This exploratory study was conducted in a Primary Health Centre which was attached to the Department of Community Medicine of a medical college which was situated in south Bangalore, India, for a period of 3 months (June – August, 2010). 210 pregnant women who attended the ANC clinic for the first time during the study period and the husbands of the NESTROFT positive women were the study subjects. All the recruited women were residents of the rural catchment area of the PHC. Ethical clearance was obtained from the Institutional Ethics Committee for conducting the study. Signed informed consents were obtained from the pregnant women who were willing to participate in the study and the details regarding the socio-demographic characteristics, the obstetric history, the past menstrual history and the family history were obtained on a structured proforma by using the interview method. With regards to the pregnant women who were positive for the NESTROF test; their respective husbands were also screened for the thalassaemia carrier status by using the same test. If both the husband and the wife were positive for the NESTROF test, then they were considered to be “at risk” of delivering a child with thalassaemia major. If the gestational age was less than 20 weeks, then the couple was counselled to undergo Hb electrophoresis to make a confirmatory diagnosis and subsequently, to make a prenatal diagnosis. If the gestational age was more than 20 weeks, the couple was counselled to undergo electrophoresis for the confirmation of the diagnosis and to make a prenatal diagnosis for the subsequent pregnancies.

The Procedure of the NESTROF Test: Two mililiters (2 ml) of 0.36% buffered saline was taken in one tube (10cm x 1cm diameter) and 2 ml distilled water was taken in another tube. 2 – 4 drops of anticoagulated blood (which was routinely collected for ANC) was added to each of the tubes. The tubes were left undisturbed for half an hour at room temperature. After half an hour, the contents of both the tubes were shaken and the tubes were held against a white paper on which a thin black line was drawn. The line would be clearly visible through the contents of the tube which contained distilled water due to complete lysis of the blood cells. If the line was visible through the contents of the tube which contained buffered saline, the test would be considered as negative, while the test would be considered as positive when the line was not visible [3,4,8].

The haemoglobin level was determined by Sahli’s haemoglobinometer method. As per the WHO, the standard cut off for the Hb concentration of pregnant woman.

As can be observed from [Table/Fig-1], out of the 210 pregnant women who were enrolled in the study, a majority 100 (47.6%) were in the age group of 21-25 years, 190 (90.5%) were literate, 148 (70.5%) were Hindus by religion and 83 (39.5%) had an upper lower socioeconomic status as per the B G Prasad classification. Among the 210 pregnant women who were subjected to the NESTROF test, 18 were positive. Thus, the Prevalence of the Beta Thalassaemia trait among the pregnant women who attended the ANC clinic in the PHC by using the NESTROF Test was 8.5%.

Among the 18 women with the beta thalassaemia trait, a majority 7 (38.9%) were in the age group of 21-25 years, 17(94.5%) were literate, 16 (88.9%) were Hindus by religion and 6(33.3%) belonged to the upper lower socioeconomic status as per the B G Prasad classification. 5(27.8%) of the pregnant women with the Beta Thalassaemia trait belonged to the Vokkaliga community and 5(6.8%) of them were born out of 2^nd degree consanguineous marriages (p=0.55) [Table/Fig-2]. 9(50%) of the pregnant women with the Beta Thalassaemia trait were primigravidae. 7(28%) had a history of one or more abortions in the past, out of which, 6(85.7%) had undergone first trimester abortions (p >0.05).

Among the pregnant women who attended the ANC clinic 118 (56.7%) had a Haemoglobin concentration of <11gm%, with a mean Hb% of 9.7+1.4gm% [Table/Fig-2]. A majority i.e 10 (55.5%) pregnant women with the Beta Thalassaemia trait had a Haemoglobin concentration of <10gm%, with a mean Hb% of 10.06+1.00gm% (p=0.88) and 8(44.4%) of the pregnant women with the Beta Thalassaemia trait belonged to the B Positive blood group.

Among the 18 pregnant women with the Beta Thalassaemia trait, only 9 (50%) had turned up with their husbands for the NESTROF test. None of the husbands of the NESTROF test positive pregnant women were found to be positive. Among the couples which were tested in the present study, no pregnancies were at a risk of delivering babies with Thalassaemia major. None (100%) of the pregnant women were aware of the disease, Thalassaemia.

In the present study, the prevalence of the beta thalassaemia trait among the pregnant women who attended the antenatal clinic at a primary health centre on using the NESTROF test was 8.5%, which is consistent with the findings of Bani Gajra et al., who had used the same test in a hospital based study which was done among pregnant women at Kolkata, India, where it was observed to be 7.1% [1]. In another study which was conducted by Deepa V et al., the overall prevalence of the beta thalassaemia trait among the Sindhi community was found to be 16.81% [3]. The results of the present exploratory study throw light on a higher existence of the carrier status, even in south Indians, which needs special attention, as much of the available published studies have been concentrated among the north Indian population.

The practice of consanguineous marriages, preferably with maternal kindreds, is an accepted socio-cultural phenomenon, irrespective of the religion, caste and the economic status, which also has an effect on the transmission of thalassaemia [9]. In the present study, it was observed that 6.8% of the carrier pregnant women were born out of 2^nd degree consanguineous marriages. In contrast, the consanguinity was found to be higher in the studies of Shivashankara AR et al., at Dharwad, north Karnataka (20%) [9] and of Hafeez M et al., in Lahore, Pakistan (56.7%) [7].

In the present study, it was observed that 55.5% of the beta thalassaemia carrier women had Hb concentrations of less than 10gm%. Similar results were observed by Deepa V et al., in their epidemiological study which was done on the beta thalasssemia trait, where 22.7% of the carrier women were diagnosed to have anaemia. The chances of misdiagnoses of the carriers of beta thalassaemia are high, as Iron deficiency anaemia itself may hinder the detection of the thalassaemia carrier status, thus giving more false negative NESTROF results. This is considered as one of the limitations of the NESTROF test.

The thalassaemia carrier status may result in abnormal pregnancy outcomes like spontaneous abortions. In the present study, 39% of the carrier women had histories of one or more abortions, of which 85.7% were first trimester spontaneous abortions. A multicentric study which was conducted by ICMR, reported a 2.8% incidence of spontaneous abortions among women with thalassaemia traits [10].

Only 50% of the women with the beta thalassaemia trait had turned up with their husbands for the NESTROF test. A similar observation was made by Bani Gajra, where only 60.3% women had turned up with their husbands for the screening to make a prenatal diagnosis for [1]. Only an improvement in the awareness of the people regarding the causes and the consequences of thalassaemia and the modes of its prevention can bring about a desirable change in the attitude of the family members, especially among the the male partners, who can volunteer for these kind of tests and procedures.

In the present study, none of the pregnancies were at a risk of delivering a Thalassaemia major child. On the contrary, Bani Gajra reported 15.8% couples to be at a risk of delivering thalassaemic children [1]. This could be because the husbands of only 50% of the antenatal mothers had turned up for the NESTROF test. Similarly, regarding the awareness about thalassaemia, we observed that none of the subjects had any knowledge about thalassaemia. This indicated that there was need to improve the awareness of the people regarding thalassaemia.

In the present study, the cost which was incurred in conducting the NESTROF test was only Rs 1.5/- only per subject, which implied that the NESTROF test was a simple and a low cost screening tool which could be used for the identification of the carrier status of beta thalassaemia. It is useful for screening large populations, especially in the remote village areas and at the primary health care centres, where laboratory facilities are not available. However, there is a need for carrying out similar studies which cover larger sample sizes and wide geographic areas, for generalizing the results, as well as the cost effectiveness of the test.