Iodine deficiency disorders and their prevention in India

Abstract

New insights on the high prevalence of functional decompensation of the thyroid among newborn and children from several states of India as well as neighbouring countries of Nepal and Bhutan helped to prevent nutritional iodine deficiency and iodine deficiency disorders through country-wide iodized salt prophylaxis. Presently on the basis of scientific studies, salt iodization in India is saving millions of children from neonatal hypothyroidism related psycho-physical retardation.

Appendix

1.1 Appropriate technology and epidemiologic strategies for organizing cost-effective screening for neonatal hypothyroidism in goiter endemia

Organizing a sophisticated programme like neonatal screening for hypothyroidism to determine its incidence in socio-economically backward endemic populations involve adopting the following technical and demographic strategies.

• 1. Development and validation of super-sensitive, specific and cost-effective immunoassays for measuring T₄ and TSH in filter paper cord blood spots obtained by post.

• 2. Ensuring adequate coverage of newborns from strictly randomized and pre-selected areas and adopting a strategy of “inclusive screening” to cover most child births in the pre-selected areas in the community (including home delivery by traditional birth attendants).

1.2 Cost-effective and appropriate development of RIA technology for use in the screening programme

Low cost and high assay sensitivity of the RIA used were two important requirements for successfully organizing NH screening in socio-economically backward endemic areas.

1.2.1 Preparation of High Specific Activity ¹²⁵I–T₄ tracer

The commercially available ¹²⁵I–T₄ tracers, besides being expensive, did not permit development of highly sensitive RIAs to measure picogram quantities of T₄. We therefore synthesized the high specific activity ¹²⁵I–T₄ required for the filter paper cord blood spot assay by adding 1 atom of ¹²⁵I (100% isotopic abundance, obtained from M. S. Amersham Radiochemical, England) to T₃. This procedure which enabled us to prepare ¹²⁵I–T₄ of predictable and high specific activity can be summarized as follows:

Substances indicated in Table 3 were added in sequence to a small glass vial fixed at eye level in a fuming cupboard. A reaction time of 45 s was permitted after the addition of chloramine T to allow optimal iodine transfer. The oxidation reaction was stopped by adding sodium metabisulphate. After adding propylene glycol, the reaction mixture was transferred on to a sephadex G-25 column and eluted with 0.05 m phosphate buffer (pH 11.9). One hundred fractions of 2 ml each were collected and the radioactivity eluted was counted in a well type gamma counter with altered geometry. A typical elution profile is shown in Fig. 2. The immunoreactive fractions of T₄ peak were pooled and preserved for use. The ¹²⁵I–T₄ preparation thus made will have a specific activity of @2008 μCi/μg of T₄ which permits 5,000 cpm per picogram tracer used in the assay. The high specific activity T₄ tracer thus prepared is stable for 4–6 weeks when stored frozen. The estimated cost of preparing 100 μCi of ¹²⁵I labeled T₄ by this method is approximately US$10 which is one tenth the cost of commercially available high specific activity T₄ tracer.

Fig. 2

Purification of T4 tracer using sephadex column

Table 3 Ingredients of ¹²⁵I–T₄ tracer

1.3 Anti-T₄ antiserum

Anti-T₄ antiserum required for highly sensitive T₄ assay was raised in rabbits. Bovine thyroglobulin (source: Sigma Chemical Co., P.B.No.14508, St. Louis, MO, 63178, USA) was the immunogen used. 200–500 μg equivalents of the immunogen were emulsified with 1 ml of Freunds Complete Adjuvent and injected biweekly in the medial aspect of the thigh of rabbits weighing approximately 2 kg. The rabbits used for immunization were treated with an ablative dose of ¹³¹I and maintained on T₃ before immunization. After the third immunization test, bleeds were done to look for T₄ antibody titre. Rabbits which showed significant T₄ titre after the fourth or fifth immunization were given booster doses of the immunogen at 10–14 days intervals till maximum antibody titres were obtained. Generally, for T₄ antisera, titres above 1:5000 dilution are considered satisfactory for harvesting antiserum. Rabbits which give a satisfactory T₄ antibody titre are repeatedly bled at fortnightly intervals to ensure adequate stock of T₄ antiserum. On repeated harvesting if there is a significant decline in the titre of antiserum, the animals are rested for 2–3 months before subsequent booster doses are administered.

1.4 T₄ RIA development and Validation

Using the high specific activity T₄ tracer and anti-T₄ antibody prepared, a T₄ RIA was developed as follows:

The assay buffer was 0.2 M Glycine Acetate (pH 8.7) fortified with 0.02% bovine serum albumin (RIA Grade). 0.02% 8-anilino naphthol sulphonic acid (sodium salt) was used to displace TBG bound T₄. Approximately 6,000 cpm of the T₄ tracer was added to assay tubes. The anti-T₄ antiserum prepared was used in final dilution of 1:10,000 in all tubes except in the “non-specific binding” tubes. Sera were assayed in 5–10 μl volumes, and equivalent volumes of hormone free sera were added to the standard tubes. The standard and assay tubes were incubated at 4°C for 24 h. The bound and free fractions were separated by dextran coated charcoal. The minimum detectability of T₄ in this assay was 12.5 pg per tube. The sensitivity of the assay was enough to measure T₄ reproducibly in concentration as low as 1 μg/dl in 1:500 dilution of patients serum. Inter and Intra assay coefficients of variations of the assay did not exceed 12%. Cross reactivity studies done, using a variety of related substances, such as T₃, rT₃, 3,3’ T₂, 3,5 T₂ and thyroglobulin did not show any significant interference with the specificity of the assay.

1.5 Adaptation of the T₄ RIA for the Filter Paper Spot Assay

The T₄ RIA described above was adapted for the dried blood spot technique essentially as described by Larsen and Broskin. The volume of whole cord blood in 3.2 mm diameter filter paper spot was estimated to be 2.4 ± 0.09 μl. The T₄ was measured in 3.2 mm diameter filter paper discs punched out in duplicate into 1 ml volume of assay cocktail. Spots were vortexed and incubated at 4°C for 24 h, before bound and free fractions were separated. Blood spots for constructing standard curves for spot assay were collected in heparinized tubes and fortified with T₄ to have a concentration that ranged from 1 to 20 μg /dl. The “standard spots” were prepared on Whatman No. 3 filter paper, using whole blood samples thus fortified. 3.2 mm discs were punched out from these “standard sports” in duplicate, to construct a standard curve. Figure 3 gives a composite standard curve obtained by assaying eight replicas of standard spots.

Fig. 3

T4 standard curve adapted for filter paper spot assay

Figure 4 shows correlation of T₄ measured by spot assay and the direct method. Very close correlation was observed between the results obtained by the two methods.

Fig. 4

Correlation between T4 direct and filter paper assays

1.6 Feasibility and cost

We have experience of screening over 30,000 newborns using the techniques described above. Most of the areas incorporated in the screening programme belonged to remote and underdeveloped regions of India where the majority of the deliveries are conducted at home by untrained traditional birth attendants. We were able to enlist the full cooperation of these personnel who conduct the majority of deliveries in Indian villages. Most of them sent very satisfactory blood spots for screening, so that 70% or more spots received in the laboratory could be successfully assayed for T₄. The newborns screened could be traced back by pre-labelling of the filter paper strip as well as the envelopes by appropriate code numbers.

1.7 Method of transportation

The cord blood spots collected at the time of delivery by the birth attendants were transported to the laboratory in Delhi in specially designed, addressed and pre-paid envelopes, distributed along with the filter paper strips. On an average it took 8–10 days for the blood spots posted to reach the laboratory in Delhi from the remote villages. There was no significant loss of T₄ immunoreactivity from the strips in conditions of postal transport up to 3 weeks.

In the existing system of postal communication, there was a 10–15% loss of strips in transit. Nevertheless, we were able to get a fairly representative sample of cord blood in adequate numbers from the region in question to obtain reliable information on the incidence of NH in that area. The cost of each child screened for neonatal hypothyroidism, using this technique, was roughly Rs.5 including Rs.1 for postal charges.