Iron deficiency is the most prevalent nutritional deficiency worldwide, affecting a significant portion of the population, particularly in developing countries. It occurs when there is inadequate iron to meet the body's needs, leading to reduced hemoglobin synthesis and anemia [1,2]. Thalassemia, on the other hand, is a genetic disorder characterized by abnormal hemoglobin production. The thalassemia trait, or carrier state, often presents with mild anemia and microcytosis, which can be easily confused with iron deficiency anemia [3]. Both conditions share similar hematological features, making differential diagnosis challenging but essential for appropriate treatment. Prevalence rates of iron deficiency do not vary much between developed and developing nations, with young women, infants, and children being the most affected populations [4]. Latent iron deficiency, characterized by iron deficiency without anemia, often goes unnoticed but can have significant health implications [5]. Latent iron deficiency is marked by a serum ferritin level of ≤15 μg/L and hemoglobin >11 g/dL. Iron deficiency anemia ensues when hemoglobin drops to ≤12 g/dL alongside low serum ferritin. Low serum ferritin levels (<12 ng/mL) are specific indicators of iron deficiency [6]. A complete blood count (CBC) might reveal a normal hemoglobin level in latent iron deficiency but indicate a decrease in serum ferritin levels, suggesting a depletion of iron stores in the body [7]. Peripheral blood smear findings in latent iron deficiency may include anisopoikilocytosis with hypochromia and microcytosis. Occasional target cells may be present, indicating early changes in red cell morphology [8]. This can lead to more severe health issues such as impaired cognitive and physical development in children and reduced work capacity in adults [9]. Latent iron deficiency is a stage when the body's iron stores are depleted, yet hemoglobin levels have not fallen below the threshold for anemia. This stage is critical as it can still affect various bodily functions and overall health [10]. Iron is a vital component of hemoglobin, which is necessary for oxygen transport in the blood. Even without anemia, low iron levels can lead to fatigue, reduced physical performance, and impaired immune function. Iron is crucial for cognitive development and function, and latent iron deficiency in children can result in long-term developmental deficits [11]. The diagnostic approach for detecting latent iron deficiency involves assessing clinical symptoms and laboratory parameters. Symptoms may include fatigue, pallor, and weakness. However, these are non-specific and can also be present in other conditions [12]. Laboratory tests are more definitive, with serum ferritin being the most reliable indicator of iron status. Ferritin reflects the body's iron stores, and low levels indicate depletion [13]. However, ferritin is also an acute-phase reactant and can be elevated in the presence of inflammation, infection, or liver disease, complicating the diagnosis [14]. The ultimate goal of such research is to enhance the accuracy and efficiency of diagnosing these conditions based on CBC and blood smear i.e. latent iron deficiency and thalassemia trait.

OBJECTIVE

To differentiate latent iron deficiency from thalassemia trait among adults presenting with mild anemia and microcytosis using complete blood count parameters, red cell indices, peripheral blood smear findings, and ferritin levels.

This was an observational cross-sectional study conducted in Watim Medical And Dental College from March 2025 to September 2025, after obtaining approval from the Institutional Review Committee of Riphah International University, Islamabad. A total of 285 participants were included in the study. The sample size was calculated using Cochran’s formula for estimating a population proportion, assuming a prevalence of 25%, a confidence level of 95%, and a margin of error of 5%. A non-probability convenience sampling technique was employed. Adult male and female patients referred from the outpatient departments of Medicine, Surgery, Gynecology, and Obstetrics with hemoglobin levels ranging from 10 to 12 g/dL were included in the study. Patients with a history of chronic diseases, those receiving hematinic therapy, and individuals with a recent history of blood transfusion were excluded. Data Collection After obtaining written informed consent, a detailed clinical history and physical examination were performed, and the findings were documented on a structured data collection proforma. Venous blood samples were collected from all participants under aseptic conditions. Complete blood count analysis was performed using the Mindray BC-5000 automated hematology analyzer to determine hemoglobin concentration, total red blood cell count, platelet count, differential leukocyte count, red cell indices, and red cell distribution width. Peripheral blood smears were prepared on clean, labeled glass slides and stained using Leishman stain. The stained smears were examined microscopically to assess red blood cell morphology and identify features suggestive of iron deficiency or thalassemia trait, including microcytosis, hypochromia, anisopoikilocytosis, and target cells. Based on the complete blood count findings and peripheral smear examination, participants were provisionally classified into either the latent iron deficiency group or the thalassemia trait group. Serum Ferritin Estimation Serum ferritin levels were measured as a confirmatory test to differentiate latent iron deficiency from thalassemia trait. Approximately 2 mL of venous blood was collected in a plain tube, and serum was separated by centrifugation at 5000 rpm for five minutes. Ferritin estimation was performed using the Rayto RT-6000 Microplate ELISA Reader according to the manufacturer's instructions. Low serum ferritin levels were considered indicative of latent iron deficiency, whereas normal or elevated ferritin levels supported the diagnosis of thalassemia trait. Fig 1: Hematology Analyzer: Mindray BC 5000 Fig 2: Mindray BC 5000 with scanner and rotator Fig 3: Slides Preparation for Peripheral Smear Data Quality Control To ensure accuracy and reliability, all blood samples were checked for correct labeling, adequate volume, absence of hemolysis, and clot-free status before processing. Standard operating procedures were followed throughout sample collection, handling, transportation, storage, and analysis. Internal quality control procedures and hematology analyzer controls were performed regularly to monitor instrument performance. All laboratory findings were recorded carefully on the data collection forms. Data Analysis Data were entered and analyzed using the Statistical Package for Social Sciences (SPSS) version 27.0. Continuous variables such as age, hemoglobin level, red blood cell indices, and serum ferritin levels were expressed as mean ± standard deviation. Categorical variables were presented as frequencies and percentages. The Chi-square test was used to compare categorical variables between groups. A p-value of less than 0.05 was considered statistically significant. Ethical Considerations Ethical approval was obtained from the Institutional Review Committee of Riphah International University, Islamabad. Written informed consent was obtained from all participants before enrollment. Participant confidentiality and anonymity were maintained by assigning unique identification codes to all study records. All collected information was kept confidential and used solely for research purposes.

Data were collected from 285 patients, mean age was 24.10 ± 3.43 years, with an age range of 18 to 29 years. There were 145 (50.9%) males and 140 (49.1%) females, indicating a nearly equal gender distribution. Regarding occupation, 128 (44.9%) participants were students, 104 (36.5%) were unemployed, and 53 (18.6%) were employed in private jobs.

Table 1. Demographic Characteristics of Study Participants (n=285)

Fig 4: Frequency of gender graph

The mean body weight of the participants was 70.51 ± 10.88 kg. The average hemoglobin level was 10.61 ± 0.63 g/dL, with values ranging from 10.00 to 12.00 g/dL. The mean white blood cell count was 7.42 ± 2.07 ×10⁹/L, while the mean red blood cell count was 4.23 ± 0.84 ×10¹²/L. The mean MCV was 73.89 ± 4.59 fL and the mean MCH was 24.22 ± 1.99 pg, indicating a predominantly microcytic hypochromic blood picture. The mean RDW was 13.04 ± 1.17%, and the mean platelet count was 296.64 ± 86.32 ×10⁹/L. The average serum ferritin level was 49.43 ± 40.98 ng/mL, with values ranging from 10.04 to 148.20 ng/mL.

Table 2. Hematological and Biochemical Profile of Study Participants (n=285)

Based on complete blood count parameters, peripheral blood smear findings, and serum ferritin levels, 175 (61.4%) participants were classified as having latent iron deficiency, while 110 (38.6%) were diagnosed with thalassemia trait.

Table 3. Classification of Participants Based on CBC, Peripheral Smear and Ferritin Findings (n=285)

All 175 participants diagnosed with latent iron deficiency demonstrated microcytosis, hypochromia, anisocytosis, and poikilocytosis on peripheral blood smear examination.

Table 4. Peripheral Smear Findings According to Diagnostic Classification

A strong positive correlation was observed between hemoglobin concentration and red blood cell count (r = 0.726, p < 0.001), indicating that higher RBC counts were associated with higher hemoglobin levels. A moderate positive correlation was also found between hemoglobin concentration and serum ferritin levels (r = 0.487, p < 0.001).

Table 5. Correlation of Hemoglobin with RBC Count and Ferritin Levels

Chi-square analysis revealed no statistically significant association between gender and hemoglobin status. The Pearson Chi-square value was 0.245 with a p-value of 0.620. Similarly, Fisher’s exact test showed a p-value of 0.629.

Table 6. Chi-Square Test for Association Between Gender and Hemoglobin Status

Fig 5: Peripheral blood smear findings crosstabulation bar chart

Our study was conducted to evaluate and determine the detection of latent iron deficiency & Thalassemia trait with red cell indices and peripheral blood smear. A total of 285 samples were collected and analyzed. Out of 285 participants, the male participants were 50.9% while the females were 49.1%. The mean age of participants recorded was 24±3 years. In our study the majority of population belong to the age group of 21-25 years with minimum age was 18 years and the maximum was 29 years. Out of 285, 122 participants belonged to age group ranging from 21-25 years conducted a study on 620 participants with majority of participants were from the age range of 21-36 years [13]. Similar to our study, all the participants underwent serum iron and ferritin with individuals showing low hemoglobin. All those participants underwent morphology with MCV, RBC count and red cell distribution width index. The study concluded a total of 135 individuals with hypochromic microcytic anemia having the normal hemoglobin F and hemoglobin A2 <3.2. out of it, 93 participants were diagnosed with iron deficiency anemia and 32 with Beta thalassemia trait. As compared to the iron deficiency anemia, the RBC count was relatively higher and MCV was much lower in beta thalassemia trait patients. In a nutshell, the RDWI (Red cell distribution width index was a reliable index in differentiating the iron deficiency with the Beta Thalassemia trait [14,15]. In our study, different parameters were recorded such as Hemoglobin, Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH) and Red cell distribution width (RDW) [16]. The mean hemoglobin calculated in our patients was 10.61 mg/dl and MCV was reported to be 73.89fl which interprets that the participants have hypochromic and microcytic blood picture. Moreover, the RDW of 13.04% indicates the variability of red cell size which supports the evidence that the participants were suffering from the iron deficiency [17]. Furthermore, the participants reported with low ferritin level of 49.43 ng indicates more of a latent iron deficiency. The thalassemia trait is distinguished from iron deficiency by normal or high ferritin levels, even in the presence of microcytic hypochromic blood findings. While low MCV with normal or high ferritin indicates thalassemia trait, low MCV and low ferritin alone is more suggestive of iron deficiency [18]. In another study conducted by on 131 individuals who underwent RBC indices such as RBC count, RDWI, MCV and MCH along with different index such as Mentzer index, Shine and Lal index, sensitivity, specificity, positive & negative predictive values and compared them with cutoff values available in the literature. As a result, all the values were reported to be higher than the cutoff values indicating 50 participants diagnosed with Iron deficiency anemia and Beta thalassemia trait and alpha thalassemia trait in 31 participants [19]. In our study, the mean of Hemoglobin levels were calculated with a low range of 10.61 g/dl indicating the majority of population as suffering from hypochromic microcytosis. The WBC count and platelet count were reported to be in the normal limit hence indicating no association in diagnosing the iron deficiency and thalassemia trait. According to a study [20], 180 participants were selected from a tertiary care hospital highly suspected of anemia. Iron studies were performed and the participants were divided in to three groups LID (Tfsat 11g/dL; n=52), IDA (Tfsat <20%, Hb <11g/dL; (n=84) and controls (Tfsat >20%, Hb >11g/dL; n=44). The comparison between the anemic group and the control group revealed that all RBC indices were found to be very significantly lower including the reticular site hemoglobin and these variables in the anemic group were lower compared to the later deficit group except the MCHC and reticulocyte count. The study concluded that the reticulocyte count index can be used as a valuable indicator in diagnosing latent iron deficiency and iron deficiency anemia. This variable was reported to as readily available and inexpensive as added benefits. Red cell indices are crucial diagnostic tools with easy accessibility for distinguishing between iron deficiency anemia (IDA) and beta thalassemia trait [21].

The analysis of the data reveals a significant prevalence of latent iron deficiency among the study population, characterized by a hypochromic microcytic blood picture and low ferritin levels. In contrast, participants classified with thalassemia trait also exhibited microcytic red blood cells but typically have normal or elevated ferritin levels. While both conditions share similar hematological features, they arise from distinct underlying causes related to iron metabolism. The findings underscore that latent iron deficiency, a major health problem in society, requires greater attention due to its widespread nature and potential long-term impact. Additionally, the absence of a significant association between gender and hemoglobin levels suggests that both males and females are equally affected by these conditions.

1. Auerbach M, Adamson JW. How we diagnose and treat iron deficiency anemia. Am J Hematol. 2015;91(1):31–38.
2. Benson CS, Shah A, Stanworth SJ, et al. The effect of iron deficiency and anaemia on women’s health. Anaesthesia. 2021;76(S4):84–95.
3. Elstrott B, Khan L, Olson S, et al. The role of iron repletion in adult iron deficiency anemia and other diseases. Eur J Haematol. 2019;104(3):153–161.
4. Antonino Davide Romano, Paglia A, Bellanti F, et al. Molecular aspects and treatment of iron deficiency in the elderly. Int J Mol Sci. 2020;21(11):3821.
5. Mensah C, Sheth S. Optimal strategies for carrier screening and prenatal diagnosis of α- and β-thalassemia. Hematology. 2021;2021(1):607–613.
6. Weyand AC, Chaitoff A, Freed GL, et al. Prevalence of iron deficiency and iron-deficiency anemia in US females aged 12–21 years. JAMA. 2023;329(24):2191.
7. Ahmad S, Zaidi N, Mehdi SR, et al. Indices in differentiating iron deficiency anemia from thalassemia trait. Asian J Med Sci. 2021;12(10):81–86.
8. Kaur S, Singh KP. Anaemia in preschool children: its correlation with pica. J Nepal Paediatr Soc. 2022;42(1):8–12.
9. Camaschella C. Iron deficiency. Blood. 2019;133(1):30–39.
10. Munkongdee T, Chen P, Winichagoon P, et al. Update in laboratory diagnosis of thalassemia. Front Mol Biosci. 2020;7:74.
11. Rashwan N, El-Abd Ahmed A, Hassan M, et al. Hematological indices in differentiation between iron deficiency anemia and beta-thalassemia trait. Int J Pediatr. 2022;10(1):15285–15295.
12. Taher AT, Weatherall DJ, Cappellini MD. Thalassaemia. Lancet. 2018;391(10116):155–167.
13. Gattermann N, Muckenthaler MU, Kulozik AE, et al. The evaluation of iron deficiency and iron overload. Dtsch Arztebl Int. 2021;118(49):847–856.
14. An R, Avanaki AA, Thota P, et al. Point-of-care diagnostic test for beta-thalassemia. Biosensors. 2024;14(2):83.
15. O’Toole F, Sheane R, Reynaud N, et al. Screening and treatment of iron deficiency anemia in pregnancy. Int J Gynecol Obstet. 2023;66(1):214–227.
16. Suria N, Kaur R, Mittal K, et al. Utility of reticulocyte haemoglobin content in early diagnosis of latent iron deficiency. Vox Sang. 2021;117(4):495–503.
17. Pasricha SR, Tye-Din J, Muckenthaler MU, et al. Iron deficiency. Lancet. 2021;397(10270):233–248.
18. Sun A, Chang JYF, Jin YT, et al. Differential diagnosis between iron deficiency anemia and thalassemia trait-induced anemia. J Dent Sci. 2023;18(4):1963–1964.
19. Benz EJ, Sankaran VG. Thalassemia. Hematol Oncol Clin North Am. 2023;37(2):xiii–xv.
20. Shalini Balendran, Forsyth C. Non-anaemic iron deficiency. Aust Prescr. 2021;44(6):193–196.
21. Tabassum S, Khakwani M, Fayyaz A, et al. Role of Mentzer index for differentiating iron deficiency anemia and beta thalassemia trait in pregnant women. Pak J Med Sci. 2022;38(4):878–882.

1. Auerbach M, Adamson JW. How we diagnose and treat iron deficiency anemia. Am J Hematol. 2015;91(1):31–38.
2. Benson CS, Shah A, Stanworth SJ, et al. The effect of iron deficiency and anaemia on women’s health. Anaesthesia. 2021;76(S4):84–95.
3. Elstrott B, Khan L, Olson S, et al. The role of iron repletion in adult iron deficiency anemia and other diseases. Eur J Haematol. 2019;104(3):153–161.
4. Antonino Davide Romano, Paglia A, Bellanti F, et al. Molecular aspects and treatment of iron deficiency in the elderly. Int J Mol Sci. 2020;21(11):3821.
5. Mensah C, Sheth S. Optimal strategies for carrier screening and prenatal diagnosis of α- and β-thalassemia. Hematology. 2021;2021(1):607–613.
6. Weyand AC, Chaitoff A, Freed GL, et al. Prevalence of iron deficiency and iron-deficiency anemia in US females aged 12–21 years. JAMA. 2023;329(24):2191.
7. Ahmad S, Zaidi N, Mehdi SR, et al. Indices in differentiating iron deficiency anemia from thalassemia trait. Asian J Med Sci. 2021;12(10):81–86.
8. Kaur S, Singh KP. Anaemia in preschool children: its correlation with pica. J Nepal Paediatr Soc. 2022;42(1):8–12.
9. Camaschella C. Iron deficiency. Blood. 2019;133(1):30–39.
10. Munkongdee T, Chen P, Winichagoon P, et al. Update in laboratory diagnosis of thalassemia. Front Mol Biosci. 2020;7:74.
11. Rashwan N, El-Abd Ahmed A, Hassan M, et al. Hematological indices in differentiation between iron deficiency anemia and beta-thalassemia trait. Int J Pediatr. 2022;10(1):15285–15295.
12. Taher AT, Weatherall DJ, Cappellini MD. Thalassaemia. Lancet. 2018;391(10116):155–167.
13. Gattermann N, Muckenthaler MU, Kulozik AE, et al. The evaluation of iron deficiency and iron overload. Dtsch Arztebl Int. 2021;118(49):847–856.
14. An R, Avanaki AA, Thota P, et al. Point-of-care diagnostic test for beta-thalassemia. Biosensors. 2024;14(2):83.
15. O’Toole F, Sheane R, Reynaud N, et al. Screening and treatment of iron deficiency anemia in pregnancy. Int J Gynecol Obstet. 2023;66(1):214–227.
16. Suria N, Kaur R, Mittal K, et al. Utility of reticulocyte haemoglobin content in early diagnosis of latent iron deficiency. Vox Sang. 2021;117(4):495–503.
17. Pasricha SR, Tye-Din J, Muckenthaler MU, et al. Iron deficiency. Lancet. 2021;397(10270):233–248.
18. Sun A, Chang JYF, Jin YT, et al. Differential diagnosis between iron deficiency anemia and thalassemia trait-induced anemia. J Dent Sci. 2023;18(4):1963–1964.
19. Benz EJ, Sankaran VG. Thalassemia. Hematol Oncol Clin North Am. 2023;37(2):xiii–xv.
20. Shalini Balendran, Forsyth C. Non-anaemic iron deficiency. Aust Prescr. 2021;44(6):193–196.
21. Tabassum S, Khakwani M, Fayyaz A, et al. Role of Mentzer index for differentiating iron deficiency anemia and beta thalassemia trait in pregnant women. Pak J Med Sci. 2022;38(4):878–882.