Anaemia is a major public health problem in developing countries, and is a cause of serious concern. It is also one of the most commonly encountered medical disorders during pregnancy. Iron deficiency is one of the important causes whereas deficiencies of other nutrients such as folic acid, protein, vitamin B-12, vitamin A and copper also contribute. The other major cause is presence of “Haemoglobinopathies” - the inherited disorders of haemoglobin, resulting in the spectrum ranging from mild anaemia to severe transfusion dependent anaemia presenting in infancy. Haemoglobinopathies are divided in to two main groups as follows:

• Thalassemia syndromes- Quantitative disorders with reduced number of globin chains.
• Structural haemoglobin variants – Qualitative disorders of globin chains.

Amongst all Haemoglobinopathies, Sickle cell syndrome and beta Thalassemia are the major constituents of public health problems.

Beta Thalassemia is detectable in almost every Indian population, most commonly seen in Sindhis, Gujaratis, Muslims, Punjabis and Bengalis. HbS is predominantly found in central India i.e., Vidarbh in Maharashtra, Madhya Pradesh, Orissa, Andhra Pradesh, Gujarat and to a lesser extent in Tamil Nadu, Karnataka, Kerala.  HbD is predominantly seen in Punjab, Uttar Pradesh, Gujarat and Jammu and Kashmir⁷.

Clinical manifestations of these Haemoglobinopathies can vary from asymptomatic state to severe, lifelong, transfusion dependant anaemia and reduced life expectancy. About 1.1% of couples around the world are at risk for having children with a haemoglobin disorder of which 2.7 per 1000 conceptions are actually affected.

It is highly recommended to screen the population at large before marriage/pregnancy– the target population mainly being senior college students, premarital age group, newly wed couples (Mass Screening). However, if this is not possible, it becomes important that the presence of haemoglobinopathies in the expectant parents is detected in a timely manner. During the antenatal period, the screening needs to be offered early in pregnancy – at the first antenatal visit/registration, to allow time for fathers to be screened so that the results of the screening tests and any prenatal

diagnosis (PND) are available sufficiently early for couples to be able to make timely informed choices.

This study was done in a tertiary care hospital, P.D.U Medical College and Hospital Rajkot, Gujarat, Western India. Mothers who attended the antenatal clinic were included and their samples were collected in OPD Laboratory, Department of Pathology, P.D.U. Medical College and Hospital, Rajkot. Two ml of peripheral blood samples were collected, in EthyleneDiaminetetraAcetic acid (EDTA) vacuum containers. Samples were initially subjected to complete blood count (CBC) in 5-part MINDRAY cell counter, peripheral blood smear examination using Leishman’s Stain and Field’s Stain. The results of Haemoglobin, Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Haemoglobin Concentration (MCHC), Red Blood Cell Count (RBC) and Red Cell Distribution Width (RDW) were correlated with Peripheral Smear examination.

The blood samples were stored at 4 degree Celsius and subjected to High Performance Liquid Chromatography (HPLC) in batches. For this study, Variant Haemoglobin Testing system (Bio-Rad variant 2) was used to perform High Performance Liquid Chromatography (HPLC). It operates on the principle of HPLC and the column comprises of a small, (3.0 x 0.46) cm cation exchange cartridge.

The status of Thalassemia’s and other haemoglobinopathies were determined depending upon the percentage of HbF and HB A2.  The below table (table 1) shows common haemoglobinopathies and their corresponding HbF and HbA2 percentage.

Table 1: Common haemoglobinopathies

For the haemoglobinopathies like sickle cell disease and HbD heterozygous, other than

see the percentage of HbS and HbD. (Table 2 and Table 3).

Table 2: Sickle cell haemoglobinopathies

Table 3: Haemoglobinopathies HbD

A total of 400 antenatal women were included in the screening for the duration of 3 months.

In the present study, out of 400 cases, 70(17.5%) cases were identified to be having haemoglobinopathies by HPLC. Out of these 70 cases, there were 36(9%) cases of beta thalassemia trait, 25(6.25%) cases of sickle cell heterozygous, 5(1.25%) cases of sickle cell

homozygous, and 4(1%) cases of HbD heterozygous. (Figure 1)                   & (table 4).

Normal adult chromatogram shows primarily HbA, small percentage of HbA2 (<3.5%) and traces of fetal Hb (<1%). Pregnancy p has no influence on HbA2 percentage but in 15-20% of women HbF percentage increases with level as high as 5% being observed.

Table 4: No. of cases distribution in percentage

From the above chart, we can say that there is high prevalence of beta thalassemia trait (9%) and sickle cell heterozygous (6.25%).

Caes that shows sickle cell homozygous state were of 1.25%, and cases with HbD heterozygous hemoglobinopathy were of 1%.

Hematological parameters in all the 400 antenatal cases showed that anemia is prevalent in pregnancy known as physiological anemia of pregnancy. Also other parameters like Hematocrit, MCV, MCH and MCHC are at lower normal limits in pregnancy. Hemoglobin values <7gm/dl- severe anemia found in 16 cases. Hemoglobin values in range of 7-9gm/dl- moderate anemia found in 70 cases. Maximum number of cases is with mild grade of anemia- 280 cases (Hb 9-11gm/dl). Cases with normal haemoglobin were found to be 34.

Table 5- Grading of anemia

We analysed the various RBC parameters and percentage of Hb fraction in various abnormal hemoglobinopathies encountered in this study.

Table 6 – showing mean of haematological profile that were seen in various haemoglobinopathies in the present study.

From the CBC and peripheral smear findings (table 5) of all the cases, we can say that beta thalassemia trait usually have a low MCV and MCH. The peripheral smear show target cells which are more characteristic of beta thalassemia trait than iron deficiency anaemia. Iron stores are normal/high in beta thalassemia. It is very important to differentiate between beta thalassemia trait and iron deficiency anemia. In all the cases of beta thalassemia trait, haemoglobin values are decreased with MCV levels <75 fl and MCH levels <27 pg. Also RDW values are <19%. MCV and RDW values show a statistically difference amongst normal pregnant women and beta thalassemia trait.

The peripheral smear in sickle cell disorders will show sickle cells and the diagnosis can be confirmed by HPLC and by sickling test.  A significant difference was noted in Hb values, PCV values amongst normal pregnant women and sickle cell homozygous case. In other haemoglobinopathies the blood picture can be non-specific and HPLC comes in as an excellent diagnostic tool for detection and quantification of several normal and abnormal haemoglobins.

 Table 7 – Fraction of Hb that are present in the individuals having different types of haemoglobinopathies in the present study.

From the above values (table 7) we can say that, all the cases of beta thalassemia trait shows HbA2 values >3.5. The HbF values are increased (>1%) in sickle cell homozygous. Whereas abnormal Hb

variant is seen in sickle cell heterozygous, sickle cell homozygous (HbS) and in HbD heterozygous (HbD). Also there is difference in the mean value of HbS and HbA2 between sickle cell homozygous and sickle cell heterozygous.  It is evident from the present study that a difference is noted for HbS/Abnormal Hb values in normal pregnant women and Sickle cell heterozygous/homozygous. In addition to HbS, HbF also significantly raised in sickle cell homozygous. Hence raised HbS can be reliably used to diagnose cases of sickle cell heterozygous cases while raised HbS and HbF can be reliably used to diagnose cases if sickle cell homozygous cases in pregnancy.

Thalassemia and other structural hemoglobinopathies are major and important cause of morbidity and mortality worldwide which can be prevented by population screening, genetic counselling and prenatal diagnosis. Screening of individuals at increased risk of being carriers can identify couples with a 25% risk of having a pregnancy with a significant genetic disorder. Antenatal screening is the important step to identify women having the risk of producing a child affected with hemoglobinopathy. In 1975, Sukumaran and a recent large study conducted in metropolitian cities of India had observed that beta thalassemia is probably the commonest inherited haemoglobin disorder in Indian sobcontinent.

The prevalence of hemoglobinopathies found in this study was about 17.5%. The WHO (world health organization) estimates that 5% of adults are carriers with 3.9% for beta thalassemia and 2.3% for sickle cell trait¹⁴, which is lower than that of the present study, the reason being the studied carried out in Gujarat, because castes like lohana, Sindhi’s have higher prevalence of hemoglobinopathies. The prevalence of beta thalassemia in the present study is 9% which is slightly higher than the study by Komal Sawaimul.et.al (2.2%) (Pune,2017)¹⁵ and Panda et. al.¹² but it is in accordance with the study of Sachdev.et.al.(Haryana,2010)¹³ in which there is high prevalence of beta thalassemia trait (8.9%). Madan N et al. found the overall gene frequency of ß-thalassemia trait in Delhi and Mumbai to be 5.47% and 2.68% respectively. The carrier rate of β-thalassemia varies from 1 to 17 % in India with an average of 3-4 %. The overall prevalence of BTT was 3.38 % among antenatal women. Conditions with borderline Hb A2 need careful interpretation. Iron deficiency may lead to a low Hb A2 and hence may mask a thalassemia trait whereas B12/folate deficiency may lead to slightly raised Hb A2 leading to a false diagnosis of a trait. Careful evaluation of indices with iron profile will usually help in such cases. Similarly, milder forms of thalassemia or a co inheritance of delta thalassemia may lead to borderline A2 levels. Genetic studies should be advised in such cases for a conclusive opinion.

The presence of sickle cell heterozygous (6.25%) is much higher that the Komal Sawaimul.et. al.(1.2%)¹⁵ and Anjali j Kelkar,et, al (Pune, 2019) (0.74%).  However it is in accordance with the other studies like Gupta et.al. and Panda et. al.

The prevalence of sickle cell homozygous in the present study (1.25%) correlates well with Gupta.et.al¹¹ study. According to Census 2001 the expected sufferers of sickle cell gene i.e. sickle cell homozygous is 0.5%. So the values in the present study are slightly higher than this.

The prevalence of HbD heterozygous in the present study is about 1% which is in higher than the study of Sachdev.et.al.¹³ in which there is 0.5% prevalence of HbD heterozygous. No other reference regarding studies for HbD heterozygous in pregnant women are found.

Table 8: Comparative studies of values of HbF, HbA2 and HbS in various hemoglobinopathies.

Our findings support that Hb-HPLC is an excellent, diagnostic tool for identification of haemoglobin variants with a high degree of precision in the quantification of HbA2, HbF, haemoglobin variants. We also endorse that antenatal screening for haemoglobinopathies is an effective method to diagnose those and with further counselling and spouse’s testing would help prevention of birth of children with transfusion dependent anaemia. Our society has a significant incidence of beta thalassemia major and other transfusion dependent disorders; while the government support systems for such patients still fall short. We are sure that the education of the patients, society at large, medical fraternity along with effective counselling, that we tried to achieve through this project, will go long way in achieving the ‘thalassemia free society.

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