Measurement of Substances Absent in Normal Serum
Tests that measure substances present in the circulation only under pathologic circumstances do not provide information on the level of thyroid gland function. They are of value in establishing the cause of the hormonal dysfunction or thyroid gland pathology.
The humoral antibodies most commonly measured in clinical practice are directed against thyroglobulin (Tg) or thyroid cell microsomal (MC) proteins. The latter is principally represented by the thyroid peroxidase (TPO). 296-298 More recently, immunoassays have been developed using purified and recombinant TPO.299, 299a, 299b Other circulating immunoglobulins, which are less frequently used as diagnostic markers, are those directed against a colloid antigen, T4 and T3. Antibodies against nuclear components are not tissue specific. Immunoglobulins possessing the property of stimulating the thyroid gland will be discussed in the next section.
A variety of techniques have been developed for the measurement of Tg and MC antibodies. These procedures include a competitive binding radioassay, complement fixation reaction,300 tanned red cell agglutination assay,301 the Coon's immunofluorescent technique,302 enzyme-linked immunosorbent assay.299,303 Although the competitive binding radioassay 304,305 is a sensitive test, agglutination methods combine sensitivity and simplicity and have now largely superceded other methods. Current commercial kits utilize synthetic gelatin beads rather than red cells.305a
In the assay of Tg and MC antibodies by hemagglutination (TgHA and MCHA), particulate material is coated with either human Tg or solubilized thyroid MC proteins (TPO) and exposed to serial dilutions of the patient's serum. Agglutination of the coated particulates occurs in the presence of antibodies specific to the antigen attached to their surface. To detect false-positive reactions, it is important to include a blank for each sample using uncoated particles. Because of the common occurrence of prozone or blocking phenomenon, it is necessary to screen all serum samples through at least six consecutive two-fold dilutions.306 Results are expressed in terms of the highest serum dilution, or titer, showing persistent agglutination. The presence of immune complexes, particularly in patients with high serum Tg levels, may mask the presence of Tg antibodies. Assays for the measurement of such Tg-anti-Tg immune complexes have been developed.307
Normally, the test response is negative but results may be positive in up to 10% of the adult population. The frequency of positive test results is higher in women and with advancing age. The presence of thyroid autoantibodies in the apparently healthy population is thought to represent subclinical autoimmune thyroid disease rather than false-positive reactions. Nonetheless, it is difficult to compare results from such studies since some laboratories using agglutination methods report low titres (1/100-1/400) as positive. It is important when reporting values that a method-specific normal range is utilized and assays calibrated against internationally available refernce preparations. The availability of such preparations allows the reporting of results in International Units.305a TPO antibodies are detectable in approximately 95% of patients with Hashimoto's thyroiditis and 85% of those with Graves' disease, irrespective of the functional state of the thyroid gland. Similarly, Tg antibodies are positive in about 60 and 30% of adult patients with Hashimoto's thyroiditis and Graves' disease, respectively.305,306,308,309 Tg antibodies are less frequently detected in children with autoimmune thyroid disease.310 Although higher titers are more common with Hashimoto's thyroiditis, quantitation of the antibody titer carries little diagnostic implication. The tests are of particular value in the evaluation of patients with atypical or selected manifestations of autoimmune thyroid disease (ophthalmopathy and dermopathy). Positive antibody titers are predicative of post partum thyroiditis.311 Low antibody titers occur transiently in some patients after an episode of subacute thyroiditis.312 There is no increased incidence of thyroid autoantibodies in patients with multinodular goiter, thyroid adenomas, or secondary hypothyroidism. In some patients with Hashimoto's thyroiditis and undetectable thyroid autoantibodies in their serum, intrathyroidal lymphocytes have been demonstrated to produce TPO antibodies.
Other antibodies directed against thyroid components or other tissues have been described in the serum of some patients with autoimmune thyroid disease. They are less frequently measured, and their diagnostic value in thyroid disease has not been fully evaluated. Circulating antibodies capable of binding T4 and T3 have also been demonstrated in patients with autoimmune thyroid diseases which may interfere with the measurement of T4 and T3 by RIA techniques.38,39,314
Antibodies reacting with nuclear components, which are not tissue specific, and with cellular components of parietal cells and adrenal, ovarian, and testicular tissues are more commonly encountered in patients with autoimmune thyroid disease.315 Their presence reflects the frequency of coexistence of several autoimmune disease processes in the same patient (see Chapter 7).
A large number of names have been given to tests which measure abnormal ?-globulins present in the serum of some patients with autoimmune thyroid disease, in particular Graves' disease.317 The interaction of these unfractionated immunoglobulins with thyroid follicular cells usually results in a global stimulation of thyroid gland activity and only rarely causes inhibition. It has been recommended that these assays all be called TSH receptor antibodies (TRAb) with a phrase "measured by .................. assay" to identify the type of method used for their determination.106 The tests will be described under three general categories: (1) those measuring the thyroid stimulating activity using in vivo or in vitro bioassays; (2) tests based on the competition of the abnormal immunoglobulin with binding of TSH to its receptor; and (3) measurement of thyroid growth promoting activity of immunoglobulins. Tests employ both human and animal tissue material or cell lines.
The earliest assays employed various modifications of the McKenzie mouse bioassay.318,319 The abnormal ?-globulin with TSH-like biological properties has relatively longer in vivo activity, hence its name, long-acting thyroid stimulator (LATS). The assay measures the LATS induced release of thyroid hormone from the mouse thyroid gland prelabeled with radioiodide. The presence of LATS in serum is pathognomonic of Graves' disease. However, depending upon the assay sensitivity, a variable percent of untreated patients will show a positive LATS response. LATS may be found in the serum of patients with Graves' disease even in the absence of thyrotoxicosis. Although it is more commonly present in patients with ophthalmopathy, especially when accompanied by pretibial myxedema,320 LATS does not appear to correlate with the presence of Graves' disease, its severity, or course of complications. LATS crosses the placenta and may be found transiently in newborns from mothers possessing the abnormal ? globulin.321
Attempts to improve the ability to detect thyroid stimulating antibodies (TSAb) in autoimmune thyroid disease lead to the development of several in vitro assays using animal as well as human thyroid tissue. The ability of the patient's serum to stimulate endocytosis in fresh human thyroid tissue is measured by direct count of intracellular colloid droplets formed. Using such a technique, human thyroid stimulator (HTS) activity has been demonstrated in serum samples from patients with Graves' disease that were devoid of LATS activity measured by the standard mouse bioassay.322 TSAb can be detected by measuring the accumulation of cyclic adenosine monophosphate (cAMP) or stimulation of adenylate cyclase activity in human thyroid cell cultures and thyroid plasma membranes, respectively.323 Accumulation of cAMP in the cultured rat thyroid cell line FRTL5 has also been used as an assay for TSAb.324 Stimulation of release of T3 from human325 and porcine326 thyroid slices is another form of in vitro assay for TSAb. An in vitro bioassay using a cytochemical technique depends upon the ability of thyroid-stimulating material to increase lysosomal membrane permeability to a chromogenic substrate, leucyl-ß-naphthylamide, which then reacts with the enzyme naphthylamidase. Quantitation is by scanning and integrated microdensitometry.327
The cloning of the TSH receptor 328,329 lead to the development of an in vitro assay of TSab using cell lines that express the recombinant TSH receptor.330,331 This assay, based on the generation if cAMP, is specific for the measurement of human TSH receptor antibodies that have thyroid stimulating activity and thus contrasts with assays based on binding to the TSH receptor (see below) that cannot distinguish between antibodies with thyroid-stimulating and TSH-blocking activity. Accordingly, the recombinant human TSH receptor assay measures antibodies relevant to the pathogenesis of autoimmune thyrotoxicosis and is more sensitive than formerly used TSab assays.331a For example, 94% of serum samples were positive for TSab compared to 74% when the same samples were assayed using FRTL5 cells.332
Thyrotropin-Binding Inhibition Assays. The principal of binding-inhibition assays dates to the discovery of another class of abnormal immunoglobulins in patients with Graves' disease; those which neutralize the bioactivity of LATS tested in the mouse.333 This material, known as LATS protector (LATS-P), is species specific having no biologic effect on the mouse thyroid gland but capable of stimulating the human thyroid.334 The original assay was cumbersome, limiting its clinical application.
Techniques used currently, which may be collectively termed radioreceptor assays, are based on the competition of the abnormal immunoglobulins and TSH for a common receptor-binding site on thyroid cells. The test is akin in principle to the radioligand assays, in which a natural membrane receptor takes the place of the binding proteins or antibodies. Various sources of TSH-receptors are employed including, human thyroid cells,335 their particulate or solubilized membrane, 336,337 and cell membranes from porcine thyroids338 or from guinea pig fat cells339 or recombinant human TSH receptor expressed in mammalian cells.340 Since the assays do not directly measure thyroid-stimulating activity, the abnormal immunoglobulins determined have been given variety of names, such as thyroid binding inhibitory immunoglobulins (TBII) or antibodies (TBIAb) and thyrotropin-displacing immunoglobulins (TDI). This type of assay has indicated that not all the antibodies detected do stimulate the thyroid, and some are inhibitory. Even using modern techniques,305a the presence of inhibitory antibody is less sensitive and specific for Graves' disease than the presence of styimulatory antibody activity.331 The stimulatory and inhibitory effects can be differentiated only by functional assays, typically measuring the production of cyclic AMP.
Assays have been also developed that measure the growth promoting activity of abnormal immunoglobulins. One such assay is based on the staining by the Feulgen reaction of nuclei from guinea pig thyroid cells in S-phase.341 Another assay measures the incorporation of 3H-thymidine into DNA in FRTL cells.342 Whether the thyroid growth stimulating immunoglobulins (TGI) measured by these assays represent a population of immunoglobulins distinct from that with stimulatory functional activity remains a subject of active debate.
Clinical Applications. Measurement of abnormal immunoglobulins that interact with thyroid tissue by any of the methods described above is not indicated as a routine diagnostic test for Graves' disease. It is useful, however, in a few selected clinical conditions: (1) in the differential diagnosis of exophthalmos, particularly unilateral exophthalmos, when the origin of this condition is otherwise not apparent; the presence of TSI would obviate the necessity to undertake more complex diagnostic procedures described elsewhere;343 (2) in the differential diagnosis of pretibial myxedema, or other forms of dermopathy, when the etiology is unclear and it is imperative that the cause of the skin lesion be ascertained; (3) in the differentiation of Graves' disease from toxic nodular goiter, when both are being considered as the possible cause of thyrotoxicosis, when other tests such as thyroid scanning and thyroid autoantibody tests have been inconclusive, and particularly when such a distinction would play a role in determining the course of therapy; (4) when non-autoimmune thyrotoxicosis is suspected in a patient with hyperthyroidism and diffuse or nodular goiter 344,345 ; (5) in Graves' disease during pregnancy, when high maternal levels of TSAb are a warning for the possible occurrence of neonatal thyrotoxicosis; (6) in neonatal thyrotoxicosis, where serial TSAb determinations showing gradual decrease may be helpful to distinguish between intrinsic Graves' disease in the infant and transient thyrotoxicosis resulting from passive transfer of maternal TSAb.321,346 Some investigators have found the persistence of TSAb's to be predicative of the relapse of Graves' thyrotoxicosis following a course of antithyroid drug therapy.347
Some patients with trophoblastic disease develop hyperthyroidism as a result of the production and release of a thyroid stimulator which has been termed molar or trophoblastic thyrotropin or big placental TSH.348 It is likely that the thyroid-stimulating activity in patients with trophoblastic disease is entirely due to the presence of high levels of human chorionic gonadotropin (hCG).350 Thus, the RIA of hCG can be useful in the differential diagnosis of thyroid dysfunction.
A variety of tests have been developed for measuring exophthalmogenic activity in serum.351-354 Although a great uncertainty still exists regarding the pathogenesis of thyroid associated eye disease, the role of the immune system appears to be central. Exophthalmogenic activity has also been detected in IgG fractions of some patients with Graves' ophthalmopathy. The role of assays to detect specific antibodies is discussed further in Chapter 7.
Delayed hypersensitivity reactions to thyroid antigens are present in autoimmune thyroid diseases (see Chapters 7). CMI was measured in several ways: (1) the migration inhibition test, which measured the inhibition of migration of sensitized leukocytes when exposed to the sensitizing antigen; (2) the lymphotoxic assay, which measured the ability of sensitized lymphocytes to kill target cells when exposed to the antigen; (3) the blastogenesis assay, which scored the formation of blast cells after exposure of lymphocytes to a thyroid antigen; and (4) thymus-dependent (T) lymphocyte subset quantitation utilizing monoclonal antibodies. More recently, measures of T-cell proliferation, determined by uptake of 3Hthymidine, has become the standard test of CMI employed in the research setting.354a, 354b The tests require fresh leukocytes from the patient, are variable in their response, and are difficult to perform.
