Is complete blood count sufficient to rule out hemoglobinopathy carrier state in females visiting antenatal care clinic: A study at one center

INTRODUCTION

In India, anemia is seen as a severe public health concern that can affect people of any age. Hemoglobinopathies are a prevalent category of hereditary illnesses worldwide. A class of diseases known as hemoglobinopathies alters the hemoglobin (Hb) molecule’s structure, which, in turn, impacts its functionality.^[1]

A heme molecule is attached to each of the two polypeptide chains that make up the tetrameric protein known as Hb. Mutations or deletions in the genes encoding the alpha (α) and beta (β) globin chains of the human Hb molecule result in hemoglobinopathies, which are monogenic disorders.^[2,3]

Thalassemias fall under the category of autosomal recessive disorder and various gene mutations are frequently seen in a wide geographic span around the world. Numerous research conducted in India have revealed that certain communities, such as the Sindhis, Kutchhi Bhanushalis, and Punjabis, have higher rates of β-thalassemia.^[4]

The α gene cluster consists of α and tau and is located on chromosome 16. Mutations cause serious variants such as HbH illness. Likewise, the β gene clusters consist of β, delta, gamma, and epsilon and are located on chromosome 11. When combined with HbE or HbD-Punjab, β thalassemia can be heterozygous or homozygous due to deletions in the β globin gene. The outcome of point mutations in sickle cell disease is the same. A fatal Hb Bart’s hydrops fetalis occurs due to deletion of all four genes.^[5,6]

Herrick first described a few peculiar, elongated, and sickle-shaped red corpuscles in a case of severe anemia, in 1910. In 1949, Pauling et al. have the first to identify sickle cell disease molecularly and demonstrate it electrophoretically.^[6,7]

Thalassemia was first identified by Cooley and Lee in 1925, as a syndrome with chronic, progressive anemia, peripheral blood erythroblastosis, splenomegaly, and presence of affected family members.^[8]

Peripheral smears (PSs) are performed when low Hb and aberrant red blood cell (RBC) indices are observed, in addition to routine complete blood count (CBC) testing. However, other confirmatory tests such as electrophoresis and liquid chromatography are used when these tests are not conclusive in determining the cause of anemia. PSs, RBC indices, and CBC all support the need for further evaluation for abnormal Hbs.

The basic, extremely sensitive, and precise method known as high-performance liquid chromatography (HPLC) aids in the quick identification of different forms of Hb.^[8]

These aforementioned techniques help in identifying and taking appropriate preventable measures to decrease transfusion dependent anemia in next generation.

MATERIAL AND METHODS

We conducted a prospective study for a period of 6 months, from February 2024 to August 2024, on females who came to our hospital for antenatal care and had abnormal Hbs diagnosed on HPLC. A blood sample was collected in a vacutainer containing ethylenediaminetetraacetic acid and processed within 24 h of collection. This sample was first processed for CBC and PSs and then analyzed in the Bio-Rad D-10 Dual Program. The values of RBCs and their various parameters were then analyzed, and a diagnosis was made for hemoglobinopathy.

PSs were then made, stained, and examined. Cases in which sickle cells were seen, a manual sickling test was performed, which showed the presence of sickle cells, on incubating the sample mixed with sodium meta-bi-sulfate.

Mindray BC 6200, the machine that is used in our laboratory for the calculation of various cellular elements in blood is a 7-part fully automated hematology analyzer. It works on the principle of electrical impedance, wherein the electrical resistance is calculated and is produced by a particle suspended in a conductive diluent, as it passes through an aperture of known dimensions. Hb is measured using the principle of the colorimetric/sodium lauryl sulphate (SLS) method.

The Bio-Rad D-10 Dual Program machine is based on the chromatographic separation of analytes by the ion exchange HPLC. The samples are automatically diluted on the D-10 and injected into the analytical cartridge. The D-10 delivers a programmed buffer gradient of increasing ionic strength to the cartridge, where various Hbs are separated based on their ionic interaction with cartridge material. The separated Hb then passes through the flow cell of the filter photometer, where absorbance at 415 nm is measured. The data collected are then transferred using the software, and graphs are formed. The retention time varies for different Hbs.

RESULTS

The abnormal Hbs in our study include, cases where the possibility of α thalassemia could not be ruled out, β-thalassemia trait, delta-β-thalassemia heterozygous, hereditary persistence of fetal hemoglobin (HPFH) heterozygous, HbS heterozygous, HbE heterozygous, and HbD Punjab heterozygous [Figure 1].

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Figure 1:

Distribution of abnormal hemoglobins.

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The hemoglobinopathies that were least prevalent in this study were HbD Punjab, of which we encountered only two cases.

Out of 40 cases, 62% (25 cases) had Beta thalassemia trait, while 18% (7 cases), 7.5% (3 cases), 7.5% (3 cases) and 5% (2 cases) of females had HbE heterozygous, HbS heterozygous, Delta-Beta thalassemia heterozygous or HPFH heterozygous and HbD Punjab heterozygous respectively [Figure 1].

The cases with S-window were confirmed with a manual sickling test.

The PSs of various abnormal Hbs showed polychromasia and anisopoikilocytosis of RBCs, most common being target cells and schistocytes.

Not all abnormal hemoglobins had a microcytic hypochromic morphology in the PS; these include HbD Punjab heterozygous, HbE heterozygous, and HbS heterozygous, as only a few among these showed a microcytic hypochromic pattern, while delta-β-thalassemia heterozygous and HPFH had normocytic normochromic cells as seen on CBC and PS.

Among the 40 cases with abnormal Hbs, only 17 cases (42.5%) were indicated for hemoglobinopathy analysis, based on RBC indices in CBC, as opposed to the remaining 23 cases (57.5%), in which the RBC indices and CBC were within normal range. The Mentzer index, which is usually used to diagnose Beta thalassemia, based on RBC parameters in CBC, such as mean corpuscular volume and total RBC count, was only indicative in 16 cases out of the 25 cases (64%) [Figure 2]. Thus, indicating that females with normal CBC and PS findings, having abnormal Hb, could be missed, and further workup should be done to prevent major hemoglobinopathies in newborns.

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Figure 2:

Beta-thalassemia trait distribution based on Mentzer index.

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Husband’s screening was advised in all the cases with abnormal Hbs.

DISCUSSION

Inherited hemoglobinopathies, which range from near-normal Hb to severe transfusion-dependent hemoglobinopathies, are common in the Indian population. Common Hb disorders in India include sickle cell anemia, HbE, HbD Punjab, and β-thalassemia.^[8]

Hemoglobinopathy carriers may present unique challenges for women who are or plan to become pregnant. This has a major effect on the fetus and the mother’s pregnancy outcomes. To avoid inheritance in the future offspring, practitioners must successfully identify and manage these illnesses. If the approach relies on RBC indices and is followed, if necessary, by HPLC testing, some carrier states cases may go unnoticed.^[6]

Few studies in other parts of the world have been done that are comparable to ours [Table 1].^[8-11]

In a related study, 423 expectant females were evaluated in Thailand in 2003 by Sanchaisuriya et al. HbE thalassemia heterozygous was the most prevalent kind of defective Hb, seen in 62.4% of the female participants in this study. Two modified tests were used on the females: the modified dichlorophenolindophenol test and the modified osmotic fragility test. The Hb patterns in the females with probable hemoglobinopathies were then identified using an automated high-performance liquid chromatography analyzer in conjunction with α- and β-globin gene polymerase chain reaction analysis.^[9]

In 2007, a comparable study was carried out in Lao. A modified dichlorophenol-indophenol test, the osmotic fragility test, and HPLC were used to screen 307 pregnant women for hemoglobinopathies and thalassemia. The results of this study were likewise in line with the earlier study carried out in Thailand, which indicated that the most prevalent hemoglobinopathy was heterozygous HbE thalassemia.^[10]

In 2008, Sachdev et al. conducted an 8-month study in North India. Hemoglobinopathies were investigated in these 2600 patients. Three hundred and twenty-seven of the patients in total had aberrant Hbs on HPLC. With 15 cases of β thalassemia major and 16 cases of thalassemia intermedia, β-thalassemia was the most prevalent abnormality. HbD-Punjab heterozygous, HbE variation (HbE homozygous, HbE heterozygous, and HbE – β-thalassemia trait double heterozygous), double heterozygous for Hb Q India and β-thalassemia trait, HbS homozygous, HbD-Iran, and HbJ are among the various abnormal Hbs.^[11]

A study was carried out in Jharkhand, India, between 2013 and 2014 by Shankar et al. involved 200 participants who underwent HPLC testing for hemoglobinopathies. Both men and women were involved in the study. The majority of the participants in our study had normal Hb levels. The results showed that sickle cell disease and sickle-β-thalassemia were the most common hemoglobinopathies, respectively.^[8]

CONCLUSION

This study shows that many times abnormal Hb carrier individuals do not always manifest with abnormal parameters of RBC indices on CBC. Majority of cases in our study had normal RBC indices and PS findings, thus not requiring any further investigations for detection of abnormal Hbs. This may in turn result in many individuals of the next generation being affected with either major or minor hemoglobinopathies. Hence, CBC, PS findings, and HPLC together can prevent and reduce the load of abnormal hemoglobinopathies in the next generation.