Erroneous Thyroid-stimulating Hormone Radioimmunoassay Results Due to Interfering Antibovine Thyroid-stimulating Hormone Antibodies ANIL SAIN, M.D., RAJASEKAR SHAM, M.D., AMARJEET SINGH, M.D., AND LAWRENCE SILVER, M.D.
SERUM thyroid-stimulating hormone (TSH) levels as determined by radioimmunoassay (RIA) are extensively used in the diagnosis and management of thyroid disorders. 3,5 The proliferation of commercially available kits makes it important to be aware of the pitfalls associated with their use. 6 Some problems specific to the radioimmunoassay of TSH, such as cross-reactivity with other glycoprotein hormones, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and human chorionic gonadotropin (HCG) are well known, 10 and most of the kits include this information in the package inserts. This case report describes an uncommon cause of erroneous TSH RIA due to the administration of bovine TSH that interfered with the assay. Report of a Case A 48-year-old woman was diagnosed as having follicular carcinoma of the thyroid after thyroidectomy for an asymptomatic cold nodule disclosed by 131I scanning. Serum thyroxine (T4) was 6.0 jitg/dl (5-11.2 jiig/dl). Postoperatively, administration of 2 gr desiccated thyroid daily as replacement therapy was started. A month after operation, a 13,I whole-body scan, done after the injection of 10 units of bovine TSH daily for three days, showed a small remnant of functioning thyroid tissue but no metastatic lesion. These findings were unchanged on repeated scanning after two Received April 6, 1977; received revised manuscript and accepted for publication April 13, 1978. Address reprint requests to Dr. Sham: Queens Hospital Center, Nuclear Medicine, Jamaica, Queens, New York 11432.
Departments of Nuclear Medicine and Endocrinology, Queens Hospital Center Affiliation of the Long Island Jewish-Hillside Medical Center, Jamaica, New York
years, during which lime the patient had continued to take 2 gr dessicated thyroid daily. Serum T4 was 11.3 /xg/dl. Serum TSH concentration was 40 ^lU/ml (normal 1-10 /nIU/ml), as determined by RIA with a Beckman kit. To confirm this unexpectedly elevated serum TSH level in the presence of an adequate replacement dose, an estimation was also done by Bio-Science Laboratories (Van Nuys, California), which reported serum TSH = 128 /itIU/ml (as high as 10 /xIU/ml), T„ = 8.2 /xg/dl, triiodothyronine (T:)) (RIA) = 122 ng/dl (110-230 ng/dl). Repeated m l scanning without prior bovine TSH injection did not show any uptake in the previously described thyroid tissue, indicating adequate suppression. The suppressive therapy was changed to 25 /xg sodium liothyronine (Cytomel0*) three times a day. After 15 days of this regimen, serum T;l(RIA) was 381 ng/dl and serum TSH was 108 |u.IU/ml. Pituitary dysfunction was thought to be unlikely due to the presence of a normal sella by roentgenogram, normal FSH and LH levels, normal responses to metyrapone stimulation, dexamethasone suppression, and growth hormone release in response to insulin hypoglycemia. Since the patient had received bovine TSH injections, the possibility of circulating antibodies to TSH interfering with the radioimmunoassay was considered.
Materials and Methods Serum TSH was estimated in our laboratory with radioimmunoassay kits, by use of the double-antibody system, obtained from Beckman Instruments, Fullerton, California, and by use of the single-antibody technic with polyethylene glycol* as the separating agent. To detect the presence of endogenous circulating antibody, extra tubes were set up while assaying, where the separating agent was added to the patient's serum without adding the first antibody (rabbit antiserum to TSH). The double-antibody method used the patient's serum 4- 125I TSH + precipitating antibody (goat antirabbit gamma globulin). The single-antibody method * Abbott Laboratories. North Chicago. Illinois.
0002-9173/79/0500/0540 $00.65 © American Society of Clinical Pathologists
540
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Sain, Anil, Sham, Rajasekar, Singh, Amarjeet, and Silver, Lawrence: Erroneous thyroid-stimulating hormone radioimmunoassay results due to interfering antibovine thyroidstimulating hormone antibodies. Am J Clin Pathol 71: 540542, 1979. Apparent elevation of serum thyroid-stimulating hormone (TSH) levels was found in a case of a patient receiving adequate replacement therapy after thyroidectomy for a follicular carcinoma. This was an artifact due to interference with the radioimmunoassay by circulating antibovine TSH antibodies. The double-antibody technic showed spuriously elevated levels, and the single-antibody technic showed low levels of serum TSH by radioimmunoassay in the presence of antibodies. (Key words: Thyroid-stimulating hormone [TSH]; Radioimmunoassay; Antibodies.)
Vol. 71 • No. 5
ERRONEOUS TSH RIA DUE TO ANTIBOVINE TSH ANTIBODIES l25
used the patient's serum + I TSH + polyethylene glycol. The rest of the assay procedure was not modified. The presence of interfering antibody was confirmed by incubating overnight the patient's serum with Coombs' antihuman globulin serumt and then repeating the double-antibody assay as described above. All counts were performed in a Beckman well counter, and the fraction bound is the number of counts in the precipitate expressed as a percentage of the total counts of 1Z5I antigen added. Results
Discussion Endogenous antibodies to hormones are known to interfere with their radioimmunoassay. For commonly administered hormones such as insulin, it is recommended that any patient who has received insulin for six weeks or longer should be screened for the presence of antibody to permit accurate interpretation of the radioimmunoassay values. 8 However, the possibility of antibodies to TSH interfering with the radioimmunoassay of TSH has not been well emphasized, although the presence of circulating antibodies has been documented after injection of bovine TSH, 2 and in some cases where there was no history of having received bovine TSH. 1 Administration of bovine TSH in minimal doses to healthy subjects has been shown to induce antibodies to bovine TSH. 4 These bovine TSH antibodies are able to bind human TSH. By contrast, antibodies to human TSH are not able to bind bovine TSH. 7 In the double-antibody assay system, the 125I-human t Hyland Laboratories, Costa Mesa, California.
TSH that is bound to the endogenous antibody is not separated by the goat antirabbit gamma globulin, since the species-specific goat serum does not react with human globulin. Therefore, the endogenous antibody + 12rT-human TSH complex separates in the free rather than the bound fraction. This leads to an interpretation of circulating TSH values higher than actually present. In the double-antibody system, using a patient blank (patient's serum + I23 I-TSH + precipitating antiserum) will not be sufficient to permit detection of any endogenous antibody. An appropriate control would be the use of antihuman gamma globulin as the precipitating agent. 9 Incubation with Coombs' serum binds the globulins interfering with the assay and allows the ,25 I-human TSH to be bound by the first antibody (rabbit antiserum to human TSH), which can then be precipitated by the second (goat) antibody. This method is suitable for showing the presence of interfering antibody but cannot be used routinely to measure serum TSH levels in the presence of antibodies. When an agent like polyethylene glycol is used to separate the bound from the free fraction, the 12"i-TSH rabbit antibody complex are both precipitated, giving a very high bound fraction and a spuriously low TSH value. Similar results will be obtained using solid-phase methods, since the l25 I-TSH + endogenous antibody complex will separate in the free fraction. In the single antibody system, setting up a patient control for nonspecific binding (patient's serum 4- 125ITSH + polyethylene glycol) will show a significantly higher number of counts in the precipitate, compared with the counts in the precipitate with a control serum, suggesting the presence of antibody. Hypothyroid patients and those who have malignancies of the thyroid are often evaluated with a TSHstimulation test using bovine TSH. When endogenous antibody is produced, it may persist for as long as 11 months, 4 thus preventing the use of serum TSH levels to monitor the adequacy of replacement therapy. The discrepancy between the TSH levels using two different methods, (single-antibody system with an agent such as polyethylene glycol and either a doubleantibody or a solid-phase system) will be sufficient to confirm the artifact due to the presence of endogenous antibodies in most cases. This is an easy method that can be performed in small laboratories that do not have the equipment to carry out more sophisticated procedures, such as chromatography and Immunoelectrophoresis, to demonstrate these antibodies. We recommend screening for the presence of anti-
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
With the double-antibody method (patient's serum + I25I TSH + rabbit anti-TSH + goat antirabbit globulin), the fraction bound was 8%. When rabbit anti-TSH was not added, the fraction bound was 7.8%. With a single-antibody method (patient's serum + 125I TSH + rabbit anti-TSH + polyethylene glycol), the fraction bound was 64%. When rabbit anti-TSH was not added, the fraction bound was 63%. After incubation with Coombs' serum, the fraction bound was 51% (patient's serum incubated with Coombs' serum + 125I TSH + rabbit anti-TSH + goat antirabbit globulin). These results showed that endogenous circulating antibody was combining with 125I TSH and was precipitated by polyethylene glycol, but not by the precipitating (goat) antibody.
541
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body to TSH in patients who have a history of having received bovine TSH and in cases where elevated levels of immunoreactive TSH are reported for patients receiving adequate replacement therapy. References 1. Chaussain JL: Evidence of antibodies to human thyrotrophin in the plasma of some pituitary dwarfs. Arch Fr Pediatr 29: 667-668, 1972 2. Greenspan FS, Lew W, Okerlund MD, et al: Falsely positive bovine TSH radioimmunoassay responses in sera from patients with thyroid cancer. J Clin Endocrinol Metab 38: 1121-1122, 1974 3. Hall R, Smith BR, Mukhtar ED: Thyroid stimulators in health and disease. Clin Endocrinol 4:213-230, 1975 4. Hays MT, Solomon DH, Beall GN: Suppression of human
ETAL.
A.J.C.P. • May 1979
thyroid function by antibodies to bovine thyrotrophin. J Clin Endocrinol 27:1540-1549, 1967 Hershman JM, Pittman JA: Utility of the radioimmunoassay of serum thyrotrophin in man. Ann Int Med 74:481-490, 1971 Kagan A: Evaluation of commercial radioimmunoassay kits. Semin Nucl Med 5:173-182, 1975 Lemarchand-Beraud TH: Comparison between antibodies to bovine and human thyrotrophin for radioimmunoassay in plasma, cross reaction studies with clinical results. Acta Endocrinol 64:610-629, 1970 Prout T: Radioisotopic measurements of insulin, Nuclear Medicine in Vitro. Edited by Rothfeld B. Philadelphia, J. B. Lippincott, 1974, p 272 Sachson RA, Martin DB: Techniques of radioimmunoassay for insulin. N Engl J Med 288:973, 1973 10. Utiger RD: Thyrotrophin, Methods of Hormone Radioimmunoassay. Edited by Jaffe BM, Behrman HR. New York, Academic Press, 1974, p 161
The appearance of the code at the bottom of the first page of an article in this journal indicates the copyright owner's consent that copies of the article may be made for personal or internal use. or for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center. Inc.. P.O. Box 765. Schenectady. New York 12301, for copying beyond that permitted by Sections 107 or 108 of the U. S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale.
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NOTICE TO COPIERS
SERUM thyroid-stimulating hormone (TSH) levels as determined by radioimmunoassay (RIA) are extensively used in the diagnosis and management of thyroid disorders. 3,5 The proliferation of commercially available kits makes it important to be aware of the pitfalls associated with their use. 6 Some problems specific to the radioimmunoassay of TSH, such as cross-reactivity with other glycoprotein hormones, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and human chorionic gonadotropin (HCG) are well known, 10 and most of the kits include this information in the package inserts. This case report describes an uncommon cause of erroneous TSH RIA due to the administration of bovine TSH that interfered with the assay. Report of a Case A 48-year-old woman was diagnosed as having follicular carcinoma of the thyroid after thyroidectomy for an asymptomatic cold nodule disclosed by 131I scanning. Serum thyroxine (T4) was 6.0 jitg/dl (5-11.2 jiig/dl). Postoperatively, administration of 2 gr desiccated thyroid daily as replacement therapy was started. A month after operation, a 13,I whole-body scan, done after the injection of 10 units of bovine TSH daily for three days, showed a small remnant of functioning thyroid tissue but no metastatic lesion. These findings were unchanged on repeated scanning after two Received April 6, 1977; received revised manuscript and accepted for publication April 13, 1978. Address reprint requests to Dr. Sham: Queens Hospital Center, Nuclear Medicine, Jamaica, Queens, New York 11432.
Departments of Nuclear Medicine and Endocrinology, Queens Hospital Center Affiliation of the Long Island Jewish-Hillside Medical Center, Jamaica, New York
years, during which lime the patient had continued to take 2 gr dessicated thyroid daily. Serum T4 was 11.3 /xg/dl. Serum TSH concentration was 40 ^lU/ml (normal 1-10 /nIU/ml), as determined by RIA with a Beckman kit. To confirm this unexpectedly elevated serum TSH level in the presence of an adequate replacement dose, an estimation was also done by Bio-Science Laboratories (Van Nuys, California), which reported serum TSH = 128 /itIU/ml (as high as 10 /xIU/ml), T„ = 8.2 /xg/dl, triiodothyronine (T:)) (RIA) = 122 ng/dl (110-230 ng/dl). Repeated m l scanning without prior bovine TSH injection did not show any uptake in the previously described thyroid tissue, indicating adequate suppression. The suppressive therapy was changed to 25 /xg sodium liothyronine (Cytomel0*) three times a day. After 15 days of this regimen, serum T;l(RIA) was 381 ng/dl and serum TSH was 108 |u.IU/ml. Pituitary dysfunction was thought to be unlikely due to the presence of a normal sella by roentgenogram, normal FSH and LH levels, normal responses to metyrapone stimulation, dexamethasone suppression, and growth hormone release in response to insulin hypoglycemia. Since the patient had received bovine TSH injections, the possibility of circulating antibodies to TSH interfering with the radioimmunoassay was considered.
Materials and Methods Serum TSH was estimated in our laboratory with radioimmunoassay kits, by use of the double-antibody system, obtained from Beckman Instruments, Fullerton, California, and by use of the single-antibody technic with polyethylene glycol* as the separating agent. To detect the presence of endogenous circulating antibody, extra tubes were set up while assaying, where the separating agent was added to the patient's serum without adding the first antibody (rabbit antiserum to TSH). The double-antibody method used the patient's serum 4- 125I TSH + precipitating antibody (goat antirabbit gamma globulin). The single-antibody method * Abbott Laboratories. North Chicago. Illinois.
0002-9173/79/0500/0540 $00.65 © American Society of Clinical Pathologists
540
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Sain, Anil, Sham, Rajasekar, Singh, Amarjeet, and Silver, Lawrence: Erroneous thyroid-stimulating hormone radioimmunoassay results due to interfering antibovine thyroidstimulating hormone antibodies. Am J Clin Pathol 71: 540542, 1979. Apparent elevation of serum thyroid-stimulating hormone (TSH) levels was found in a case of a patient receiving adequate replacement therapy after thyroidectomy for a follicular carcinoma. This was an artifact due to interference with the radioimmunoassay by circulating antibovine TSH antibodies. The double-antibody technic showed spuriously elevated levels, and the single-antibody technic showed low levels of serum TSH by radioimmunoassay in the presence of antibodies. (Key words: Thyroid-stimulating hormone [TSH]; Radioimmunoassay; Antibodies.)
Vol. 71 • No. 5
ERRONEOUS TSH RIA DUE TO ANTIBOVINE TSH ANTIBODIES l25
used the patient's serum + I TSH + polyethylene glycol. The rest of the assay procedure was not modified. The presence of interfering antibody was confirmed by incubating overnight the patient's serum with Coombs' antihuman globulin serumt and then repeating the double-antibody assay as described above. All counts were performed in a Beckman well counter, and the fraction bound is the number of counts in the precipitate expressed as a percentage of the total counts of 1Z5I antigen added. Results
Discussion Endogenous antibodies to hormones are known to interfere with their radioimmunoassay. For commonly administered hormones such as insulin, it is recommended that any patient who has received insulin for six weeks or longer should be screened for the presence of antibody to permit accurate interpretation of the radioimmunoassay values. 8 However, the possibility of antibodies to TSH interfering with the radioimmunoassay of TSH has not been well emphasized, although the presence of circulating antibodies has been documented after injection of bovine TSH, 2 and in some cases where there was no history of having received bovine TSH. 1 Administration of bovine TSH in minimal doses to healthy subjects has been shown to induce antibodies to bovine TSH. 4 These bovine TSH antibodies are able to bind human TSH. By contrast, antibodies to human TSH are not able to bind bovine TSH. 7 In the double-antibody assay system, the 125I-human t Hyland Laboratories, Costa Mesa, California.
TSH that is bound to the endogenous antibody is not separated by the goat antirabbit gamma globulin, since the species-specific goat serum does not react with human globulin. Therefore, the endogenous antibody + 12rT-human TSH complex separates in the free rather than the bound fraction. This leads to an interpretation of circulating TSH values higher than actually present. In the double-antibody system, using a patient blank (patient's serum + I23 I-TSH + precipitating antiserum) will not be sufficient to permit detection of any endogenous antibody. An appropriate control would be the use of antihuman gamma globulin as the precipitating agent. 9 Incubation with Coombs' serum binds the globulins interfering with the assay and allows the ,25 I-human TSH to be bound by the first antibody (rabbit antiserum to human TSH), which can then be precipitated by the second (goat) antibody. This method is suitable for showing the presence of interfering antibody but cannot be used routinely to measure serum TSH levels in the presence of antibodies. When an agent like polyethylene glycol is used to separate the bound from the free fraction, the 12"i-TSH rabbit antibody complex are both precipitated, giving a very high bound fraction and a spuriously low TSH value. Similar results will be obtained using solid-phase methods, since the l25 I-TSH + endogenous antibody complex will separate in the free fraction. In the single antibody system, setting up a patient control for nonspecific binding (patient's serum 4- 125ITSH + polyethylene glycol) will show a significantly higher number of counts in the precipitate, compared with the counts in the precipitate with a control serum, suggesting the presence of antibody. Hypothyroid patients and those who have malignancies of the thyroid are often evaluated with a TSHstimulation test using bovine TSH. When endogenous antibody is produced, it may persist for as long as 11 months, 4 thus preventing the use of serum TSH levels to monitor the adequacy of replacement therapy. The discrepancy between the TSH levels using two different methods, (single-antibody system with an agent such as polyethylene glycol and either a doubleantibody or a solid-phase system) will be sufficient to confirm the artifact due to the presence of endogenous antibodies in most cases. This is an easy method that can be performed in small laboratories that do not have the equipment to carry out more sophisticated procedures, such as chromatography and Immunoelectrophoresis, to demonstrate these antibodies. We recommend screening for the presence of anti-
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
With the double-antibody method (patient's serum + I25I TSH + rabbit anti-TSH + goat antirabbit globulin), the fraction bound was 8%. When rabbit anti-TSH was not added, the fraction bound was 7.8%. With a single-antibody method (patient's serum + 125I TSH + rabbit anti-TSH + polyethylene glycol), the fraction bound was 64%. When rabbit anti-TSH was not added, the fraction bound was 63%. After incubation with Coombs' serum, the fraction bound was 51% (patient's serum incubated with Coombs' serum + 125I TSH + rabbit anti-TSH + goat antirabbit globulin). These results showed that endogenous circulating antibody was combining with 125I TSH and was precipitated by polyethylene glycol, but not by the precipitating (goat) antibody.
541
SAW
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body to TSH in patients who have a history of having received bovine TSH and in cases where elevated levels of immunoreactive TSH are reported for patients receiving adequate replacement therapy. References 1. Chaussain JL: Evidence of antibodies to human thyrotrophin in the plasma of some pituitary dwarfs. Arch Fr Pediatr 29: 667-668, 1972 2. Greenspan FS, Lew W, Okerlund MD, et al: Falsely positive bovine TSH radioimmunoassay responses in sera from patients with thyroid cancer. J Clin Endocrinol Metab 38: 1121-1122, 1974 3. Hall R, Smith BR, Mukhtar ED: Thyroid stimulators in health and disease. Clin Endocrinol 4:213-230, 1975 4. Hays MT, Solomon DH, Beall GN: Suppression of human
ETAL.
A.J.C.P. • May 1979
thyroid function by antibodies to bovine thyrotrophin. J Clin Endocrinol 27:1540-1549, 1967 Hershman JM, Pittman JA: Utility of the radioimmunoassay of serum thyrotrophin in man. Ann Int Med 74:481-490, 1971 Kagan A: Evaluation of commercial radioimmunoassay kits. Semin Nucl Med 5:173-182, 1975 Lemarchand-Beraud TH: Comparison between antibodies to bovine and human thyrotrophin for radioimmunoassay in plasma, cross reaction studies with clinical results. Acta Endocrinol 64:610-629, 1970 Prout T: Radioisotopic measurements of insulin, Nuclear Medicine in Vitro. Edited by Rothfeld B. Philadelphia, J. B. Lippincott, 1974, p 272 Sachson RA, Martin DB: Techniques of radioimmunoassay for insulin. N Engl J Med 288:973, 1973 10. Utiger RD: Thyrotrophin, Methods of Hormone Radioimmunoassay. Edited by Jaffe BM, Behrman HR. New York, Academic Press, 1974, p 161
The appearance of the code at the bottom of the first page of an article in this journal indicates the copyright owner's consent that copies of the article may be made for personal or internal use. or for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center. Inc.. P.O. Box 765. Schenectady. New York 12301, for copying beyond that permitted by Sections 107 or 108 of the U. S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
NOTICE TO COPIERS
