected from a total of 3,249 pregnant women who attended the antenatal clinic of the hospital. The study included all women with hemoglobin levels below 11.0 g/dL from October 2022 to September 2023. These patients were routinely tested for anemia during pregnancy as per hospital protocol, which included a complete blood count (CBC), peripheral blood smear (PBS), serum iron, serum ferritin, total iron-binding capacity (TIBC), high-performance liquid chromatography (HPLC), liver function tests (LFT), renal function tests (urea and creatinine), thyroid function tests (TFT), serum folate, and serum vitamin B12 levels.

The study population comprised a moving patient cohort with diverse ethnic backgrounds. The ethnicity of the patients was documented as part of the data collection process. Various hematological indices, including the Mentzer index, Shine & Lal index, Srivastava index, and red cell distribution width index (RDWI), were retrospectively recalculated for the BTT patient group to assess whether BTT could be detected in low-resource settings without HPLC availability. Patients with other causes of anemia, including other hemoglobinopathies, and those with a history of blood transfusion within the last three months were excluded from the study.

Maternal medical records were reviewed to obtain demographic and clinical variables. Since this study involved a retrospective evaluation of automated red cell indices, ethical clearance was not obtained. No additional samples were collected, and no extra tests were performed beyond routine hospital protocols. All methods adhered to relevant guidelines and institutional regulations.

Of the 3,249 patients, a total of 165 were diagnosed with hemoglobinopathies. Among them, 103 patients (3.36%) had beta-thalassemia trait (BTT) (Hb A2 > 3.5%), 35 patients (1.14%) were hemoglobin E heterozygous, 13 patients (0.42%) were hemoglobin E homozygous, 9 patients (0.29%) had hemoglobin S heterozygous, and 4 patients (0.11%) had hemoglobin D (Punjab). Additionally, one patient was diagnosed with hemoglobin E-beta thalassemia trait double heterozygous. Among other causes of anemia, 4 patients (0.13%) had anemia of chronic diseases (e.g., tuberculosis, breast cancer, systemic lupus erythematosus), 15 patients (0.48%) had folate or vitamin B12 deficiencies, and the remaining 2,881 patients (93.9%) were diagnosed with iron deficiency anemia (IDA) (serum ferritin < 12 g/mL).

Among the 165 hemoglobinopathy-positive patients, BTT formed the largest subgroup (62.4%). Patients with both IDA and BTT were excluded from the study to prevent misinterpretation of hematological indices. The mean age of BTT patients was 29 ± 2.9 years. Most were multigravida, yet BTT was identified for the first time in the current pregnancy. The regional distribution of BTT patients included 33 from Bengal, 28 from Bihar, 13 from Punjab, 8 from Assam, 7 each from Odisha and Maharashtra, 4 from Tamil Nadu, and 3 from Nepal. Only 9 patients reported a family history of thalassemia, while 25 reported symptoms such as fatigue and palpitations; the rest were asymptomatic.

The ROC analysis of various hematological indices used for diagnosing Beta Thalassemia Trait (BTT) highlights significant variations in their diagnostic accuracy. The Shine & Lal Index demonstrated the highest sensitivity (99.01%), making it the most effective index for identifying BTT cases. However, its specificity (73.8%) was lower than other indices, indicating a higher rate of false positives. In contrast, the Mentzer Index, Srivastav Index, and RDW Index exhibited high specificity (~99%), meaning they were better at ruling out BTT but had lower sensitivity, reducing their effectiveness in detecting positive cases. The area under the curve (AUC) values indicate that while the Shine & Lal Index is the most reliable for identifying BTT, other indices may still be useful in specific clinical settings where specificity is prioritized over sensitivity. The findings emphasize the importance of selecting an appropriate index based on the diagnostic objective, particularly in resource-limited settings where advanced diagnostic tests are not readily available. (Graph 1)

The diagnostic accuracy of various hematological indices was evaluated using HPLC as the gold standard. Mentzer's Index correctly identified 24 out of 103 BTT patients (23.3%), Srivastava Index detected 21 cases, RDWI identified 33 cases, and the Shine & Lal Index accurately diagnosed 102 of the BTT patients.

Graph 1: ROC curve analysis of hematological indices

Table 1: hematological Indices calculators

Table 2: Causes of Anemia among the recruited patients

Table 3 : Diagnostic accuracies of hematological indices.

Table 4: Hematological indices diagnostic values

Table 5 : Specificity/ Sensitivity of hematological indices

India has the highest prevalence of Beta Thalassemia Major (BTM) globally, with an estimated 42 million cases of Beta Thalassemia Trait (BTT), accounting for a carrier frequency of 3–4%. Annually, approximately 10,000 to 15,000 infants are born with BTM (7). Carrier screening, both pre-marital and during the antenatal period, remains the most effective strategy for reducing the incidence of BTM births. The present study was conducted in a hospital in eastern India with a mixed population, including military personnel families. High-Performance Liquid Chromatography (HPLC) and iron studies, while effective, are relatively expensive, require technical expertise, and are time-consuming. Consequently, it is cost-effective and convenient to rely on simple and existing hematological indices, particularly in remote areas with low-resource settings. Numerous researchers have employed various hematological indices to differentiate BTT from iron deficiency anemia (IDA) using complete blood count (CBC) values. This scenario motivated us to compare the efficacy of different hematological indices.

Bhargava et al. (2020) observed that the Shine & Lal index demonstrated nearly 100% accuracy in diagnosing BTT, which is consistent with the findings of our study (8). However, their study focused on a non-pregnant population with anemia, whereas our research involved pregnant women. Similarly, Arora et al. (2017) conducted a validation study of these hematological indices in a high-volume medical college in New Delhi on pregnant patients and reported that Mentzer’s index was the most reliable for accurately diagnosing BTT. In contrast, our study found the Shine & Lal index to be the most reliable (9, 10). Shukla et al. (2018) examined 2,000 pregnant women with microcytic hypochromic anemia, identifying 63 cases of BTT, and demonstrated that the Shine & Lal index accurately detected nearly all BTT cases, aligning with our findings (11).

In the present study, the Shine & Lal index exhibited a sensitivity of 99% in detecting BTT. This finding aligns with studies conducted by Habib et al. (2022) in Pakistan, as well as research by Wickramaratne et al. and Suman et al., where the Shine & Lal index showed approximately 99% sensitivity, along with high specificity (73.8%), a positive predictive value (PPV) of 35%, and a negative predictive value (NPV) of 0% (12, 13, 14). Additionally, Singh et al. (2023) conducted a study on 460 pregnant women and reported that the Shine & Lal index had higher sensitivity compared to other hematological indices (15). In contrast, Kumari et al. found that 82% of patients exhibited high sensitivity for the Mentzer index (16,17).

BTT follows an autosomal recessive pattern of inheritance, making screening the most effective strategy for reducing its incidence. All individuals diagnosed with BTT and their partners should receive genetic counseling and undergo screening. This practice was followed at our institution (18,19) and yielded favorable results. To optimize outcomes, most patients were screened during the first trimester, allowing for timely partner screening and, if necessary, consideration of pregnancy termination. However, awareness of thalassemia remains limited among at-risk populations in India. Pre-marital screening is neither widely accepted nor encouraged, as also reported by Colah et al. (20). To address this gap, increased efforts by governmental and non-governmental organizations are essential in enhancing public awareness through media campaigns and community outreach programs.

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