INTRODUCTION

Anemia is defined as a reduction of the hemoglobin concentration or red blood cell (RBC) volume below the range of values occurring in healthy persons. “Normal” hemoglobin and hematocrit (packed red cell volume) vary substantially with age and sex. There are also racial differences. Anemia is a significant global health problem affecting children and reproductive age women¹. It has been estimated that 30% of the global population suffers from iron deficiency anemia (IDA) , and most of those affected live in the developing countries^2,3. According to the World Health Organization (WHO), it is reported that 30% of children in the 0 to 4 years age group and 48% of children in the 5 to 14 years age group are anemic in developing countries^4,5.

An initial step in classification of anemia separates them into three groups on the basis of average cell size: the macrocytic, microcytic, and normocytic anemia. Anemia is classified as macrocytic if Mean corpuscular volume (MCV) exceeds 100 femtolitre ( fl). Usually, the Mean corpuscular hemoglobin (MCH) is also increased whereas the Mean corpuscular hemoglobin concentration (MCHC) remains within normal limits. Microcytic anemia is identified when the MCV is less than 80 fl in adults. The anemia is normocytic when the indices are within normal limits, with an MCV between 80 and 100 fl. In children, MCV values vary as a function of age, and, correspondingly, the definition of microcytic, normocytic, and macrocytic differ accordingly⁶. Hypochromia is a reduction of the staining of the red cell. There is an increase in central pallor, which occupies more than the normal approximate one‐third of the red cell diameter⁷.   

β thalassemia trait and Iron deficiency anemia (IDA) are the most commonly encountered with hypochromic microcytic anemia^8,9. Other diseases result in the production of microcytic hypochromic red blood cells, such as lead poisoning, chronic inflammation, sideroblastic anemia^10-13. The morphological findings in both the IDA and β thalassemia trait are at times so close that it is really difficult to differentiate one from the other¹⁴. The differentiation between IDA and β thalassemia trait is important because of two main reasons, first, because hemoglobin (Hb) won’t improve in β thalassemia trait if it is misdiagnosed as IDA and unnecessary iron being prescribed by the attending physician that leads to iron overload. The second grave reason is that misdiagnosed β thalassemia trait as IDA may get married to a β thalassemia trait, resulting in homozygous or thalassemia major in the offspring^14-16. Though both of them are microcytic and hypochromic so, it is difficult to differentiate one from another only by simple hematological parameters obtained from automated blood cell analyzers. Additional complicated and expensive tests are necessary, including serum iron (SI), serum ferritin (SF), transferrin saturation, total iron binding capacity, and levels of hemoglobin A2 (HbA2)^17-20. Hemoglobin analysis and iron study are costly and not available routinely in low resource settings, whereas the automated blood cell counter is widely used in routine practice. Thus, based on red cell indices and formulas, the screening of β thalassemia trait and iron deficiency anemia can be done without additional cost to the medical system^21,22. These kind of discrimination indices have been established since 1970s^23-34.

The thalassemias are a group of congenital anemias that have in common deficient synthesis of one or more of the globin subunits of the normal human hemoglobins (Hb). According to the chain whose synthesis is impaired, the most common thalassemias are called alpha-, beta-, gamma-, or delta beta-thalassemias. These subgroups have in common an imbalanced globin synthesis, with the consequence that the globin produced in excess is responsible for ineffective erythropoiesis (intramedullary destruction of erythroid precursors) and hemolysis (peripheral destruction of red cells)³⁵. β thalassemia are more common than other forms and usually produce severe anemia in their homozygous and compound heterozygous state³⁶. But β thalassemia trait are symptom free with mild anemia except in periods of stress such as pregnancy³⁶. The β thalassemias are distributed widely in Mediterranean populations, the Middle East, parts of India and Pakistan, and throughout Southeast Asia³⁷ .

Iron deficiency is the state in which the content of iron in the body is less than normal. Iron deficiency anemia, the most advanced stage of iron deficiency, is characterized by absent iron stores, low serum iron concentration, low transferrin saturation, and low blood hemoglobin concentration^38,39. Iron deficiency is particularly common in young children and pregnant women. Iron deficiency was detected in 9% of infants and toddlers, with anemia in approximately one third of those children. In infancy, the occurrence of iron deficiency was equal in both sexes. It is usually detected between the ages of 6 and 20 months. The peak incidence was at a younger age in infants born prematurely than in those born at term, because premature infants do not have full opportunity to acquire maternal iron during the third trimester⁴⁰.

A definitive differential diagnosis between ?-thalassemia trait and IDA is based on the result of hemoglobin A2 (HbA2) electrophoresis, serum iron levels, and a ferritin calculation⁴¹. The iron study including the measurement of serum iron (SI), total iron biding capacity (TIBC), serum ferritin and transferrin saturation⁴². Ferritin reflects body iron stores, measurement of serum ferritin has been widely adopted as a test for iron deficiency and iron overload⁴³. In most normal adults, serum ferritin concentrations lie within the range of 15–300 mg/l. In children, mean levels of storage iron are lower and a 12-15 mg/l threshold for serum ferritin has been found to be appropriate for detecting iron deficiency⁴³. Estimation of serum iron concentration is recommended by the International Council for Standardization in Haematology (ICSH) and is based on the development of a coloured complex when ferrous iron released by serum^44,45. The iron-binding capacity is a measure of the amount of transferrin in circulating blood. Normally, there is enough transferrin present in 100 mL serum to bind 4.4 to 8.0 μmol (250 to 450 mcg) of iron; because the normal serum iron concentration is approximately 1.8 μmol/dL (100 mcg/dL), transferrin may be found to be approximately one-third saturated with iron. The unsaturated or latent iron-binding capacity (UIBC) is easily measured with radioactive iron or by spectrophotometric techniques. The sum of the UIBC and the plasma iron represents total iron-binding capacity (TIBC). In iron-deficiency anemia, TIBC are often increased and serum iron concentrations are decreased so that transferrin saturation of 15 percent or less is usually found³⁸.

The confirmation test for ?-thalassemia trait diagnosis needs to be performed by quantitation of HbA2 levels on high performance liquid chromatography (HPLC)⁴⁶. HPLC is a sensitive and precise method for the identification of  HbA2, fetal hemoglobin (HbF) and abnormal haemoglobins. It has become the method of choice for thalassaemia screening because of its speed and reliability⁴⁷.

Electronic cell counters have been used to determine red cell indices as a first indicator of ?-thalassemia trait. The purpose of using indices to discriminate anemia is to detect subjects who have a high probability of requiring appropriate follow-up and to reduce unnecessary investigative costs. Since 1970, a number of complete blood count indices have been proposed as simple and inexpensive tools to determine whether a blood sample is more suggestive of ?-thalassemia trait or IDA^48-51. Those indices we used here are mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW) and Mentzer index. Mean corpuscular volume (MCV) is average volume of a red blood corpuscle⁵².Mentzer index is a discrimination index between IDA & ?-thalassemia trait. It is calculated by MCV/RBC count⁵³.

There are only a few studies conducted in pediatric group comparing reliability of these methods. The aim of this study is to evaluate retrospectively the diagnostic reliability of some RBC indices and formulas in the differentiation of TT and IDA in pediatric age group patients^53,54.

                                AIMS AND OBJECTIVES 

1. To compare the RBC indices ( MCV, MCH, MCHC ), RDW and Mentzer index between iron deficiency anemia and beta thalassemia trait.

• To evaluate the suspected cases of early ?-thalassemia trait by those hematological profile in resource poor setting.

                             REVIEW OF LITERATURE

Anemia defined as decrease in total number of circulating red cells with decrease in hemoglobin when compared with normal for that age group and sex.

According to WHO criteria, adult males hemoglobin (Hb) < 13g/dl, adult females Hb < 12g/dl, infant and children upto 12 years Hb < 11g/dl and pregnant women Hb < 11g/dl.

For children aged 1- 12 years, cut-off value of hemoglobin to define anemia are (Brugnara C, Oski FJ, Nathan DG: Nathan and Oski’s hematology of infancy and childhood, ed 7, Philadelphia, 2009, WB Saunders, p. 456)  –

Age group ( year )	Hemoglobin ( g/dl )
1	< 11
2 – 4	< 11
5 – 7	< 11.5
8 – 11	< 12

CLASSIFICATION OF ANEMIA :

Two types of classifications are there – Etiological and morphological.

According to etiological classification anemia is subdivided into two groups – A) Anemia due to impaired red cell production and B) Anemia due to increased red cell destruction.

According to morphological classification it can be subdivided into three groups depends on mean corpuscular volume (MCV) values. Those are – microcytic, normocytic and macrocytic anemia. Microcytic anemia defined as MCV < 80 femtolitre (fl). In normocytic anemia MCV values are with in (81-99) fl range and in macrocytic anemia MCV > 100 fl. But for pediatric population the cut off value of MCV is different for different age group. According to pediatric age group definition of microcytic anemia (Brugnara C, Oski FJ, Nathan DG: Nathan and Oski’s hematology of infancy and childhood, ed 7, Philadelphia, 2009, WB Saunders, p. 456)  –                                                   

Age group ( year )	MCV values ( fl )
1	< 70
2 – 4	< 73
5 – 7	< 75
8 – 11	< 76

Hypochromia defined as reduction of the staining of the red cell and increase in central pallor, which occupies more than one‐third of the red cell diameter.

The most common types of microcytic hypochromic anemia are iron deficiency anemia (IDA) and beta thalassemia trait (TT). Other diseases result in the production of microcytic hypochromic red blood cells, such as lead poisoning, chronic inflammation and sideroblastic anemia.

BETA THALASSEMIA TRAIT :

Thalassemia is not a single disease but a group of disorders, each resulting from an inherited abnormality of globin production⁵⁵ .The conditions form part of the spectrum of diseases known collectively as the hemoglobinopathies, which can be classified broadly into two types.

1. The first subdivision consists of conditions, such as sickle cell anemia, that result from an inherited structural alteration in one of the globin chains. Although such abnormal hemoglobins may be synthesized less efficiently or broken down more rapidly than normal adult hemoglobin, the associated clinical abnormalities result from the physical properties of the abnormal hemoglobin.
2. The second major subdivision of the hemoglobinopathies, the thalassemias, consists of inherited defects in the rate of synthesis of one or more of the globin chains. The result is imbalanced globin chain production, ineffective erythropoiesis, hemolysis, and a variable degree of anemia.

According to the chain whose synthesis is impaired, the common thalassemias are called alpha-, beta-, gamma-, or delta beta-thalassemias. These subgroups have in common an imbalanced globin synthesis, with the consequence that the globin produced in excess is responsible for ineffective erythropoiesis (intramedullary destruction of erythroid precursors) and hemolysis (peripheral destruction of red cells). Two major categories are the alpha- and beta- thalassemias.

Functionally, some thalassemia mutations cause a complete absence of globin chain synthesis and these are called alpha0- or beta0- thalassemias; in others, the globin chain is produced at a reduced rate and these are designated as alpha+ or beta+ thalassemias³⁶.

This beta+ thalassemia are  the heterozygous state of beta–thalassemia/beta thalassemia trait which is usually identified during family studies of patients with more severe forms of beta–thalassemia, population surveys, or, most frequently, by the chance finding of the characteristic hematologic changes during a routine study⁵⁶.

History and Geographic Distribution:

Whipple coined the phrase thalassic anemia^57,58 and condensed it to thalassemia, from θαλασσα(“the sea”), because early patients were all of Mediterranean background. In 1925, Cooley and Lee⁵⁹ first described a form of severe anemia that occurred early in life and was associated with splenomegaly and bone changes. In 1932, George H. Whipple and William L. Bradford⁶⁰ published a comprehensive account of the pathologic findings in this disease.

The true genetic character of the disorder became fully appreciated after 1940. The disease described by Cooley and Lee is the homozygous state of an autosomal gene for which the heterozygous state is associated with much milder hematologic changes. The severe homozygous condition became known as thalassemia major. The heterozygous states, thalassemia trait, were designated according to their severity as thalassemia minoror minima. Later, the term thalassemia intermediawas used to describe disorders that were milder than the major form but more severe than the traits^61,62.

The beta-thalassemias are distributed widely in Mediterranean populations, the Middle East, parts of India and Pakistan, and throughout Southeast Asia. The disease is common in Tajikistan, Turkmenistan, Kyrgyzstan, and the People’s Republic of China. Because of the extensive migration from areas of high gene frequency such as the Mediterranean region (e.g., Italy, Greece), Africa, and Asia to the Americas, the alpha– and beta-thalassemia genes and clinical disease are relatively common, especially in North, but also South, America. The beta-thalassemias are rare in Africa, except for isolated pockets in West Africa, notably Liberia, and in parts of North Africa. However, beta-thalassemia occurs sporadically in all racial groups and has been observed in the homozygous state in persons of pure Anglo-Saxon heritage. Thus, a patient’s racial background does not preclude the diagnosis^63,64.

It has been described in high frequencies (10% to 20%) in Indian and Kurdish Jews⁶⁵. Among Indians, frequencies between 3.5% and 14.9% have been reported⁶⁶.

Genetic Basis Of Disease:

Synthesis of hemoglobin, the molecule used for oxygen transport, is directed by two gene clusters: The alphalocus, which contains the embryonic zetagene, plus the two adult alphagenes; and the betacluster, which contains the embryonic epsilon, the fetal Ggammaand Agamma, and the adult delta and betagenes (Fig. 1). Different hemoglobins are produced during development and two globin gene switches take place: the embryonic to fetal switch (epsilonto gammaand zetato alpha), which starts very early in pregnancy and is completed at 10 weeks of gestation; and the fetal to adult switch (gammato beta), which occurs during the perinatal period⁶⁷.

The beta-globin gene is located in the short arm of chromosome 11 in a region containing also the deltagene, the embryonic epsilongene, the fetal Ggammaand Agammagenes, and the pseudogene beta1 (Fig.1).The five functional globin genes are arranged in the order of their developmental expression. The complete sequencing of the beta- globin gene complex has shown interspersed repetitive sequences (microsatellite repeats of [CA]n, an [ATTTT]n repeat, AluI and KpnI families of repeat DNA sequences), which may play a role in the generation of the deletions of the betacluster⁶⁸. The appropriate expression of the different beta-like globin genes in erythroid tissues during development depends on a major regulatory region highly conserved in mammals named the locus control region (LCR), located 5 to 25 kb upstream from the epsilon globin gene⁶⁹.Five DNAase hypersensitive sites (HSs) have been described in this region and each HS contains one or more binding motifs for erythroid-specific transcriptional activator1 (GATA-1 and NF-E2) and for ubiquitous DNA-binding proteins⁷⁰.The importance of the LCR for the control of beta-like globin geneexpression has also been suggested by a series of naturally occurring deletions that totally or partially remove the HS sites and result in the inactivation of the intact downstream beta-globin gene⁷¹.

Beta-Thalassemia mutations result in either a complete absence of beta-globin chains (beta 0-thalassemia) or in a largely variable reduction of beta-globin output (beta +-thalassemia). More than 200 different mutations producing beta-thalassemia are there; the large majority are point mutations in functionally important sequences of the beta-globin gene(Table-3).

Chr 11

 

3’

5’

Alpha2Delta2HbA2Alpha2beta2HbA

Zeta2epsilon2Hb Gower I  Alpha2epsilon2Hb Gower II

Zeta2Ggamma2Zeta2Agamma2Hb Portland Alpha2Ggamma2Alpha2Agamma2HbF

Chr 16

Adult

Fetus

Embryos

3’

5’

Fig 1 – Alpha- and beta-globin gene cluster and hemoglobins (Hbs) produced during development. LCR- Locus Control Region. ( Wintrobe’s Clinical Hematology; Thirteenth edition; Caterina Borgna Pignatti)

    Table 3 – Mutations causing beta thalassemia. ( Wintrobe’s Clinical Hematology; Thirteenth edition; Caterina Borgna Pignatti )

Pathogenesis:

The molecular defects in beta thalassemia result in absent or reduced beta chain production while alpha globin synthesis is unaffected. The imbalance in globin chain production leads to an excess of alpha chains. The free alpha globin chains are highly unstable and precipitate in red cell precursors, forming intracellular inclusions  that interfare with red cell maturation. There is variable degree of intramedullary destruction of erythroid precursors( i.e. ineffective erythropoiesis ) that characterizes all beta thalassemias. Those red cells that mature and enter the circulation contain alpha chain inclusions that interfare with their passage through the microcirculation, particularly spleen. The degradation products of excess alpha chains , particularly haem and iron, produce a wide range of deleterious effects on red cell membrane proteins and lipids, manifested by marked abnormalities of  electrolyte hemostasis and membrane deformability. The end result is an extremely rigid red cell with a shortened survival.

Thus, the anemia from beta thalassemia results from combination of ineffective erythropoiesis and hemolysis. It stimulates erythropoietin production, which causes expansion of the bone marrow and may lead to serious deformities of the skull and long bones. Because the spleen is being constantly bombarded with abnormal red cells , it hypertrophies. The resulting splenomegaly, together with bone marrow expansion, causes a major increase in plasma volume, which also contributes to the anemia.

HbF production almost ceases after birth. However some adult red cell precursors retain the ability to produce a variable amount of gamma chains. Because the latter can combine with excess alpha chains to form HbF, cells which make relatively more gamma chains in the bone marrow are partly protected against the deleterious effect of alpha chain precipitation. These F cells come under selection in the marrow and peripheral blood and thus individuals with beta thalassemia have variable increases in HbF due to selective survival of these F cells. In some cases there is also a genuine increase in HbF production, as well as selection of F cells due to coinheritance of genetic determinant or quantitative trait loci, which increase HbF production.

It follows therefore that if the anemia is corrected with blood transfusion, the erythropoietin drive is shut off, growth and development are normal, bone deformities do not occur and splenomegaly is less marked. On the other hand each unit of blood contains 200-250 mg iron, and with regular transfusion there is steady accumulation of iron in the liver, endocrine glands and myocardium. Thus, although well transfused thalassaemic children grow and develop normally, they die of iron overload unless steps are taken to remove iron.

                                          Fig 2 – Pathogenesis of beta thalassemia

                      ( Williams Hematology; Nineth edition; David J Weatherall )

Clinical Features:

The classical heterozygous carrier of beta-thalassemia is usually asymptomatic, and the diagnosis is made by chance, because of positive family history or during population screening⁷³. Anemia is mild or absent. These patients have some of the complications characterized in untransfused thalassemia major patients, but the severity varies depending on the degree of ineffective erythropoiesis. They can develop medullary hyperplasia, hepatosplenomegaly, hematopoietic pseudotumors, pulmonary hypertension, thrombotic events and growth failure. Many patients develop hemosiderosis secondary to increased gastrointestinal absorption of iron requiring chelation. Extramedullary hematopoiesis can occur in the vertebral canal, compressing the spinal cord and causing neurologic symptoms⁷⁴.

 Serum bilirubin levels present considerable variation. Increased risk of gallstones has also been observed with the beta-thalassemia trait. A partially improved cardiovascular risk profile has been observed in terms of low hematocrit, low-density lipoprotein (LDL) cholesterol, and apo-B in carriers of beta-thalassemia⁷⁵.

Laboratory Diagnosis:

1. Routine blood tests-  Hemoglobin(Hb) – 6-10 g/dl

                                  Total red blood cell count – Normal/ Elevated

                                  MCV(Mean corpuscular volume) – Low

                                  MCH(Mean corpuscular hemoglobin) – Low

                                  RDW (Red cell Distribution Width) – Normal

• Peripheral blood smear – Microcytic hypochromic red cells. Few red cells show basophilic stippling
• Reticulocyte count – Increased
• HPLC(High Performance Liquid Chromatography) – (Diagnostic test)

HbA2 – Increased (3.5-7%)

HbF – Increased in 50% cases (1-3%).

IRON DEFICIENCY ANEMIA:

Iron deficiency is the state in which the content of iron in the body is less than normal.

Iron depletion is the earliest stage of iron deficiency, in which storage iron is decreased or absent but serum iron concentration, transferrin saturation, and blood hemoglobin levels are normal. Iron deficiency without anemia is a more advanced stage of iron deficiency, characterized by absent storage iron, usually low serum iron concentration and transferrin saturation, but without frank anemia.

Iron-deficiency anemia, the most advanced stage of iron deficiency, is characterized by absent

iron stores, low serum iron concentration, low transferrin saturation, and low blood hemoglobin concentration.

Iron-deficiency anemia is the most common anemia worldwide, and is especially prevalent in women and children in regions where meat intake is low, food is not fortified with iron, and malaria, intestinal infections, and parasitic worms are common^76-78.

Iron Metabolism:

1. Iron Absorption – Much of the dietary iron is non-haem iron from cereals with a lesser component f haem iron from meat and fish. Iron is released from protein complexes by acid and proteolytic enzymes in the stomach and small intestine. Iron is maximally absorbed from duodenum and less well from the jejunum. It is regulated both the stages-

1. Mucosal uptake – Non-haem iron is released from the food as Fe³⁺ and reduced by duodenal cytochrome b₁(DCytb) to Fe²⁺. This is transported across the brush border membrane by DMT1(Divalent Metal Transporter) which is upregulated in iron deficiency. Iron enters into the labile pool and some may be incorporated into ferritin and lost when the cells are exfoliated.

Haem iron is initially bound by haem receptors at the brush border membrane and released intracellularly by haem oxygenase which entering into the labile iron pool and ultimately follow the common pathway with iron of non-haem origin.

• Transfer into blood – Iron is transported across the serosal membrane by ferroportin. Hephaestin presents in baso-lateral surface which converts Fe²⁺ to Fe³⁺. The iron is brought out by transferrin as Fe³⁺ and transported into portal blood⁷⁹.

Fig 3 – Pathways of iron absorption. Wintrobe’s Clinical Hematology; Thirteenth edition; John P Greer.

 

• Iron Transport – Iron in the blood is carried all over the body by transferrin. Each molecule of transferring carries two atoms of iron. Iron is released from transferrin through transferrin receptor pathway in the marrow for erythropoiesis and transferrin is reutilized to carry iron⁸⁰.

About 85% of transferrin iron normally enters developing red cells via transferrin receptors. This transferrin-receptor complex is taken up by a process of receptor mediated endocytosis. The iron is released at the low pH of the endosome , reduced from Fe³⁺ to Fe²⁺ by STEAP3 ( a ferrireductase ), before the apotransferrin and receptor are recycled to the plasma and the cell membrane, respectively. Iron release from the endosome is via DMT1 and it is transported into mitochondria by mitoferritin or enters ferritin. 80-90% iron taken into developing erythroblast is converted to haem within one hour⁷⁹. 

Fig 4 – Cellular uptake of iron-transferrin.( TfR- Transferrin receptor ). Wintrobe’s Clinical Hematology; Thirteenth edition; John P Greer.

• Storage of Iron – Iron is stored in the body in two forms.

1. Ferritin – Serum ferritin concentrations correlate with iron stores. Concentrations below 15 microg/L are specific for storage iron depletion.
2. Haemosiderin – Iron is stored with in the reticuloendothelial cells of bone marrow, spleen and liver and also in the erythroblasts. Staining of bone marrow in a case of iron deficiency anemia shows no stainable iron with in macrophages and erythroblasts.

Pathogenesis :

Three pathogenic factors are implicated in the anemia of iron deficiency. First, hemoglobin synthesis is impaired as a consequence of reduced iron supply. Second, there is a generalized

defect in cellular proliferation. Third, survival of erythroid precursors and erythrocytes is reduced, particularly when the anemia is severe.

When transferrin saturation falls below 15%, the supply of iron to the marrow is inadequate to meet basal requirements for hemoglobin production (generally approx. 25 mg of iron daily in average adults). As a result, the amount of free erythrocyte protoporphyrin increases, reflecting the excess of protoporphyrin over iron in heme synthesis. Globin protein synthesis is reduced and each cell that is produced contains less hemoglobin, resulting in microcytosis and hypochromia⁴⁰. Globin synthesis is controlled by heme availability at both the transcriptional and

translational levels. On the translational level, heme regulates globin synthesis by binding to and controlling the activity of heme-regulated eIF2 alphakinase (HRI)⁸¹. With high intracellular heme concentrations, heme binds to HRI and renders it inactive, but in heme deficiency, heme dissociates from HRI, and the kinase is activated by autophosphorylation. HRI then phosphorylates eIF2 alpha, preventing the recycling of eIF2 for another round of protein synthesis initiation, resulting in reduced globin protein synthesis and preventing formation of

toxic globin precipitates in the absence of heme.

Cellular proliferation is also restricted in iron deficiency, and red blood cell numbers fall. Although there is relative erythroid hyperplasia in the bone marrow, both the degree of erythroid

hyperplasia and the reticulocyte count are low for the degree of anemia. There is a significant component of “ineffective erythropoiesis” in iron deficiency, and a proportion of immature

erythroid cells in iron-deficient subjects are so defective that they are rapidly destroyed. Their iron is reused within the bone marrow, making the interpretation of ferrokinetic studies more

complicated.

In iron deficiency, survival of circulating erythrocytes is normal or somewhat shortened. There is a strong correlation between the degree to which red cell survival is shortened and the proportion of morphologically abnormal cells on blood smear.The principal site of destruction is the spleen⁸².

Clinical Features :

Most of the children are asymptomatic. Pallor is the most important clinical sign of iron deficiency but is not visible until the hemoglobin falls to 7-8 g/dl. Growth in infancy is impaired. Iron deficient children are irritable and demonstrate lack of interest in surroundings. When iron deficiency occurs in the critical period of neurodevelopment, changes are irreversible and development of the child is affected.

Other non-hematologic consequences are pica ( desire to ingest non nutritive substances ), pagophagia ( desire to ingest ice ).

This is also associated with impaired neurocognitive function in infancy. Irreversible cognitive defects are there in later^80,83.

Laboratory Diagnosis :

In progressive iron deficiency a sequence of biochemical and hematologic events occurs. A presumptive diagnosis of iron-deficiency anemia is most often made by a complete blood count demonstrating a microcytic anemia with a high red cell distribution width, reduced red blood cell count, normal white blood cell count and normal or elevated platelet count. Reticulocyte percentage may be normal or moderately elevated, but absolute reticulocyte counts indicate an insufficient response to the degree of anemia. Elliptocytic or cigar shaped red cells are also seen in peripheral blood smear.

Other laboratory studies, such as reduced serum ferritin, reduced serum iron and increased total iron binding capacity also needed⁸³.

The indicators of iron- deficiency anemia – (  Zimmermann MB, Hurell RF: Nutritional iron deficiency, Lancet 370: 511-520, 2007 )

1. Hemoglobin (g/dl) – <11 for non-hispanic whites ages 0.5-4 year
2. MCV (microm³) – <70 from 6-24 months
3. Serum ferritin ( microg/L) – <=5 year <12

                                             >5 year    <15

                                                         In all age groups in the presence of infection <30

• Reticulocyte hemoglobin content (CHr) – In infants and young children <27.5

                                                                    In adults <=28.0

• Serum transferrin receptor (sTfR) – Cutoff varies with assay and with patient’s age and

                                                          ethnic origin

• Transferrin saturation – <16%
• Erythrocyte zinc protoporphyrin (ZPP) (micromol/mol heme) – <=5 year  >70

                                                                                                                  >5 year >80

                                                                                                                  >5 year on washed red cells  >40

     8.   Hepcidin – usually <=10 ng/ml

In differentiating between beta thalassemia trait and iron deficiency anemia, HPLC (High Performance Liquid Chromatography) hemoglobin electrophoresis and iron profile ( Serum iron, Total iron binding capacity, serum ferritin, serum transferrin saturation ) estimation are the best methods. But we can differentiate them through routine blood tests as HPLC is a costly test and not available in resource poor settings. There are lot of RBC indices ( MCV, MCH, MCHC, RDW, Mentzer index = MCV/RBC count, RDW index, Green and King index, England and Fraser index, Shine and Lal index and Srivastava index ) use previously in different studies to differentiate between these two diseases. Few study reviews are shown comparing their outcomes regarding our study parameters – MCV, MCH, MCHC, RDW and Mentzer index to differentiate between iron deficiency anemia and beta thalassemia trait.

Mohamed M. Eldibany, Kameel F. Totonchi, NinosJ. Joseph, and Douglas Rhone M in 1999, a retrospective study done between 383 adults. It showed the data of hemogram indices, HbA2 and F concentrations, and iron studies for iron deficiency anemia and beta thalassemia trait ( Table 1). Mentzer index showed sensitivity (90.2% and 51%) to diagnose iron deficiency anemia and beta thalassemia trait respectively⁸⁴.

In another study Cengiz Beyan, Kurs at Kaptan, Ahmet Ifran done in Turkey in 2007 among 45 IDA and 66 Beta thalassemia trait cases aged 17-57 years showed that sensitivity (88.9%), specificity (84.8%), PPV (80%) and NPV (91.8%) of RBC count in cases of iron deficiency anemia whereas sensitivity (84.8%), specificity (88.9%), PPV (91.8%) and NPV (80%) in cases of beta thalassemia trait. RDW values showed sensitivity (84.4%), specificity (18.2%), PPV (41.3%) and NPV (63.1%) in cases of iron deficiency anemia and sensitivity (18.2%), specificity (84.4%), PPV (63.1%) and NPV (41.3%) in cases of beta thalassemia trait. Mentzer index showed sensitivity (88.9%), specificity (75.7%), PPV (71.4%) and NPV (90.9%) in cases of iron deficiency anemia  and sensitivity (75.7%), specificity (88.9%), PPV (90.9%) and NPV (71.4%) in cases of beta thalassemia trait.

Therefore RBC count only showed sensitivity and specificity more than 80% among other indices done in this study. Discrimination indices were also came out from this study – ( RBC count: <5 = IDA and >5 = Beta thalassemia trait, RDW: <14 = Beta thalassemia trait and >14 = IDA, Mentzer index: <13 = Beta thalassemia trait and >13 = IDA )

         They concluded that none of these different formulations were superior to RBC value obtained from automated analyzers in adult cases with IDA and Beta -TT. Serum ferritin, serum iron, transferrin saturation and hemoglobin A2 level should be obtained for accurate differential diagnosis of IDA and Beta -TT until more efficient tools develop. Everyone with low MCV and low MCH must had the above studies. If resources were scarce, then hemoglobin electrophoresis should be performed only and preferably on the individual’s partner as well. Once a person was found to have iron deficiency, then once this has been treated, the individual should be retested with hemoglobin electrophoresis as there are concerns that low iron levels result in suppression of HbA2⁸⁵.

In a study by Fakher Rahim and Bijan Keikhaei in 2009 showed that among the 323 patients, 170 (114 children, 56 adults) were diagnosed to have IDA and 153 (59 children, 94 adults) were diagnosed to have β-TT. The different indices for patients younger or older than 10 years were calculated individually. All of the indices showed overlapping in the patients with β-TT and IDA. The overlaps were between 12.5 and 21.1% in RDW and between 4.3 and 5.49 × 10¹² in RBC for patients younger than 10 years, while these respective values were between 11.4 and 25.1% and between 4.38 and 5.94 × 10¹² in those older than 10 years. None of the indices was completely sensitive or specific in differentiation between β-TT and IDA. Indices in decreasing order in patients younger than 10 years were as follows: RBC > SI > MI > RDW and in patients older than 10 years were: RBC > MI > SI > RDW. The scatter-gram of the hematological data of all patients showed significant differences between these two disorders.

            They concluded that patients with microcytic anemia, either of β-TT or IDA was shown in a patient younger than 10 years with correct measures on RBC and in a patient older than 10 years with correct measures on RBC and RDW indices, the diagnoses are likely to be correct. However, in a small number of patients, it would still be necessary to study body iron status or HbA2 for accurate diagnosis⁵³.

In a study done by Chuan Shen et al. in China in 2010 Among the 300 children included in the study, evaluation of indices showed that RBC, Hb, MCV, MCH, MCHC, RDW and SF (Serum Ferritin) were different between IDA and beta -TT (P<0.001). In these parameters, RBC, Hb, MCH, MCHC and SF of the beta -TT patients were higher than those of the IDA patients whereas MCV and RDW were lower than those of the IDA patients. ROC curves were plotted to establish new cut-off values with higher sensitivity and specificity for each index. About 41.7% children with Hb less than 10g/dL were in b-TT group. RBC showed low sensitivity (78.7%) and high specificity (90.8%) at a cut-off value of 5.17*10¹²/L. While RDW showed relatively a little higher sensitivity (81.9%) and specificity (79.8%) at a cut-off value of 18.05%.

        They concluded that RDW, which was an indicator anisocytosis, was accordingly high in patients who were more anemic such as the IDA. However, compared with other indices in their study, RDW was not the best one¹⁷.

In a study done by Burcin Nalbantoglu et. al. in Turkey in 2012, Among the 100 children of both sexes (52girls) aged between 2 and 14 years included in the study. The values of RBC, Hb, hematocrit, MCV, and RDW in patients with IDA and TT are shown inTable1. Significant differences are observed in the RBC and MCV values.  

The discriminant indices as well as sensitivity (SENS), specificity (SPEC), positive predictive value (PPV) and negative predictive value (NPV) were calculated. Their data showed that sensitivity of RBC (77%), MI (67.8%) and RDW (16.1%). Furthermore, the specificity of  RDW was (79.2%), MI(73.6%) and RBC (10.5%). The PPV was 80.8% for MI,65.8% for RBC, and 55.5% for RDW. The NPV was 22.2% for RBC, followed by RDW (36.6%) and MI (58.3%).                    

           They concluded that none of the formulas appears reliable in discriminating between TT and IDA patients. The evaluation of iron status and measurement of HbA2 remain the most reliable investigations to differentiate between TT and IDA patients⁷.

Aziz Batebi, Abolghasem Pourezza, Reza Esmailian in 2012 with study population included 440 women and 460 men. They showed that 444 persons had BTM (Beta thalassemia minor) and the remaining 457 persons had non-thalassemic causes of hypochromia and microcytosis such as IDA, chronic diseases, and rare causes such as lead poisoning and sideroblastic anemia. According to Table 1, while the MCV in thalassemic patients was in the range of 50.6-78.8 fL, with an average of 66.4 fL, the corresponding values for non-thalassemics were 67.3-84.5 fL, with an average of 79 fL. The significant difference (P < 0.001) between these values indicates that, in each person whose MCV is lower than the arbitrary microcytic cut-off  point, the type of anemia, either thalassemic or non-thalassemic, could be recognized easily. This is why, in some texts, a MCV of less than 60 fL and a MCH value of less than 20 pg/cell are considered as indicators of a very low probability of having IDA and a higher probability of thalassemia minor

The sensitivity, specificity, PPV and NPV values of MCV and Mentzer index were shown in table 2.

        They concluded that IDA and BTM were recognized as the most important causes of hypochromia and microcytosis. In order to avoid much more expensive, time-consuming, and complicated procedures for discrimination between these disorders, researchers attempted to employ either RBC indices such as MCV, MCH, and red blood cell distribution width (RDW), or formulas derived from these indices. This process helped to select appropriate individuals for more detailed examination⁸⁶.

In a study by M. Ferrara et al. in 2013 found that significant differences were observed among all ( RBC, hemoglobin, hematocrit, MCV ) these parameters, except for RDW values ( reported in Table 1).

Discriminant indices as well as SENS (Sensitivity), SPEC (Specificity), PPV (Positive predictive value), NPV (Negative predictive value), EFF (Efficiency) were reported. Their data showed that 69.7%  SENS of RBC, 59% for MI (Mentzer index) formula and 2.79% for RDW. Furthermore, the value of SPEC showed 97.9% for MI, 95.8% for RBC and 92.5% for RDW. PPV was (96.5%) for MI, (96.2%) for RBC count, (20%) for RDW. NPV was (36.9%) for MI, followed by RDW (51.1%), RBC (76%). Efficiency was 79.6% for MI. Gaussian curves are obtained from beta – TT and IDA subjects with various formulas or indices show a different degree of overlap.

                They concluded that the wide spectrum of beta chain molecular alterations in the world and the degree of anemia may influence the discrimination function and reliability of RBC indices and formulas. The evaluation of iron status and measurement of HbA2 remain the most reliable investigations to differentiate between beta TT and IDA subjects⁸⁷.

Januária Fonseca Matos et al. in 2013 in a cross-sectional study , which was done in 289 patients aged 18-89 years, showed that median values and interquartile values of some hematological parameters for the two groups, as well as the difference between these groups, are presented in Table 1.

Among the other indices tested here Mentzer index showed sensitivity (87.9%), specificity (76.6%), PPV (18.2%), NPV (99.1%) and efficiency (86.3%) in case of iron deficiency anemia and sensitivity (76.6%), specificity (87.9%), PPV (7.7%) and NPV (99.6%) in case of beta thalassemia trait.

          They concluded that one cannot reach a definitive diagnosis of IDA or β TT based merely on the discriminant functions, these simple calculations were potentially useful in screening patients with microcytic anemia. These indices were a useful tool in the doctor’s guidance

about the initial approach to be adopted, but did not relieve patient monitoring that may eventually required confirmatory tests to elucidate the strong suspicion initially raised by the application of these simple indices. Furthermore, these formulas were the only differential tool in situations where other specific confirmatory tests were not available¹⁰.

Aysel Vehapoglu et al. 2014 in a study performed retrospectively to assess reliability of hematological indices for differential diagnosis of beta thalassemia trait and iron deficiency anemia. It showed Hb values in the ?-TT group were10.39 ± 0.69,and those in the IDA group were 10.23±0.95 (? > 0.05). MCV was 60.11± 3.49 in the ?-TT group, and this value was lower than those in the IDA group (67.49 ± 7.14; ? < 0.05). RBC count was higher in the ?-TT (5.56±0.4) group than that in the IDA (4.84 ± 0.59; ? < 0.05). A high erythrocyte count (RBC > 5.0 × 106/?L) was a common feature of IDA and ?-TT. The RBC count was one of the most accurate indices available. The RBC count provided the best sensitivity (94.8%) but had low specificity (70.5%). According to result none of the indices studied demonstrated 100% precision in recognizing ?-TT. Therefore these indices could not be used as screening tools for?-TT , as using them could result in a significant number of false –negative results. The Mentzer indices demonstrated the highest specificities at 85.3%.  None of the indices were completely sensitive or specific in distinguishing ?-TT and IDA. The Mentzer index showed good sensitivity, specificity  and values were 98.7% and 82.3%.When the Mentzer index was calculated, 264 children with microcytic anemia (91%) were correctly diagnosed. The difference between the results of all of these indices and the gold standard  (HbA2) was significant (? < 0.001).

        They concluded that the cell-count-based indices, particularly the Mentzer index, was easily available and reliable methods for detecting ?-TT. According to their results, the percentage of correctly diagnosed patients was the highest with the Mentzer index (91%) . Cell-count-based parameters and formulas, particularly the MCV and RBC counts and their related indice (Mentzer index) have good discrimination ability in diagnosing ?-TT².

Sakorn Pornprasert et al. in a study done in Thailand in 2014, showed that based on the HPLC and molecular analyses, 21 of 265 children (7.9%) were diagnosed as beta-thalassemia trait, seven carried the codon 17 mutation, four carried the codons 71/72 mutation, six carried the IVS-I-1 mutation, and four carried unidentified mutations. The transferrin saturation of less than 16.0% and/or the serum ferritin level of less than 16.0 ng/mL was found in 56 children (21.1%); thus, they were diagnosed as having an iron deficiency. The children with the beta-thalassemia trait had significantly higher mean levels of serum iron, transferrin saturation, and serum ferritin than those with iron deficiency (82.5 vs. 48.3 mg/dL, 23.6 vs. 12.4% and 77.8 vs. 56.3 ng/mL, p <0.001, <0.001 and 0.02, respectively). On the other hand, the children with beta-thalassemia trait had significantly lower mean level of TIBC than those with iron deficiency (353.0 vs. 388.0 mg/dL, p = 0.01). There were no statistical differences in mean age, gender, levels of white blood cells (WBCs) and platelets between the beta-thalassemia trait and iron deficiency groups. However, the beta-thalassemia trait had significantly higher levels of RBCs and RDW than iron deficiency. On the other hand, mean levels of Hb, hematocrit [or packed cell volume (PCV)], MCV, MCH, and MCHC of the beta-thalassemia trait were significantly lower than those of the iron deficiency group. All red cell indices and formulas, except the MCHC and RDW, had 100.0% sensitivity and negative predictive value. However, only RDW had specificity and positive predictive value higher than 90.0%.

              They concluded that none of the red cells indices and formulas provided 100.0% sensitivity, specificity and efficiency for the discrimination of beta-thalassemia trait from iron deficiency. However, Hb analysis and iron study should be performed as confirmation tests²¹.

Ebrahim Miri-Moghaddam et. al. in 2014 did a cross-sectional study to determine cut off of discrimination indices for differentiating between beta thalassemia trait and iron deficiency anemia and took 77 IDA and 100 beta thalassemia trait patients as sample who came for premarital screening in the reference laboratory of Zhedan University of Medical Sciences in southern Iran. They showed that mean and standard deviation of the various hematological parameters of IDA and BTM (Beta thalassemia trait) groups are shown in Table 1.

     Among the other indices tested here Mentzer index showed sensitivity (72%), specificity (82%), PPV (68%), NPV (67%), published cut-off for beta thalassemia trait (<13) and proposed cut-off of beta thalassemia trait from their study (<13).

            Therefore they concluded that microcytic hypochromic anemia cases could be easily screened out in mass screening in the absent of other improve procedures. Should be considered, none of these indices shows the sensitivity and specificity of 100%.  

            The spectrum thalassemia mutations in each population can affect on of various RBC indices, as a result, the cutoff point of discrimination formulas varies. Therefore, it is suggested to determine the cut-off point for every formula in different populations⁸.

Tahir Jameel et al. in 2017 in a study performed to differentiate between iron deficiency anemia and thalassemia by hematological indices in people aged 21-36 years ; done in soudi arabia showed that out of 620 individuals undergoing premarital screening, 135 revealed low Hb and low MCV (Hb < 9 gram/dl & MCV <80fl) and Hb F was undetectable in their blood. These (38 males & 97 females) individuals were enrolled for the study. Their age ranged from 21 to 36 years with the mean of 24 years ±1.5. According to the criteria mentioned in subjects and methods, ninety-three individuals (20 males and 73 females) were diagnosed having IDA, whereas thirty-two of them (15 males and 17 females) as having β TT. Ten individuals revealed the presence of other causes such as chronic disease and sideroblastic anemia. The RBC count was found to be higher in patient of β TT (6.8-7.7 x10¹² /l with the mean of 7.3 x10¹² /l ±1.12) as compared to IDA patients in which it ranged from 3.6-4.9 x10¹² /l.  The range of MCV in β TT was in the range of 51.1-57.9 fl with a mean value of 53.2fl ±0.53, the corresponding values for IDA were 62.3 – 79.4 fl, with the mean value of 73.5 79 fl ±0.85. MCH and MCHC values did not show much difference among both the groups. Serum ferritin was remarkably low in patients diagnosed as IDA (2.6-9.7 with the mean of 5.02 whereas its levels were on the higher side in β TT patients. Though in some the patients having hemoglobin A2 above the cutoff limits, the serum ferritin levels were below the cutoff limit of 15µg/l, indicating the coexistence of IDA and β TT. Significant differences of Hb level and MCV, found in between β TT and IDA.

Another important RBC parameter for detection of IDA and β TT is RDW. In their study, the mean RDW was found 16.4% in case of β TT and 16.9% in case of IDA. The results were not statistically significant (p=0.269) whereas the derived index i.e. RDWI (RDW index) showed better discriminative effect between β TT and IDA, as this index had both sensitivity and specificity more than 85% in detection of β TT and IDA. Sensitivity and specificity of RDWI for detection of β TT was found 94.0% and 88.0%. Again for IDA, sensitivity and specificity were found 88.0% and 86.0% respectively. Youden’s index (YI) takes into account both sensitivity and specificity and gives an appropriate measure of the validity of a particular technique. YI of RDWI was found 83.0, which could be a reliable discriminator between βTT and IDA.Though several indexes had been mentioned for the purpose of discrimination between β TT and IDA, in this study they tried to evaluate a simple, effective and user-friendly index which doesn’t require mathematical calculations.

              They concluded that RDWI appears to be a reliable and useful index for initial screening of microcytic hypochromic anemia and is better than RDW in differentiating IDA from β TT¹⁴.

In another study by Yeter Düzenli Kar et. al. in Turkey done at 2019 found that Hb, Hct, RBC, MCH, MCHC, transferrin saturation, and serum iron and ferritin concentrations were significantly lower in the IDA group than in the β-TT (P=0.004 for MCH, P<0.001 for others) groups. There was no difference between the β-TT and IDA groups in terms of MCV values (P>0.05). RDW values were significantly higher in the IDA group than in the β-TT groups (P<0.001). The mean HbA2 level in the β-TT group was 5.06±0.6% (min-max: 3.6% to 6.40%). Accordingly, the indices with the highest sensitivity and specificity for distinguishing IDA and β-TT was identified as RBC. The revised cutoff values of the erythrocyte indices and formulas used to differentiate IDA and TT were determined with a 95% confidence interval range. In differentiating IDA from β-TT, the ranking of the erythrocyte indices and formulas from the highest to lowest according to their area under curve (AUC) values was RBC, MI (Mentzer Index and RDW.

      They concluded that erythrocyte indices and formulas can be used as initial tests for the differential diagnosis of TT and IDA. The results of their study showed that RBC was the most useful indices in the differential diagnosis of IDA and TT, whereas RDW and MI were not useful. Cutoff values vary among populations; therefore, it would be more beneficial for different communities to use erythrocyte indices by determining their specific and appropriate cutoff values⁴.

MATERIALS AND METHODS

METHOD & MATERIALS:

1. Study Setting: Department of Pathology and Pediatrics, Ramakrishna Mission Seva Pratisthan, Vivekananda Institute Of Medical Sciences.

• Study Population: All indoor and OPD patients being attended this hospital and who met the inclusion criteria.

• Study Period: January 2019 to June 2020

• Study Design: A prospective institution based case control study in which comparative observation will be made.

• Sample Size & Design: Participants will be stratified into two groups.

Group I: Children ( age group 1-12 years ) having microcytic hypochromic anemia with beta thalassemia trait.

Group II: Children ( age group 1-12 years ) having microcytic hypochromic anemia with iron deficiency anemia.

These population will be further evaluated for RBC indices , RDW, Mentzer index.

Total sample size will be 100 cases of microcytic hypochromic anemia in 1-12 years age group. 50 patients are taken from each group.

• Inclusion Criteria:
• Children 1 to 12 years of age group having microcytic hypochromic anemia with beta thalassemia trait.
• Children 1 to 12 years of age group having microcytic hypochromic anemia with iron deficiency anemia.

• Exclusion Criteria:

i)Children with microcytic hypochromic anemia ( 1 to 12 years ) having both beta thalassemia trait and iron deficiency anemia.

ii) Children with severe anemia ( Hb levels < 8 g/dl )

                  iii)Children having chronic illness.

• Required Parameter:

i)RBC indices ( MCV, MCH, MCHC ) & RDW by automated haematology analyzer.

ii)Mentzer index by calculating from MCV and RBC count.

    Mentzer index = MCV/RBC count

    RBC count will also obtain from automated haematology analyzer.

iii)HbF, HbA2 & HbA value by HPLC technique.

iv)Iron profile (serum iron, total iron binding capacity, ferritin, transferrin saturation).

1. Instruments used:

SYSMEX XN-550 – Auto analyzer is used for complete blood count (CBC).

• Sample used – A 2 mL blood sample from each patient was collected into a vial containing 2.0 mg/ml K₂EDTA and preserved at 37^oC for complete blood count by a qualified phlebotomist using 24G needle.
• Specimen volume required – Optimal draw is a tube drawn to capacity. The collection tube must be filled to a minimum of one-half full for acceptable results. A minimum of 1 ml of whole blood is required for sample analysis. To maintain the proper anticoagulant ratio tube has been filled to the 250 microlitre line at the time of collection.
• Unacceptable specimens – 1. Clotted samples or those containing clots or fibrin strands.

                                                              2.Samples drawn above an IV (intra venous)

• Principle – It is a multi parameter quantitative automated hematology analyzer for in vitro diagnostic use in determining whole blood diagnostic parameters. The device performs hematology analyses based on hydrodynamically focused impedance measurement, the flow cytometry method (using a semiconductor laser) and the SLS-hemoglobin method. The device counts and sizes red blood cells (RBC) and platelets (PLT) using hydrodynamic impedance counting. At the same time the hematocrit (HCT)  is measured as a ratio of the total RBC volume to whole blood via the RBC pulse height detection method. Cytometry is used to analyze physiological and chemical characteristics of cells and other biological particles. Flow cytometry is a method used to analyze those cells and particles as they pass through extremely small flow cells.

BIORAD VARIANT II – It is a fully automated, high throughput hemoglobin analyzer. It is used to detect thalassemia.

• Sample used: A venous whole blood specimen collected in EDTA is required.
• Specimen volume required: 1 ml whole blood or minimum 50 microlitre whole blood
• Unacceptable specimen: Clotted samples or those containing clots and fibrin strands.
• Principle: The variant II uses the principles of high performance liquid chromatography (HPLC) for the separation and determination of normal and abnormal hemoglobin. Two dual piston pumps in the VCS (Variant II chromatographic station) deliver a buffer solution to the analytical cartridge and the detector. Primary sample tubes are spun in the VSS (Variant II sampling station); a sample is then drawn, diluted and introduced to the analytical flow path using automatic injection. Between sample injections, the sample needle is rinsed with wash solution to minimize sample carryover. The sample is carried by the buffer through analytical cartridge, where the sample is separated into its individual components. The separated components then pass through the dual wavelength detector, where absorbance of the sample components is measured at 415 nm. Background noise is reduced with the use of a secondary wavelength at 690 nm. The absorbance data is transmitted from the detector to the PC and displayed by CDM as a real time chromatogram (graph of absorbance vs. time ). The processed data is incorporated into a printed report, which contains the following: 1)A complete summary of the sample’s detected components (peak identification, retention time, area), 2)The sample’s chromatogram, 3)Date and time of analysis, 4)Vial number and sample identification.
• Reference range:

WINDOW	RETENTION TIME (min)
P1	0.63-0.85
F	0.98-1.20
P2	1.24-1.40
P3	1.40-1.90
A0	1.90-3.10
A2	3.30-3.90
D	3.90-4.30
S	4.30-4.70
C	4.90-5.30

For diagnosing beta thalassemia trait, HbA2 levels increased (between 4-9%)

                  COBAS – Immunoassay for the in vitro quantitative determination of ferritin in

                  human serum and plasma.

• Test Principle: Sandwich principle. Total duration of assay – 18 minutes.

1^st incubation: 10 microlitre of sample, a biotinylated monoclonal ferritin specific antibody , and a monoclonal ferritin specific antibody labeled with a ruthenium complex form a sandwich complex.

2^nd incubation: After addition of streptavidin coated microparticles , the complex becomes bound to the solid phase via interaction of biotin and streptavidin.

The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed with Procell/Pro cell M. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier.

Results are determined via a calibration curve which is instrument specifically generated by 2-point calibration and a master curve provided via the reagent barcode.

VITROS – Quantitatively measure serum iron and total iron binding capacity (TIBC).

• Principle for serum iron measurement: It is a multilayered analytical element coated on a polyester support. A drop of patient sample is deposited on the slide and is evenly distributed by the spreading layer to the underlying layers. Iron (as ferric iron) is removed from transferrin at acidic pH and migrates to the reducing layer, where ascorbic acid reduces iron to the ferrous form. The ferrous ion then is bound to the dye and forms a colored complex in the reagent layer. Following addition of the sample, the slide is incubated and the reflection density measured after 1 and 5 minutes. The difference in reflection density is proportional to the iron concentration in the sample.

Transferrin- Fe⁺³————-à transferrin + Fe⁺³

Fe⁺³ + Ascorbic acid ———à Fe⁺²

Fe⁺² + dye ———-à Fe⁺²-dye (colored complex)

• Principle for TIBC measurement: Excess iron citrate reagent is added to the sample to saturate all available apotransferrin sites. After an incubation period of 5 minutes , the treated sample is applied to an alumina column where iron that is not bound to transferrin is absorbed. The transferrin bound iron contained in the eluate represents the total iron binding capacity of the sample. TIBC is determined with VITROS Fe slides. A drop of patient sample is deposited on the slide and is evenly distributed by the spreading layer to the underlying layers. After the addition of the sample, the slide is incubated at 37^oC . Two reflection density measurements at 600nm are made at approximately 1 and 5 minutes. The difference in reflection density is proportional to the iron concentration in the sample.

                              Fig 5 – HPLC chromatogram shows normal study.

           Fig 6 – HPLC chromatogram shows high A2 window (Beta thalassemia carrier state).

RESULTS

In our study total 100 patients, between 1-12 years of age group, were taken in which 50 patients were diagnosed with iron deficiency anemia and another 50 patients were diagnosed with beta thalassemia trait. HPLC and iron profile were done in all patients to rule out co-existance of diseases. Then RBC indices (MCV,MCH,MCHC,RDW and Mentzer index) of these two groups compared for analysis.

Statistical Methods

Continuous variables are expressed as Mean and Standard Deviation and compared across the groups using unpaired t test.

Categorical variables are expressed as Number of patients and percentage of patients and compared across the groups using Pearson’s Chi Square test for Independence of Attributes/ Fisher’s Exact Test as appropriate.

The statistical software SPSS version 20 has been used for the analysis.

An alpha level of 5% has been taken, i.e. if any p value is less than 0.05 it has been considered as significant.

Table 1: Distribution of age between IDA and B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
AGE(Year)	6.18	3.73	5.60	4.16	0.465	Not Significant

Fig.7 – Distribution of age between IDA and B-TT

The mean age group of iron deficiency anemia is 6.18 years with standard deviation 3.73 and in cases of beta thalassemia trait is 5.60 years with standard deviation 4.16. As per this test p value is 0.465 which is not significant.

Table 2: Group wise distribution of age between IDA and B-TT

		GROUP		Total
		IRON DEFICIENCY ANEMIA	BETA THALASSEMIA TRAIT		p Value	Significance
AGE(Year)	<=5	24(48)	30(60)	54(54)	0.070	Not Significant
	6-10	18(36)	8(16)	26(26)
	11-12	8(16)	12(24)	20(20)
Total		50(100)	50(100)	100(100)

Fig.8 – Group wise distribution of age between IDA and B-TT

Out of 50 iron deficiency anemia patients, 24 are in <=5 years of age, 18 are in 6-10 years of age and 8 are in 11-12 years of age. Out of 50 beta thalassemia trait patients, 30 are in <=5 years of age, 8 are in 6-10 years of age and 12 are in 11-12 years of age. As per this test p value is 0.070 which is not significant.

Table 3: Distribution of gender of the patients

		GROUP		Total
		IRON DEFICIENCY ANEMIA	BETA THALASSEMIA TRAIT		p Value	Significance
SEX	FEMALE	21(42)	22(44)	43(43)	0.840	Not Significant
	MALE	29(58)	28(56)	57(57)
Total		50(100)	50(100)	100(100)

Fig.9 – Distribution of gender of the patients

Out of 50 iron deficiency anemia patients, 21 are female and 29 are male. Out of 50 beta thalassemia trait patients, 22 are female and 28 are male. As per this test, p value is 0.840 which is not significant.

Table 4: Comparison of mean HbF values between IDA and B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
HbF	0.60	0.21	1.01	0.43	<0.001	Significant

Fig.10 – Comparison of mean HbF values between IDA and B-TT

The mean value of HbF in cases of iron deficiency anemia is 0.60 with standard deviation 0.21 and in cases of beta thalassemia trait is 1.01 with standard deviation 0.43.

Table 5: Comparison of mean HbA2 value between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
HbA2	2.46	0.24	4.92	0.33	<0.001	Significant

Fig.11 – Comparison of mean HbA2 value between IDA & B-TT

The mean value of HbA2 in cases of iron deficiency anemia is 2.46 with standard deviation 0.24 and in cases of beta thalassemia trait is 4.92 with standard deviation 0.33.

Table 6: Comparison of mean serum iron value between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
SERUM IRON(micromol/l)	34.92	7.85	88.40	18.41	<0.001	Significant

Fig. 12 – Comparison of mean serum iron value between IDA & B-TT

The mean value of serum iron in cases of iron deficiency anemia is 34.92 with standard deviation 7.85 and in cases of beta thalassemia trait is 88.40 with standard deviation 18.41.

Table 7: Comparison of mean TIBC value between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
TIBC(micromol/l)	477.56	43.77	308.88	63.86	<0.001	Significant

Fig.13 – Comparison of mean TIBC value between IDA & B-TT

The mean value of TIBC in cases of iron deficiency anemia is 477.56 with standard deviation 43.77 and in cases of beta thalassemia trait is 308.88 with standard deviation 63.86.

Table 8: Comparison of mean ferritin value between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
FERRITIN(microgm/l)	8.77	2.98	71.78	15.22	<0.001	Significant

Fig.14 – Comparison of mean ferritin value between IDA & B-TT

The mean value of ferritin in cases of iron deficiency anemia is 8.77 with standard deviation 2.98 and in cases of beta thalassemia trait is 71.78 with standard deviation 15.22.

Table 9: Comparison of mean transferrin saturation between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
TRANSFERRIN SATURATION(%)	7.00	2.06	29.32	5.44	<0.001	Significant

Fig. 15 – Comparison of mean transferrin saturation between IDA & B-TT

The mean value of transferrin saturation in cases of iron deficiency anemia is 7 with standard deviation 2.06 and in cases of beta thalassemia trait is 29.32 with standard deviation 5.44.

Table 10: Comparison of mean haemoglobin values between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
HAEMOGLOBIN(g/dl)	9.69	0.76	10.00	1.05	0.097	Not Significant

Fig.16 – Comparison of mean haemoglobin values between IDA & B-TT

The mean value of hemoglobin in cases of iron deficiency anemia is 9.69 with standard deviation 0.76 and in cases of beta thalassemia trait is 10 with standard deviation 1.05. As per this test p value is 0.097 which is not significant.

Table 11: Comparison of mean RBC count between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
TOTAL RBC COUNT(million/cmm)	4.42	0.36	5.40	0.56	<0.001	Significant

Fig.17 – Comparison of mean RBC count between IDA & B-TT

The mean value of total RBC count in cases of iron deficiency anemia is 4.42 with standard deviation 0.36 and in cases of beta thalassemia trait is 5.40 with standard deviation 0.56. As per this test p value is less than 0.001 which is significant.

Table 12: Comparison of mean MCV values between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
MCV(fl)	67.94	2.19	62.69	3.40	<0.001	Significant

Fig.18 – Comparison of mean MCV values between IDA & B-TT

The mean value of MCV in cases of iron deficiency anemia is 67.94 with standard deviation 2.19 and in cases of beta thalassemia trait is 62.69 with standard deviation 3.40. As per this test p value is less than 0.001 which is significant.

Table 13: Comparison of mean values of MCH between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
MCH(pg)	20.56	3.04	18.50	1.45	<0.001	Significant

Fig.19 – Comparison of mean values of MCH between IDA & B-TT

The mean value of MCH in cases of iron deficiency anemia is 20.56 with standard deviation 3.04 and in cases of beta thalassemia trait is 18.50 with standard deviation 1.45. As per this test p value is less than 0.001 which is significant.

Table 14: Comparison of mean value of MCHC between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
MCHC(%)	29.60	1.74	29.46	1.06	0.628	Not Significant

Fig.20 – Comparison of mean values of MCHC between IDA & B-TT

The mean value of MCHC in cases of iron deficiency anemia is 29.60 with standard deviation 1.74 and in cases of beta thalassemia trait is 29.46 with standard deviation 1.06. As per this test p value is 0.628 which is not significant.

Table 15: Comparison of mean values of RDW between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
RDW	18.00	1.77	16.59	1.42	<0.001	Significant

Fig.21 – Comparison of mean values of  RDW between IDA & B-TT

The mean value of RDW in cases of iron deficiency anemia is 18 with standard deviation 1.77 and in cases of beta thalassemia trait is 16.59 with standard deviation 1.42. As per this test p value is less than 0.001 which is significant.

Table 16: Comparison of mean values of Mentzer index between IDA & B-TT

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
MENTZER INDEX	15.45	1.37	11.74	1.49	<0.001	Significant

Fig.22 – Comparison of mean values of Mentzer index between IDA & B-TT

The mean value of Mentzer index in cases of iron deficiency anemia is 15.45 with standard deviation 1.37 and in cases of beta thalassemia trait is 11.74 with standard deviation of 1.49. As per this test p value is less than 0.001 which is significant.

By taking conventional cut off 13 of Mentzer index value, the tests were done.

Table 17: Distribution status of Mentzer index ( cutoff 13 ) in cases of IDA and B-TT

		MENTZER INDEX		Total
		<13	>13
GROUP	IRON DEFICIENCY ANEMIA BETA THALASSEMIA TRAIT	0	50	50
		37	13	50
Total		37	63	100

Out of 50 iron deficiency anemia all cases show the value of Mentzer index is above 13. Out of 50 beta thalassemia trait 37 cases show less than 13 Mentzer index whereas 13 cases show greater than 13 Mentzer index.

Table 18: Diagnostic ability of Mentzer index ( Cutoff 13 )

Scoring system	TP	TN	FP	FN	sensitivity	specificity	PPV	NPV	Diagnostic accuracy
MENTZER INDEX	37	50	0	13	74.00	100.00	100.00	79.37	87.00

Out of 100 cases, 37 cases are true positive, 50 cases are true negative, no false positive cases and 13 cases are false negative. As per this test, sensitivity is 74%, specificity is 100%, positive predictive value (PPV) is 100% and negative predictive value (NPV) is 79.37%. Diagnostic accuracy of the test is 87%.

Fig.23 – ROC- curve to compare the efficacy of Mentzer index in cases of IDA and B-TT

                         Table 19: Efficacy of Mentzer index

Area	p Value	Asymptomtic 95% Confidence Interval
		Lower Bound	Upper Bound
0.971	<0.001	0.946	0.996

The area under the curve is 0.971, p value is less than 0.001 with 95% of confidence interval.

The cut off from ROC is 14.   

Table 20: Distribution status of Mentzer index ( Cutoff 14 ) in cases of IDA & B-TT          

		MENTZER INDEX		Total
		<=14	>14
GROUP	IRON DEFICIENCY ANEMIA	7	43	50
	BETA THALASSEMIA TRAIT	47	3	50
Total		54	46	100

Table 21: Diagnostic ability of Mentzer index (Cutoff 14)

Scoring system	TP	TN	FP	FN	sensitivity	specificity	PPV	NPV	Diagnostic accuracy
MENTZER INDEX 14	47	44	6	3	94.00	88.00	88.68	93.62	91.00

So, taking 14 cut off as per the ROC, out of 50 iron deficiency anemia 7 cases show less than 14 and 43 cases show greater than 14. Out of 50 beta thalassemia trait 47 cases show less than 14 and 3 cases show greater than 14.

Out of 100 cases, 47 are true positive (TP), 44 are true negative (TN), 6 are false positive (FP) and 3 are false negative (FN). As per this test, sensitivity is 94%, specificity is 88%, positive predictive value (PPV) is 88.68% and negative predictive value (NPV) is 93.62%. Diagnostic accuracy is 91%.

                                                   DISCUSSION

β Thalassemia trait (B-TT) and Iron deficiency anemia (IDA) are the most commonly encountered microcytic hypochromic anemia. The differential diagnosis between IDA and B-TT is an important concern for every physician, to avoid unnecessary iron therapy and false diagnosis of B-TT.  B-TT and IDA present similar pattern as hypochromic microcytic anemia. Definitive methods for differential diagnosis between B-TT and IDA include quantitative estimation of HbA2 via HPLC (High Performance Liquid Chromatography) test and iron profile study⁸. The objective of our study is to compare the RBC indices (MCV, MCH, MCHC), RDW & Mentzer index between IDA & B-TT and also to evaluate the suspected cases of β Thalassemia trait (B-TT). The definitive tests (HPLC & iron profile) are time consuming and costly & also an economical burden in resource poor setting, so we perform this study to differentiate them easily by routine investigations.

                   Our study is focused on whether RBC indices (MCV, MCH, MCHC), RDW & Mentzer index can differentiate between IDA & B-TT. We took previously diagnosed IDA and B-TT patients and did the routine blood tests. Both the outcomes were compared and analyzed.

                   In our study out of 100 children (1-12 years of age group ) 50 children were previously diagnosed with IDA and another 50 children were previously diagnosed with B-TT. HPLC & iron profile were done in all patients to rule out co-existance of diseases. We did routine blood tests among those 100 diagnosed children and compared their results. In our sample, out of 100 children, 43 were female and 57 were male. Out of 43 female children, 42% (n=21) were diagnosed with IDA & 58% (n=22) were diagnosed with B-TT. Out of 57 male children, 44% (n=29)  were diagnosed with IDA & 56% (n=28)  were diagnosed with B-TT (Table 3; Fig. 9).  In this population, 54% were between 1-5 years, 24% were between 6-10 years and 20% were between 11-12 years. Out of 50 IDA cases, 48% (n=24) were between 1-5 years, 36% (n=18) were between 6-10 years, 16% (n=8) were between 11-12 years. Out of 50 B-TT cases, 60% (n=30) were between 1-5 years, 16% (n=8) were between 6-10 years, 24% (n=12) were between 11-12 years (Table 2; Fig.8). The mean age (mean + SD) of IDA patients was 6.18 + 3.73 years and B-TT patients was 5.60 + 4.16 years (Table 1; Fig. 7).

The diagnosis was done by HPLC technique (for B-TT cases) and iron profile study (for IDA cases). At diagnosis the mean HbF value in B-TT was 1.01 + 0.43 (Range 0.3 – 2.1) & in IDA was 0.60 + 0.21 (Range 0.1 – 1.1) (Table 4; Fig.10). The mean HbA2 value in B-TT was 4.92 + 0.33 (Range 4.3 – 5.5) & in IDA was 2.46 + 0.24 (Range 2.1 – 3.1) (Table 5; Fig.11). The mean  serum iron value in IDA was 34.92 + 7.85 (Range 20 – 50 micromol/l) & in B-TT was 88.40 + 18.41 (Range 46 – 155 micromol/l) (Table 6; Fig. 12). The mean TIBC value in IDA was 477.56 + 43.77 (Range 389 – 575 micromol/l) & in B-TT was 308.88 + 63.86 (Range 187 – 450) (Table 7; Fig.13). The mean ferritin value in IDA was 8.77 + 2.98 (Range 4 – 14 microgm/l) & in B-TT was 71.78 + 15.22 (Range 34 – 98 microgm/l) (Table 8; Fig.14). The mean transferrin saturation value in IDA was 7 +  2.06 (Range 3 – 12 %) & in B-TT was 29.32 + 5.44 (Range 20 – 39 %) (Table 9; Fig.15).

                           In IDA cases, the mean Hb value was 9.69 + 0.76 (Range 8.4 – 10.9 g/dl). The mean Total RBC count value was 4.42 + 0.36 (Range 3.56 – 5.21 million/cmm). The mean MCV value was 67.94 + 2.19 (Range 64.7 – 72 fl). The mean MCH value was 20.56 + 3.04 (Range 13.2 – 26.6 pg). The mean MCHC value was 29.60 + 1.74 (Range 23.6 – 31.8%). The mean RDW value was 18 + 1.77 (Range 15.8 – 24.3) and mean Mentzer index value was 15.45 + 1.37 (Range 13.1 – 19.3).

                           In B-TT cases, the mean Hb value was 10 + 1.05 (Range 8.1 – 12.2 g/dl). The mean Total RBC count was 5.40 + 0.56 (Range 4.52 – 6.48 million/cmm). The mean MCV value was 62.69 + 3.40 (Range 57 – 73.9 fl). The mean MCH value was 18.50 + 1.45 (Range 13.2 – 26.6 pg). The mean MCHC value was 29.46 + 1.06 (Range 26.8 – 31.3%). The mean RDW value was 16.59 + 1.42 (Range 15.8 – 24.3) and mean Mentzer index value was 11.74 + 1.49 (Range 9.4 – 14.3).

                              Comparing the values of IDA & B-TT – The mean value of MCV in IDA is 67.94 + 2.19 & B-TT is 62.69 + 3.40. As per the unpaired t test, the p value is <0.001 (Table 12; Fig.18). So, MCV can be a significant parameter in distinguishing between IDA & B-TT. The mean value of MCH in IDA is 20.56 + 3.04 & B-TT is 18.50 + 1.45. As per the unpaired t test, the p value is <0.001 (Table 13; Fig.19). So, MCH can be a significant parameter in distinguishing between IDA & B-TT.

The mean value of MCHC in IDA is 29.60 +1.74 & in B-TT is 29.46 + 1.06. As per the unpaired t test, p value is 0.628 (Table 14; Fig.20). So MCHC cannot be a significant parameter in distinguishing between IDA & B-TT according to our study.

                        The mean value of RDW in IDA is 18 + 1.77 & in B-TT is 16.59 + 1.42. As per the unpaired t test, p value is <0.001 (Table 15; Fig.21). So, RDW can be a significant parameter in distinguishing between IDA & B-TT.

                        The mean value of TRBC in IDA is 4.42 + 0.36 & in B-TT is 5.40 + 0.56. As per the unpaired t test, the p value is <0.001(Table 11; Fig.17). So, TRBC can be a significant parameter in differentiating between IDA & B-TT.

                        The mean value of Mentzer index in IDA is 15.45 + 1.37 & in B-TT is 11.74 + 1.49. As per the unpaired t test, the p value is <0.001 (Table 16; Fig.22). So, Mentzer index can be a significant parameter in differentiating between IDA & B-TT.

                         Table 22: Comparison of different parameters   

	GROUP
	IRON DEFICIENCY ANEMIA		BETA THALASSEMIA TRAIT
	Mean	Std. Deviation	Mean	Std. Deviation	p Value	Significance
AGE(Year)	6.18	3.73	5.60	4.16	0.465	Not Significant
HAEMOGLOBIN(g/dl)	9.69	0.76	10.00	1.05	0.097	Not Significant
TOTAL RBC COUNT(million/cmm)	4.42	0.36	5.40	0.56	<0.001	Significant
MCV(fl)	67.94	2.19	62.69	3.40	<0.001	Significant
MCH(pg)	20.56	3.04	18.50	1.45	<0.001	Significant
MCHC(%)	29.60	1.74	29.46	1.06	0.628	Not Significant
RDW	18.00	1.77	16.59	1.42	<0.001	Significant
MENTZER INDEX	15.45	1.37	11.74	1.49	<0.001	Significant

By taking conventional cutoff 13 of Mentzer index, all 50 IDA cases show Mentzer value greater than 13. Out of 50 B-TT cases, 37 cases show Mentzer value less than 13  & 13 cases show Mentzer value greater than 13 (Table 17). So, the sensitivity is 74%, specificity is 100%, positive predictive value (PPV) is 100%, negative predictive value (NPV) is 79.37% & diagnostic accuracy is 87% (Table 18). The ROC curve shows AUC 0.971. The p value of Mentzer index is <0.001 with CI 95% (Table 19). So, the Mentzer index can be a very useful test in differentiating between IDA & B-TT. But in our study, ROC curve shows 14 is better cut off for Mentzer index than 13 (Fig.23). Out of 50 IDA cases, 7 cases show Mentzer value less than 14 & 43 cases show Mentzer value greater than 14. Out of 50 cases B-TT cases, 47 cases show Mentzer value less than 14 & 3 cases show Mentzer value greater than 14 (Table 20). So, the sensitivity is 94%, specificity is 88%, positive predictive value (PPV) is 88.68%, negative predictive value (NPV) is 93.62% & diagnostic accuracy is 91% (Table 21).

                 While comparing with other studies by we found that the outcomes of RBC indices and RDW are almost similar with the study done by Aysel Vehapoglu et. al. in Turkey². The overall outcome also matches with other studies by different authors^{2, 4, 7, 17, 21, 87}(Table 23 & 24).

Table 23: Comparison of different similar studies with mean value of RBC indices & RDW in IDA

Parameter	Our study	Aysel vehapoglu2	Yeter Dujenli Kar4	Burcin Nalbantoglu7	Chuan Shen17	Sakorn Porn- Prasert21	M. Ferrara87
MCV	67.94 + 2.19	67.49 + 7.14	59.29 + 5.03	70.02	62.65 + 7.40	82 + 2.1	63.9 + 2.5
MCH	20.56 + 3.04	21.33 + 3.09	18.36 + 2.67	22.45	17.46 + 2.91	26.1 + 2.1	———–
MCHC	29.60 + 1.74	———–	30.16 + 4.28	—————	27.5 + 2.40	31.9 + 6.0	———-
RDW	18 + 1.77	17.4 + 3.48	20 + 2.39	15.77	20.08 + 3.09	12.5 + 1.0	18.2 + 2.1

Table 24: Comparison of different similar studies with mean value of RBC indices & RDW  in B-TT

Parameter	Our study	Aysel vehapoglu2	Yeter Dujenli Kar4	Burcin Nalbantoglu7	Chuan Shen17	Sakorn Porn- Prasert21	M. Ferrara87
MCV	62.69 + 3.40	60.11 + 3.49	59.97 + 3.9	63.46	58.84 + 3.79	63.0 + 3.0	72.1 + 2
MCH	18.50 + 1.45	18.9 + 1.37	19.33 + 1.44	20.79	18.65 + 1.24	19.3 + 1.1	———–
MCHC	29.46 + 1.06	———–	32.16 + 0.66	—————	31.7 + 0.95	30.5 + 4.0	———-
RDW	16.59 + 1.49	16.76 + 1.83	17.18 + 2.04	16.59	17.03 + 1.80	15.0 + 1.3	17.2 + 2.1

From the analysis of our study, we can compare the efficacy of Mentzer index with other studies by different authors as mentioned in table 25^{2, 4, 7, 17, 21, 87}.

Table 25: Comparison of efficacy of Mentzer index with other similar studies

Efficacy (in %)	Our study	Aysel vehapoglu2	Yeter Dujenli Kar4	Burcin Nalbantoglu7	Chuan Shen17	Sakorn Porn- Prasert21	M. Ferrara87
Sensitivity	74	86.36	80	67.8	84.3	100	59
Specificity	100	98.24	92	73.6	79.2	92.9	97.9
PPV	100	98.7	90.9	80.8	74.8	84	96.2
NPV	79.37	82.35	82.1	58.3	87.3	100	36.9
Diagnostic accuracy	87	91	—–	—–	——	94.8	79.6