LAST WEEK

Finally it’s the end of SIP and I guest everyone would be busy with their MP now. This would be the last entry and we are done with it! Alright here goes..

This week I would introduce a test done in serology called SERODIA-ATG .

Intended use

SERODIA-ATG is a semi-quantitative microtiter particle agglutination test use for detection and titration of thyroglobulin Abs in human serum.

Introduction

Autoimmune disease such as chronic thyroiditis (Hashimoto’s disease) may commonly produce Abs to thyroglobulin or microsomal Ag of the thyroid. Other than being found in thyroiditis, these Abs may be found in other thyroid disorders such as primary myxedema, hyperthyroidism, goiter and thyroid tumors.

Thyroglobulin Abs can be demonstrated by several procedures, such as passive agglutination. SERODIA-ATG is prepared using gelatin particles sensitized with purified thyroglobulin. As thyroid autoimmune disease may demonstrate an immunological response to Ags other than throglobulin, SERODIA-ATG would be recommended to use in conjunction with clinical findings or other immunological thyroid test.

Principle of the test

This test is based on gelatin particle carriers sensitized with thyroglobulin, extracted and purified from human thyroid tissue. Serum containing specific Abs will react with the thyroglobulin-sensitized coloured gelatin particlesto form a smooth mat of agglutinated particles in the microtitration plate. Negative reactions are characterized by a compact button formed by the settling of the nonagglutinated particles. The test is designed to be used with microtitration techniques.

Materials

- Reconstituting solution: for reconstituting the sensitized and unsensitized particles
- Sample diluents: for diluting human serum in assay
- Sensitized particles
- Unsenitized particles
- Positive control
- Dropper: to dispense approximately 25µL per well

Test procedure

1. Use a ‘U’ shaped microplate sideways. One row consisting 12 wells is necessary to test one
patient sample.
2. Drop 2 drops of sample diluents into the first 2 wells and 3 drops into the third to twelfth
well.
3. Using a pipette, add 10µL of positive control or patient’s serum into the first well. Mix well
by pipetting u and down several times. Then transfer 25µL of the diluted serum or control
from the 1st well to the 2nd well. Mix well again and transfer 25µL to the 3rd well. Repeat
mixing and transferring till the twelfth well.
4. Drop one drop of Unsensitized Particles into the 2nd well and drop one drop of Sensitized
Particles into the 3rd to 12th well.
5. Repeat the above step for every patient sample and positive control.
6. Mix the contents in the well by tapping the plate with finger. Then cover the plate and
place on a level surface. Allow it to stand for 3 hrs at room temperature.

Final dilution of each well:
Well 1- 1:6
Well 2- 1:27
Well 3- 1:100 (10)2
Well 4- 1:400 (20)2
Well 5- 1:1600 (40)2
Well 6- 1:6400 (80)2
.
.
.
Well 12- 1: 26214 (5120)2

Interpretation of results

Settling patterns of particles:
1. Non-reactive (-): particles are concentrated in the shape of a button at the center of well.
There is a smooth round outer margin.
2. Indeterminate (+): particles are concentrated to form a compact-ring shape with smooth
outer margin.
3. Reactive (+): particles form large ring with a rough multiform outer margin. Peripheral
agglutination occurs.
4. Reactive (++): firmly agglutinated particles spread out covering the bottom of the well
uniformly.

Expected results

Thyroid Abs are seldom found in serum of normal patient. However, 2 to 17% of the normal population may show signs of low titers of these Abs with no symptoms of disease. This happen more on women and increases with age. The occurrence of these Abs may also indicate that there’s previous autoimmune disorders. Patients with low titer of Ab should be tested periodically as the presence of the Ab may be an early sign of autoimmune disease.

In active cases of thyroid autoimmune disease and in some cases of thyrotoxicosis, moderate (1:1600) to very high (1:25600) Ab titer may be observed. The observation of very hugh Ab titers in an individual with a firm, hard, fast-growing, symmetrical goiter strongly suggest Hashimoto’s goiter.

Note: in any case of reactive result at any dilution should be interpreted in accordance to the clinical findings. Diagnosis of thyroid autoimmune disease should not be based on the test alone but in conjunction with other immunological tests, physical examination, familiar studies and if necessary, biopsy.

Sharon
Tg01