- Email: [email protected]
The use of filter paper-dried blood spots for thyroid-antibody screening in adults
The use of filter paper– dried blood spots for thyroid-antibody screening in adults LINDSAY F. HOFMAN, THOMAS P. FOLEY, JUDY J. HENRY, and EDWIN W. NAYLOR BRIDGEVILLE, PENNSYLVANIA
Thyroid-antibody screening is recommended for several adult populations, including the elderly and pregnant and postpartum women, and analysis of filter paper– dried blood spots (DBSs) has been a cost-effective means of screening newborns for hypothyroidism for more than 30 years. The aim of this study was to show that DBS specimens can be used to test adults for thyroid-stimulating hormone (TSH) antibodies and thyroid antibodies during screening for thyroid disease in a community setting. Thyroid peroxidase antibody and thyroglobilin antibody enzyme-linked immunosorbent assay kits for serum have been adapted for use with DBSs. A TSH assay for newborn DBSs was adapted for the screening of adults. Parallel specimens, serum and DBSs, were collected during routine care of patients attending a thyroid clinic. In addition, 962 DBS specimens were collected from volunteers at community centers and nursing homes. Twelve months later, a second specimen was collected from each of 411 original volunteers. Antibody results are reported as normal and positive. Ninety-seven percent of the serum/DBS results correlated exactly. Of 962 volunteers from nursing homes and community centers, testing for antibodies was positive or indeterminate (borderline) in 266 (27%). Eighty percent of the patients with an initially normal TSH reading who were positive on the 12-month collection were positive for at least one antibody in the first collection. The use of DBSs is a convenient and efficient way to screen for thyroid antibody as a means of detecting occult thyroid disease in adults. (J Lab Clin Med 2004;144:307-12) Abbreviations: CV ⫽ coefficient of variation; DBS ⫽ dried blood spot; ELISA ⫽ enzyme-linked immunosorbent assay; NHANES III ⫽ National Health and Nutrition Examination Survey; TAb ⫽ thyroid antibody; TGAb ⫽ thyroglobulin antibody; TPOAb ⫽ thyroid peroxidase antibody; TSH ⫽ thyroid-stimulating hormone (thyrotropin)
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creening for TAbs is advised in several populations, including the elderly, pregnant and postpartum women, and individuals at increased risk for autoimmune hypothyroidism and thyrotoxicosis, From Pediatrix Screening, PO Box 219, 90 Emerson Lane, Bridgeville, PA 15017. Supported by Small Business Innovation Research (SBIR) grants R43DK-53666 and R44-DK-53666 from the National Institute for Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, United States Public Health Service, Department of Health and Human Services. Drs Hofman and Foley are consultants to Pediatrix Screening. Submitted for publication May 15, 2004; revision submitted September 20, 2004; accepted for publication September 22, 2004. Reprint requests: Lindsay F. Hofman, PO Box 771, Vashon, WA 98070; e-mail: [email protected]. 0022-2143/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lab.2004.09.009
particularly women older than 35 years.1–3 Although DBS specimens have been used for screening in newborns for more than 30 years and analysis of DBS specimens has been shown to be a cost-effective, accurate means of screening for thyroid diseases,4 there has been little use of the DBS in the screening of adults. Recently we showed that the use of DBS specimens is a cost-effective, accurate method of TSH screening for hypothyroidism in adults.5 We undertook the study reported here to show that a DBS assay for TAbs involving the same specimen collected for TSH screening is sufficiently sensitive and specific to be used in the screening of adults for thyroid disease. TGAb and TPOAb have been found in the presence of autoimmune thyroid disease, including overt and mild (subclinical) hypothyroidism. Because autoimmune thyroiditis (Hashimoto’s disease) is the most common cause of primary hypothyroidism among 307
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adults in the United States,2,6 assessment of TPOAb and TGAb concentrations can provide information about both the cause of a high TSH reading and help determine an individual’s risk for hypothyroidism. We previously reported an adaptation of a neonatal assay to measure TSH in adults5 and presented preliminary results with a DBS assay for TAbs in pregnant women.7 It would be advantageous to use the same DBS specimen for both TSH and antibody testing. Previously published methods for the measurement of TAbs in DBS specimens have involved the use of a hemagglutination assay8 or recombinant immunoassay.9,10 We describe an ELISA assay for the measurment of TPOAb and TGAb in DBS and report our experience in screening a large population of adult volunteers. METHODS Reagents. Reagents for both serum and DBS TPOAb and TGAb assays were purchased from KRONUS (Boise, Idaho) in the form of Kalibre ELISA TGAb (KR7280) and TPOAb (KR7260) kits. Filter paper (number 903; Schleicher & Schuell, Keene, NH) was used for the collection of specimens and preparation of controls. We took TSH reagents from the Auto Delfia Neonatal hTSH kit (Perkin-Elmer Wallac, Turku, Finland), using standards made in-house especially for the adult assays.5 DBS controls were prepared from heparin-treated whole blood collected from individuals with and without high antibody titers. DBS controls spotted on filter paper, which were stored in plastic bags with desiccant, were stable for more than 2 years at ⫺15°C. Sample collection. To compare serum and DBS levels of antibody, we collected specimens prospectively from children, adolescents, and adults during evaluation for thyroid disease. Serum antibody levels were measured as part of the routine clinical thyroid evaluation with the use of the Kronus recombinant-immunoassay TPOAb and TGAb assays in the Pediatric Endocrine Laboratory of Children’s Hospital (Pittsburgh, Pa). Whole blood, left in the tubing of the scalpvein needle after the collection of clinical specimens in 1998 and 1999, was spotted onto filter paper. The paper was allowed to dry horizontally at room temperature for 4 hours, placed in a self-sealing plastic bags with desiccant, and stored at ⫺20°C. In addition, we collected 962 samples on filter paper by means of fingerstick from volunteers recruited in nursing homes and community centers in western Pennsylvania from 2000 through 2002. The study, which was carried out in accordance with the principles of the Declaration of Helsinki, was approved by the Western International Review Board. A nurse explained the study to each volunteer before asking him or her to sign an informed-consent form that had been approved by the Western International Review Board. The nurse then pricked the volunteer’s finger and collected a blood specimen on a specially designed Schleicher & Schuell filterpaper card on which identifying and demographic informa-
Table I. Kronus reference ranges in adults’ serum Reading (U/mL) Antibody
TPOAb TGAb
Normal
Indeterminate
Positive
⬍1 ⬍2
1–2 2–3
⬎2 ⬎3
tion was entered. Specimens were dried and stored as described above and assayed within a month of their collection. Analysis. The DBS antibody assays were similar to those described in the Food and Drug Administration–approved Kronus package inserts for TPOAb and TGAb ELISAs, with the addition of an extraction step at the beginning.11 We used 100 L of kit diluent to extract antibodies from each 6-mm DBS. The specimen was incubated for 30 minutes on a plate shaker at 800 rpm. We used 50 L eluate in the assay and continued the procedure as described in the package insert. Absorbance was read at 405– 650 nm. Results were calculated from a logit log plot of the standard concentration against absorbance. All assays were run by one of four technicians in a diagnostic clinical laboratory. Serum results were received after the completion of the DBS assay. The DBS assay for TSH was an adaptation of the AutoDelfia Neonatal TSH kit.5 Standards were made in-house to cover the decision level for adults, which is lower than that for neonates. RESULTS
Because the reagents we used were from Food and Drug Administration–approved serum assays for the antibodies, the focus of the validation was comparison of DBS results with the results of the serum assay. Liquid standards from the kit were used in the DBS assay. Eluate (50 L) from normal DBSs was added to the standards. The eluate had very little effect on the results of the assay. The correlation for absorbance of TPOAb standards with and without eluate was as follows: Liquid standard ⫽ 0.88 (standard ⫹ eluate) ⫹ .022 (r ⫽ .998) (Y⫽ MX ⫹ B). Blank absorbance (no DBS present), averaged over 10 assays, was 0.042 A405– 630nm units (SD 0.035) less than the DBS 0 standard for TPOAb and 0.048 A405– 630nm units (SD 0.036) less than the DBS 0 standard for TGAb. The sensitivity of the DBS assay described as the lowest standard corrected for volume factors was 0.68 U/mL for TPOAb and 1.4 U/mL for TGAb. Kronus reference ranges in serum for adults are set forth in Table I. The DBS values reported here have been corrected to yield the level in serum. We found no correlation between the quantitative value of the antibody and the TSH level (TSH vs TGAb [r ⫽ .16], TSH vs TPOAb [r ⫽ .35]) or the quantitative value of TPOAb versus
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Fig 1. Initial and 12-month populations: distribution by sex and age. DBS specimens were collected from 962 volunteers at nursing homes and community centers in western Pennsylvania; 788 were female, 176 male. Volunteers from whom a specimen was collected received a postcard reminding them to have a second specimen collected 12 months after the first. Of the 411 volunteers who returned for a second collection, 323 were female and 89 were male.
that of TGAb (r ⫽ .26). Because the actual antibody level does not correlate with clinical outcome (ie, an antibody level of 55 is no more likely to denote a high TSH value than is an antibody level of 8), we consider the antibody-test results semiquantitative and report them as normal or positive. We reported indeterminate (borderline) results as normal because this category did not exist at the start of the study. Each specimen was run singly in each assay. If the test results for both antibodies were positive or negative, the specimen was reported as such. If the result for one antibody was positive and that for the other was normal, the test that yielded the positive result was rerun. If the positive value was confirmed, the specimen was reported as positive. In only one case did repeat testing yield a negative result, and a second specimen was requested. Serum and DBS thyroid-antibody tests were run on 33 specimens for both antibodies. All patients but one fell into the same diagnostic category for serum and
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DBS on the basis of the results of both tests; this patient had normal TPOAb results on both DBS and serum testing, as well as a normal DBS TGAb result. Only the testing for serum TGAb yielded a positive result. We evaluated interassay precision by running high and low DBS controls in 18 assays. Because this assay was designed to be run as a screening assay, we calculated precision from singlewell values. In the TPOAb assay, the high control, with a mean of 35 U/mL, had a CV of 9.95% and the low control with a mean of 8.5 U/ml had a CV of 11.4%. In the TGAb assay, the high control, with a mean of 48 U/mL, had a CV of 13.4% and the low control, with a mean of 13 U/mL, had a CV 16.3%. These results compare favorably with the CVs calculated from the ranges supplied with the kit for kit-high controls for the serum assay which were 25% for both antibodies. One advantage of using DBS specimens is that the filter-paper cards can be sent by regular mail at ambient temperatures. We performed stability studies designed to simulate conditions that specimens would be subject to during in shipping in glassine (transparent glazed paper, resistant to the passage of air and grease) envelopes. DBS specimens were mailed across the country under conditions similar to those used to send newborns’ specimens to the laboratory. We compared the results with a sample kept at refrigerator temperature. Specimens spent five days in transit. Shipped specimens measured in the same assay as the refrigerated specimen differed by less than 13% at a mean TPOAb level of 44 U/mL. DBS controls were stored in glassine envelopes at room temperature. Two, 7, 10, and 14 days after sampling, the specimens were put in the freezer. All samples were tested in one assay and run in duplicate. At 14 days, the TPOAb specimen was found to have gone from 35 to 32 U/mL and the TGAb specimen was found to have gone from 32 to 30 U/mL. The variation was well within the precision of the assay. The specificity data for serum apply to DBS. The antigens used to coat the wells are highly (affinity) purified.11 Consequently, the thyroglobulin-antigen preparation was free of contaminating thyroid peroxidase antigen and the thyroid peroxidase antigen preparation was free of contaminating thyroglobulin antigens. TGAb, TPOAb, and TSH were measured in eluates from DBS specimens collected from 962 individuals. Sex and age distribution is depicted in Fig 1. Fliers announcing the opportunity to participate were distributed to community centers and nursing homes. Any individual who presented himself or herself at these locations on the announced day and was willing to read and sign a consent form was accepted into the study.
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Table II. Comparison of first- and second-specimen results, TPOAb Specimen
TPOAb-positive specimen 2 TPOAb-indeterminate specimen 2 TPOAb-normal specimen 2 Total
TPOAb-positive specimen 1
TPOAb-indeterminate specimen 1
TPOAb-normal specimen 1
71 1 7 79
2 1 20 23
5 11 293 309
Table III. Comparison of first- and second-specimen results, TGAb Specimen
TGAb-positive specimen 2 TGAb-indeterminate specimen 2 TGAb-normal specimen 2 Total
TGAb-positive specimen 1
TGAb-indeterminate specimen 1
TGAb-normal specimen 1
37 3 6 46
5 1 25 31
4 2 328 334
We did not exclude people who were taking thyroid medication. Of the 962 individuals tested, 266 (27%) tested positive or indeterminate for one or both antibodies. Of the 266, 36 (14%) had increased TSH levels (⬎5mU/L) and 56 (21%) reported taking thyroid medication. Of the 36 who tested positive or indeterminate for antibodies and had increased TSH levels, 13 were of the group reported to be taking thyroid medication. Increased TSH concentration had not previously been reported in the remaining 23 individuals. In summary, of the total population we tested, 23 (2.4%) had unrecognized clinical or subclinical hypothyroidism.12–14 Normal antibody results were reported for six individuals with increased TSH readings. One was a 94year-old woman who died a few months later. All others had TSH concentrations of less than 10 mU/L. One had a normal TSH reading two months later, two had indeterminate TGAb levels, and from two no further information could be obtained. Although most positive antibody specimens demonstrated positive TPOAb results, 8% of the specimens demonstrating increased TSH concentrations and 4% of the total specimens had normal TPOAb and high TGAb titers. It was therefore necessary to run both antibody tests. Because their formats are similar, the tests can be run at the same time to reduce the costs of technician time. When the assays are used to confirm each other, reagent costs can be minimized. Repeat testing 12 months after the initial blood test was included in the study design. Testing was offered to all adult volunteers who were originally recruited from nursing homes and community centers in western Pennsylvania and had given consent. Study participants were contacted by phone or mail. Of the 962 volunteers who participated in the baseline study, 411 returned for collection of a second filter-paper specimen by a fin-
gerstick 12 months later. Specimen collection was performed by the same nurse, using the identical procedure, and specimen handling was conducted as done initially. Fig 1 shows the age and sex distributions of the 411 individuals from whom second specimens were collected 12 months after the first. Tables II and III show the initial and repeat outcomes for TPOAb and TGAb. Of those patients whose samples were positive or normal for TPOAb or TGAb on the first testing, 86% remained in the same category. Most of the specimens that tested indeterminate the first time were normal on the second testing (83%), but seven (13%) were positive on the second test. A much smaller proportion of specimens that were normal on the first test were positive on the second (1.3%). Eight specimens had normal TSH readings on the first test and positive TSH on the second. Three of these individuals were known to be hypothyroid and were taking thyroid medication. Of the remaining five, three had positive TPOAb results on the first test and one was positive for both antibodies (Table IV). In summary, the antibody result predicted the development of hypothyroidism in 80% of cases. DISCUSSION
Screening for autoimmune thyroid diseases and primary hypothyroidism in adults, especially pregnant and postpartum women and members of the general population 50 years and older, is recommended by several national and international groups because of the high prevalence of disease.1–3,6,12–14 Our studies support these recommendations and provide an inexpensive method for specimen collection and analysis with which to screen large populations, comparable to older
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Table IV. Increased TSH only on second specimen, TGAb and TPOAb Patient
TSH 1
TGAb 1
TPOAb 1
TSH 2
TGAb 2
TPOAb 2
1 2 3 4 5
2.1 ⬍2 3.5 2.7 4.8
⬍1.4 11 1.9 1.9 3.7
0.7 7.3 14 26 ⬎68
5.1 6.4 6.7 5.7 5.7
⬍1.4 7.1 ⬍1.4 1.6 9
⬍0.68 13 6.9 ⬎68 81
Data expressed as units per milliliter. Five patients who were classified as having normal TSH concentrations in the first specimen were positive for TSH on the second specimen. In this study, a positive TSH reading was defined as a concentration greater than 5 mU/L in the DBS specimens. These results were confirmed with serum TSH test. The number 1 refers to the hormone or antibody level on the first specimen. The number 2 refers to the level on the 12-month specimen.
studies whose findings justified the screening of newborns for congenital hypothyroidism.4 Thyroid-antibody data were reported from a recently published nationwide survey, the NHANES III, in which specimens from adolescents and adults were tested with the use of the same TAb-assessment methods reported here.6,15 Of 16,533 individuals ages 12 years and older with no thyroid disease or goiter who were not taking thyroid medication (termed the “disease-free populations”), TPOAbs were present in 11.3 % and TGAbs were found in 10.4 %.6 Eighteen percent tested positive for one or both of the TAbs. The authors concluded that a significant proportion of the thyroid disease–free populations in the United States have evidence of thyroid disease on TSH and TAb testing. These data support the usefulness of screening in the early detection of thyroid disease.6 In our study, for which we recruited an older population, we found a TAb prevalence of 27%. In the NHANES III study, the prevalence of positivity for positive TPOAbs increased from 16% in women ages 40 to 49 years to 27% in women older than 70 years, comparable to the prevalence in our population. In addition, our study included individuals taking thyroid medication who probably would test positive for antibodies, given that autoimmune thyroiditis is the most common cause of hypothyroidism in adults. Further analysis of the NHANES III data to determine the prevalence of TPOAbs at specific serum TSH ranges revealed that the TPOAb prevalence was lowest (4.9% in male subjects, 6.1% in females) in male subjects whose TSH concentration ranged from 0.4 to 2.0 mU/L and female subjects whose concentrations ranged from 0.4 to 1.5 mU/L. TPOAb prevalence progressively increased in both sexes as serum TSH concentration increased.15 The conclusion drawn from these studies: Occult thyroid disease is common in normal reference populations. From the TPOAb-prevalence data, the normal serum TSH adult reference range for subjects with no evidence of thyroid disease
ranges from 0.4 to 2.0 mU/L. Patients with TSH values greater than 2.0 mU/L should be tested for TPOAb, and this new reference range should be used as the optimal endpoint for thyroid hormone–replacement therapy.15 The prevalence of primary hypothyroidism unknown to the volunteers before TSH screening was 2.4% among the entire study population. Members of the group included in this study include subjects with known hypothyroidism and those in whom hypothyroidism was detected for the first time. The total number with hypothyroidism includes both groups, volunteers who were undergoing thyroid therapy, and those found to have primary hypothyroidism de novo in this study. When this number is factored into the calculations, the prevalence of hypothyroidism is 8.2%, a value comparable to the 4% to 10% reported for adult populations in the NHANES III study and other reports of adult populations with mild (subclinical) hypothyroidism.6,13 In the United States, nearly all cases of subclinical adult hypothyroidism are caused by early or mild autoimmune thyroiditis, which can be detected through measurement of TPOAbs and TGAbs.12–14 We report good agreement between the serum and DBS assays for TSH5 and thyroid antibodies. Samples collected on filter paper at nursing homes and community centers can be transported at ambient temperatures to central facilities for inexpensive testing. Reagent and labor costs for the 3 DBS tests total less than $20. Even with a 50% overhead and a 20% profit, the tests could be offered at a price of $36, considerably less than the estimated serum costs for the 3 tests of $69.1,16 We have shown that the same DBSs collected for TSH screening can be tested for TPOAb and TGAb to yield information about the risk and probable cause of thyroid disease. In summary, our results achieved with the use of DBS specimens for screening are comparable to reports in the literature involving serum specimens to detect TAbs in adult populations ages 50 years and older. In this study, TAbs were detected in 266 (27%) of
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972 subjects screened, and TSH values were greater than 5 mU/L in 36 of these subjects, or 3.7%. Application of the techniques used in quantitation of TAbs in eluates from DBS filter-paper specimens greatly expands the possibilities for population screening to identify diseases before their clinical expression. The use of DBS specimens for the detection of immunoglobulins can be applied to population screening of women before and at the time of diagnosis of pregnancy for many routinely obtained tests, as well as tests for additional diseases that are valuable in the clinical management of woman and her fetus. Furthermore, this cost-effective specimen can be applied to the early detection of autoimmune thyroid diseases among populations at increased risk for the subsequent development of these diseases (eg, type I diabetes mellitus, Down syndrome, Turner syndrome, other autoimmune diseases). Pregnant and postpartum women, adults ages 35 (women) to 50 (men) years and older, and patients with existing autoimmune and genetic diseases would greatly benefit from TSH and TAb screening. This low-cost, accurate population-screening technology can be used to prevent significant morbidity through the early detection of inexpensively treatable diseases. We thank Pierce Hofman and Pam Polana for their technical assistance. REFERENCES
1. Ladenson PW, Singer PA, Ain KB, Bagchi N, Bigos ST, Levy EG, et al. American Thyroid Association guidelines for detection of thyroid dysfunction. Arch Intern Med 2000;160:1573–5. 2. Wiersinga WM. Adult hypothyroidism: screening for hypothyroidism. In: De Groot LJ, Hennemann G, editors. The thyroid and its diseases: the thyroid manager. Available at: www. thyroidmanager.org/Chapter9/9-frame.htm. Accessed: August 2004. 3. Muller AF, Drexhage HA, Berghout A. Postpartum thyroiditis and autoimmune thyroiditis in women of childbearing age: recent insights and consequences for antenatal and postnatal care. Endocrine Rev 2001;22:605–30.
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4. Fisher DA, Dussault JH, Foley TP Jr, Klein AH, LaFrenchi S, Larsen PR, et al. Screening for congenital hypothyroidism: Results of screening one million North American infants. J Pediatr 1979;94:700 –5. 5. Hofman LF, Foley TP Jr, Henry JJ, Naylor EW. TSH assay using dried blood spotted filter paper specimens to screen for hypothyroidism in older children and adults. J Med Screening 2003;10: 5–10. 6. Hollowell JG, Staehling NW, Flanders WD, et al. Serum TSH, T4, and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2002:87:489 –99. 7. Hofman LF, Foley TP, Henry JJ, Naylor EW. Screening for thyroid antibodies in pregnant women with DBS specimens [abstract]. Genoa, Italy: Fifth Meeting of the International Society for Neonatal Screening, June 25–29, 2002. 8. Amino N, Kuro R, Miayi K, Kumahara Y. Measurement of anti-thyroid antibodies in dried blood spots. Clin Exp Immunol 1979;35:158 – 60. 9. Phillips DIW, McLachlan SM, Rees Smith B, Bradbury J, Beeve K. Autoantibodies to thyroglobulin and thyroid peroxidase in blood spots measured with a sensitive, direct radioimmunoassay. Clin Chem 1990;36:823– 4. 10. Mitchell ML, Hermos RJ, Larson CA. Thyroid peroxidase antibodies in dried blood specimens of newborns. Thyroid 2002;12: 609 –11. 11. Package insert. Kronus Kalibre ELISA kit for quantificative determination of serum antibodies to thyroglobulin and thyroid peroxidase. Boise, Idaho; Kronus: 2000. 12. Sherman SI. The laboratory approach to thyroid disorders. In: Cooper DS, ed. Medical management of thyroid disease. New York, NY: Marcel Dekker; 2001:19 –21. 13. McDermott MT, Ridgway EC. Diagnosis and treatment of hypothyroidism. In: Cooper DS, ed. Medical management of thyroid disease. New York, NY; Marcel Dekker; 2001:149 –52. 14. Mandel SJ. Thyroid disease and pregnancy. In: Cooper DS. Medical management of thyroid disease. New York, NY: Marcel Dekker; 2001:411–12. 15. Spencer C, Hollowell J, Nicoloff J, Braverman L. NHANES III: Impact of TSH:TPOAb relationships on redefining the serum TSH normal reference range. In: Abstracts of the 74th Annual Meeting of the American Thyroid Association; October 10 –13, 2002; Los Angeles, Calif. Program 2. p. 111. 16. Danese MD, Powe NR, Sawin CT, Ladenson PW. Screening for mild thyroid failure at the periodic health examination: a decision and cost-effective analysis. JAMA 1996;276:285–92.
Thyroid-antibody screening is recommended for several adult populations, including the elderly and pregnant and postpartum women, and analysis of filter paper– dried blood spots (DBSs) has been a cost-effective means of screening newborns for hypothyroidism for more than 30 years. The aim of this study was to show that DBS specimens can be used to test adults for thyroid-stimulating hormone (TSH) antibodies and thyroid antibodies during screening for thyroid disease in a community setting. Thyroid peroxidase antibody and thyroglobilin antibody enzyme-linked immunosorbent assay kits for serum have been adapted for use with DBSs. A TSH assay for newborn DBSs was adapted for the screening of adults. Parallel specimens, serum and DBSs, were collected during routine care of patients attending a thyroid clinic. In addition, 962 DBS specimens were collected from volunteers at community centers and nursing homes. Twelve months later, a second specimen was collected from each of 411 original volunteers. Antibody results are reported as normal and positive. Ninety-seven percent of the serum/DBS results correlated exactly. Of 962 volunteers from nursing homes and community centers, testing for antibodies was positive or indeterminate (borderline) in 266 (27%). Eighty percent of the patients with an initially normal TSH reading who were positive on the 12-month collection were positive for at least one antibody in the first collection. The use of DBSs is a convenient and efficient way to screen for thyroid antibody as a means of detecting occult thyroid disease in adults. (J Lab Clin Med 2004;144:307-12) Abbreviations: CV ⫽ coefficient of variation; DBS ⫽ dried blood spot; ELISA ⫽ enzyme-linked immunosorbent assay; NHANES III ⫽ National Health and Nutrition Examination Survey; TAb ⫽ thyroid antibody; TGAb ⫽ thyroglobulin antibody; TPOAb ⫽ thyroid peroxidase antibody; TSH ⫽ thyroid-stimulating hormone (thyrotropin)
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creening for TAbs is advised in several populations, including the elderly, pregnant and postpartum women, and individuals at increased risk for autoimmune hypothyroidism and thyrotoxicosis, From Pediatrix Screening, PO Box 219, 90 Emerson Lane, Bridgeville, PA 15017. Supported by Small Business Innovation Research (SBIR) grants R43DK-53666 and R44-DK-53666 from the National Institute for Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, United States Public Health Service, Department of Health and Human Services. Drs Hofman and Foley are consultants to Pediatrix Screening. Submitted for publication May 15, 2004; revision submitted September 20, 2004; accepted for publication September 22, 2004. Reprint requests: Lindsay F. Hofman, PO Box 771, Vashon, WA 98070; e-mail: [email protected]. 0022-2143/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lab.2004.09.009
particularly women older than 35 years.1–3 Although DBS specimens have been used for screening in newborns for more than 30 years and analysis of DBS specimens has been shown to be a cost-effective, accurate means of screening for thyroid diseases,4 there has been little use of the DBS in the screening of adults. Recently we showed that the use of DBS specimens is a cost-effective, accurate method of TSH screening for hypothyroidism in adults.5 We undertook the study reported here to show that a DBS assay for TAbs involving the same specimen collected for TSH screening is sufficiently sensitive and specific to be used in the screening of adults for thyroid disease. TGAb and TPOAb have been found in the presence of autoimmune thyroid disease, including overt and mild (subclinical) hypothyroidism. Because autoimmune thyroiditis (Hashimoto’s disease) is the most common cause of primary hypothyroidism among 307
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adults in the United States,2,6 assessment of TPOAb and TGAb concentrations can provide information about both the cause of a high TSH reading and help determine an individual’s risk for hypothyroidism. We previously reported an adaptation of a neonatal assay to measure TSH in adults5 and presented preliminary results with a DBS assay for TAbs in pregnant women.7 It would be advantageous to use the same DBS specimen for both TSH and antibody testing. Previously published methods for the measurement of TAbs in DBS specimens have involved the use of a hemagglutination assay8 or recombinant immunoassay.9,10 We describe an ELISA assay for the measurment of TPOAb and TGAb in DBS and report our experience in screening a large population of adult volunteers. METHODS Reagents. Reagents for both serum and DBS TPOAb and TGAb assays were purchased from KRONUS (Boise, Idaho) in the form of Kalibre ELISA TGAb (KR7280) and TPOAb (KR7260) kits. Filter paper (number 903; Schleicher & Schuell, Keene, NH) was used for the collection of specimens and preparation of controls. We took TSH reagents from the Auto Delfia Neonatal hTSH kit (Perkin-Elmer Wallac, Turku, Finland), using standards made in-house especially for the adult assays.5 DBS controls were prepared from heparin-treated whole blood collected from individuals with and without high antibody titers. DBS controls spotted on filter paper, which were stored in plastic bags with desiccant, were stable for more than 2 years at ⫺15°C. Sample collection. To compare serum and DBS levels of antibody, we collected specimens prospectively from children, adolescents, and adults during evaluation for thyroid disease. Serum antibody levels were measured as part of the routine clinical thyroid evaluation with the use of the Kronus recombinant-immunoassay TPOAb and TGAb assays in the Pediatric Endocrine Laboratory of Children’s Hospital (Pittsburgh, Pa). Whole blood, left in the tubing of the scalpvein needle after the collection of clinical specimens in 1998 and 1999, was spotted onto filter paper. The paper was allowed to dry horizontally at room temperature for 4 hours, placed in a self-sealing plastic bags with desiccant, and stored at ⫺20°C. In addition, we collected 962 samples on filter paper by means of fingerstick from volunteers recruited in nursing homes and community centers in western Pennsylvania from 2000 through 2002. The study, which was carried out in accordance with the principles of the Declaration of Helsinki, was approved by the Western International Review Board. A nurse explained the study to each volunteer before asking him or her to sign an informed-consent form that had been approved by the Western International Review Board. The nurse then pricked the volunteer’s finger and collected a blood specimen on a specially designed Schleicher & Schuell filterpaper card on which identifying and demographic informa-
Table I. Kronus reference ranges in adults’ serum Reading (U/mL) Antibody
TPOAb TGAb
Normal
Indeterminate
Positive
⬍1 ⬍2
1–2 2–3
⬎2 ⬎3
tion was entered. Specimens were dried and stored as described above and assayed within a month of their collection. Analysis. The DBS antibody assays were similar to those described in the Food and Drug Administration–approved Kronus package inserts for TPOAb and TGAb ELISAs, with the addition of an extraction step at the beginning.11 We used 100 L of kit diluent to extract antibodies from each 6-mm DBS. The specimen was incubated for 30 minutes on a plate shaker at 800 rpm. We used 50 L eluate in the assay and continued the procedure as described in the package insert. Absorbance was read at 405– 650 nm. Results were calculated from a logit log plot of the standard concentration against absorbance. All assays were run by one of four technicians in a diagnostic clinical laboratory. Serum results were received after the completion of the DBS assay. The DBS assay for TSH was an adaptation of the AutoDelfia Neonatal TSH kit.5 Standards were made in-house to cover the decision level for adults, which is lower than that for neonates. RESULTS
Because the reagents we used were from Food and Drug Administration–approved serum assays for the antibodies, the focus of the validation was comparison of DBS results with the results of the serum assay. Liquid standards from the kit were used in the DBS assay. Eluate (50 L) from normal DBSs was added to the standards. The eluate had very little effect on the results of the assay. The correlation for absorbance of TPOAb standards with and without eluate was as follows: Liquid standard ⫽ 0.88 (standard ⫹ eluate) ⫹ .022 (r ⫽ .998) (Y⫽ MX ⫹ B). Blank absorbance (no DBS present), averaged over 10 assays, was 0.042 A405– 630nm units (SD 0.035) less than the DBS 0 standard for TPOAb and 0.048 A405– 630nm units (SD 0.036) less than the DBS 0 standard for TGAb. The sensitivity of the DBS assay described as the lowest standard corrected for volume factors was 0.68 U/mL for TPOAb and 1.4 U/mL for TGAb. Kronus reference ranges in serum for adults are set forth in Table I. The DBS values reported here have been corrected to yield the level in serum. We found no correlation between the quantitative value of the antibody and the TSH level (TSH vs TGAb [r ⫽ .16], TSH vs TPOAb [r ⫽ .35]) or the quantitative value of TPOAb versus
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Fig 1. Initial and 12-month populations: distribution by sex and age. DBS specimens were collected from 962 volunteers at nursing homes and community centers in western Pennsylvania; 788 were female, 176 male. Volunteers from whom a specimen was collected received a postcard reminding them to have a second specimen collected 12 months after the first. Of the 411 volunteers who returned for a second collection, 323 were female and 89 were male.
that of TGAb (r ⫽ .26). Because the actual antibody level does not correlate with clinical outcome (ie, an antibody level of 55 is no more likely to denote a high TSH value than is an antibody level of 8), we consider the antibody-test results semiquantitative and report them as normal or positive. We reported indeterminate (borderline) results as normal because this category did not exist at the start of the study. Each specimen was run singly in each assay. If the test results for both antibodies were positive or negative, the specimen was reported as such. If the result for one antibody was positive and that for the other was normal, the test that yielded the positive result was rerun. If the positive value was confirmed, the specimen was reported as positive. In only one case did repeat testing yield a negative result, and a second specimen was requested. Serum and DBS thyroid-antibody tests were run on 33 specimens for both antibodies. All patients but one fell into the same diagnostic category for serum and
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DBS on the basis of the results of both tests; this patient had normal TPOAb results on both DBS and serum testing, as well as a normal DBS TGAb result. Only the testing for serum TGAb yielded a positive result. We evaluated interassay precision by running high and low DBS controls in 18 assays. Because this assay was designed to be run as a screening assay, we calculated precision from singlewell values. In the TPOAb assay, the high control, with a mean of 35 U/mL, had a CV of 9.95% and the low control with a mean of 8.5 U/ml had a CV of 11.4%. In the TGAb assay, the high control, with a mean of 48 U/mL, had a CV of 13.4% and the low control, with a mean of 13 U/mL, had a CV 16.3%. These results compare favorably with the CVs calculated from the ranges supplied with the kit for kit-high controls for the serum assay which were 25% for both antibodies. One advantage of using DBS specimens is that the filter-paper cards can be sent by regular mail at ambient temperatures. We performed stability studies designed to simulate conditions that specimens would be subject to during in shipping in glassine (transparent glazed paper, resistant to the passage of air and grease) envelopes. DBS specimens were mailed across the country under conditions similar to those used to send newborns’ specimens to the laboratory. We compared the results with a sample kept at refrigerator temperature. Specimens spent five days in transit. Shipped specimens measured in the same assay as the refrigerated specimen differed by less than 13% at a mean TPOAb level of 44 U/mL. DBS controls were stored in glassine envelopes at room temperature. Two, 7, 10, and 14 days after sampling, the specimens were put in the freezer. All samples were tested in one assay and run in duplicate. At 14 days, the TPOAb specimen was found to have gone from 35 to 32 U/mL and the TGAb specimen was found to have gone from 32 to 30 U/mL. The variation was well within the precision of the assay. The specificity data for serum apply to DBS. The antigens used to coat the wells are highly (affinity) purified.11 Consequently, the thyroglobulin-antigen preparation was free of contaminating thyroid peroxidase antigen and the thyroid peroxidase antigen preparation was free of contaminating thyroglobulin antigens. TGAb, TPOAb, and TSH were measured in eluates from DBS specimens collected from 962 individuals. Sex and age distribution is depicted in Fig 1. Fliers announcing the opportunity to participate were distributed to community centers and nursing homes. Any individual who presented himself or herself at these locations on the announced day and was willing to read and sign a consent form was accepted into the study.
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Table II. Comparison of first- and second-specimen results, TPOAb Specimen
TPOAb-positive specimen 2 TPOAb-indeterminate specimen 2 TPOAb-normal specimen 2 Total
TPOAb-positive specimen 1
TPOAb-indeterminate specimen 1
TPOAb-normal specimen 1
71 1 7 79
2 1 20 23
5 11 293 309
Table III. Comparison of first- and second-specimen results, TGAb Specimen
TGAb-positive specimen 2 TGAb-indeterminate specimen 2 TGAb-normal specimen 2 Total
TGAb-positive specimen 1
TGAb-indeterminate specimen 1
TGAb-normal specimen 1
37 3 6 46
5 1 25 31
4 2 328 334
We did not exclude people who were taking thyroid medication. Of the 962 individuals tested, 266 (27%) tested positive or indeterminate for one or both antibodies. Of the 266, 36 (14%) had increased TSH levels (⬎5mU/L) and 56 (21%) reported taking thyroid medication. Of the 36 who tested positive or indeterminate for antibodies and had increased TSH levels, 13 were of the group reported to be taking thyroid medication. Increased TSH concentration had not previously been reported in the remaining 23 individuals. In summary, of the total population we tested, 23 (2.4%) had unrecognized clinical or subclinical hypothyroidism.12–14 Normal antibody results were reported for six individuals with increased TSH readings. One was a 94year-old woman who died a few months later. All others had TSH concentrations of less than 10 mU/L. One had a normal TSH reading two months later, two had indeterminate TGAb levels, and from two no further information could be obtained. Although most positive antibody specimens demonstrated positive TPOAb results, 8% of the specimens demonstrating increased TSH concentrations and 4% of the total specimens had normal TPOAb and high TGAb titers. It was therefore necessary to run both antibody tests. Because their formats are similar, the tests can be run at the same time to reduce the costs of technician time. When the assays are used to confirm each other, reagent costs can be minimized. Repeat testing 12 months after the initial blood test was included in the study design. Testing was offered to all adult volunteers who were originally recruited from nursing homes and community centers in western Pennsylvania and had given consent. Study participants were contacted by phone or mail. Of the 962 volunteers who participated in the baseline study, 411 returned for collection of a second filter-paper specimen by a fin-
gerstick 12 months later. Specimen collection was performed by the same nurse, using the identical procedure, and specimen handling was conducted as done initially. Fig 1 shows the age and sex distributions of the 411 individuals from whom second specimens were collected 12 months after the first. Tables II and III show the initial and repeat outcomes for TPOAb and TGAb. Of those patients whose samples were positive or normal for TPOAb or TGAb on the first testing, 86% remained in the same category. Most of the specimens that tested indeterminate the first time were normal on the second testing (83%), but seven (13%) were positive on the second test. A much smaller proportion of specimens that were normal on the first test were positive on the second (1.3%). Eight specimens had normal TSH readings on the first test and positive TSH on the second. Three of these individuals were known to be hypothyroid and were taking thyroid medication. Of the remaining five, three had positive TPOAb results on the first test and one was positive for both antibodies (Table IV). In summary, the antibody result predicted the development of hypothyroidism in 80% of cases. DISCUSSION
Screening for autoimmune thyroid diseases and primary hypothyroidism in adults, especially pregnant and postpartum women and members of the general population 50 years and older, is recommended by several national and international groups because of the high prevalence of disease.1–3,6,12–14 Our studies support these recommendations and provide an inexpensive method for specimen collection and analysis with which to screen large populations, comparable to older
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Table IV. Increased TSH only on second specimen, TGAb and TPOAb Patient
TSH 1
TGAb 1
TPOAb 1
TSH 2
TGAb 2
TPOAb 2
1 2 3 4 5
2.1 ⬍2 3.5 2.7 4.8
⬍1.4 11 1.9 1.9 3.7
0.7 7.3 14 26 ⬎68
5.1 6.4 6.7 5.7 5.7
⬍1.4 7.1 ⬍1.4 1.6 9
⬍0.68 13 6.9 ⬎68 81
Data expressed as units per milliliter. Five patients who were classified as having normal TSH concentrations in the first specimen were positive for TSH on the second specimen. In this study, a positive TSH reading was defined as a concentration greater than 5 mU/L in the DBS specimens. These results were confirmed with serum TSH test. The number 1 refers to the hormone or antibody level on the first specimen. The number 2 refers to the level on the 12-month specimen.
studies whose findings justified the screening of newborns for congenital hypothyroidism.4 Thyroid-antibody data were reported from a recently published nationwide survey, the NHANES III, in which specimens from adolescents and adults were tested with the use of the same TAb-assessment methods reported here.6,15 Of 16,533 individuals ages 12 years and older with no thyroid disease or goiter who were not taking thyroid medication (termed the “disease-free populations”), TPOAbs were present in 11.3 % and TGAbs were found in 10.4 %.6 Eighteen percent tested positive for one or both of the TAbs. The authors concluded that a significant proportion of the thyroid disease–free populations in the United States have evidence of thyroid disease on TSH and TAb testing. These data support the usefulness of screening in the early detection of thyroid disease.6 In our study, for which we recruited an older population, we found a TAb prevalence of 27%. In the NHANES III study, the prevalence of positivity for positive TPOAbs increased from 16% in women ages 40 to 49 years to 27% in women older than 70 years, comparable to the prevalence in our population. In addition, our study included individuals taking thyroid medication who probably would test positive for antibodies, given that autoimmune thyroiditis is the most common cause of hypothyroidism in adults. Further analysis of the NHANES III data to determine the prevalence of TPOAbs at specific serum TSH ranges revealed that the TPOAb prevalence was lowest (4.9% in male subjects, 6.1% in females) in male subjects whose TSH concentration ranged from 0.4 to 2.0 mU/L and female subjects whose concentrations ranged from 0.4 to 1.5 mU/L. TPOAb prevalence progressively increased in both sexes as serum TSH concentration increased.15 The conclusion drawn from these studies: Occult thyroid disease is common in normal reference populations. From the TPOAb-prevalence data, the normal serum TSH adult reference range for subjects with no evidence of thyroid disease
ranges from 0.4 to 2.0 mU/L. Patients with TSH values greater than 2.0 mU/L should be tested for TPOAb, and this new reference range should be used as the optimal endpoint for thyroid hormone–replacement therapy.15 The prevalence of primary hypothyroidism unknown to the volunteers before TSH screening was 2.4% among the entire study population. Members of the group included in this study include subjects with known hypothyroidism and those in whom hypothyroidism was detected for the first time. The total number with hypothyroidism includes both groups, volunteers who were undergoing thyroid therapy, and those found to have primary hypothyroidism de novo in this study. When this number is factored into the calculations, the prevalence of hypothyroidism is 8.2%, a value comparable to the 4% to 10% reported for adult populations in the NHANES III study and other reports of adult populations with mild (subclinical) hypothyroidism.6,13 In the United States, nearly all cases of subclinical adult hypothyroidism are caused by early or mild autoimmune thyroiditis, which can be detected through measurement of TPOAbs and TGAbs.12–14 We report good agreement between the serum and DBS assays for TSH5 and thyroid antibodies. Samples collected on filter paper at nursing homes and community centers can be transported at ambient temperatures to central facilities for inexpensive testing. Reagent and labor costs for the 3 DBS tests total less than $20. Even with a 50% overhead and a 20% profit, the tests could be offered at a price of $36, considerably less than the estimated serum costs for the 3 tests of $69.1,16 We have shown that the same DBSs collected for TSH screening can be tested for TPOAb and TGAb to yield information about the risk and probable cause of thyroid disease. In summary, our results achieved with the use of DBS specimens for screening are comparable to reports in the literature involving serum specimens to detect TAbs in adult populations ages 50 years and older. In this study, TAbs were detected in 266 (27%) of
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972 subjects screened, and TSH values were greater than 5 mU/L in 36 of these subjects, or 3.7%. Application of the techniques used in quantitation of TAbs in eluates from DBS filter-paper specimens greatly expands the possibilities for population screening to identify diseases before their clinical expression. The use of DBS specimens for the detection of immunoglobulins can be applied to population screening of women before and at the time of diagnosis of pregnancy for many routinely obtained tests, as well as tests for additional diseases that are valuable in the clinical management of woman and her fetus. Furthermore, this cost-effective specimen can be applied to the early detection of autoimmune thyroid diseases among populations at increased risk for the subsequent development of these diseases (eg, type I diabetes mellitus, Down syndrome, Turner syndrome, other autoimmune diseases). Pregnant and postpartum women, adults ages 35 (women) to 50 (men) years and older, and patients with existing autoimmune and genetic diseases would greatly benefit from TSH and TAb screening. This low-cost, accurate population-screening technology can be used to prevent significant morbidity through the early detection of inexpensively treatable diseases. We thank Pierce Hofman and Pam Polana for their technical assistance. REFERENCES
1. Ladenson PW, Singer PA, Ain KB, Bagchi N, Bigos ST, Levy EG, et al. American Thyroid Association guidelines for detection of thyroid dysfunction. Arch Intern Med 2000;160:1573–5. 2. Wiersinga WM. Adult hypothyroidism: screening for hypothyroidism. In: De Groot LJ, Hennemann G, editors. The thyroid and its diseases: the thyroid manager. Available at: www. thyroidmanager.org/Chapter9/9-frame.htm. Accessed: August 2004. 3. Muller AF, Drexhage HA, Berghout A. Postpartum thyroiditis and autoimmune thyroiditis in women of childbearing age: recent insights and consequences for antenatal and postnatal care. Endocrine Rev 2001;22:605–30.
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4. Fisher DA, Dussault JH, Foley TP Jr, Klein AH, LaFrenchi S, Larsen PR, et al. Screening for congenital hypothyroidism: Results of screening one million North American infants. J Pediatr 1979;94:700 –5. 5. Hofman LF, Foley TP Jr, Henry JJ, Naylor EW. TSH assay using dried blood spotted filter paper specimens to screen for hypothyroidism in older children and adults. J Med Screening 2003;10: 5–10. 6. Hollowell JG, Staehling NW, Flanders WD, et al. Serum TSH, T4, and thyroid antibodies in the United States population (1988 to 1994): National Health and Nutrition Examination Survey (NHANES III). J Clin Endocrinol Metab 2002:87:489 –99. 7. Hofman LF, Foley TP, Henry JJ, Naylor EW. Screening for thyroid antibodies in pregnant women with DBS specimens [abstract]. Genoa, Italy: Fifth Meeting of the International Society for Neonatal Screening, June 25–29, 2002. 8. Amino N, Kuro R, Miayi K, Kumahara Y. Measurement of anti-thyroid antibodies in dried blood spots. Clin Exp Immunol 1979;35:158 – 60. 9. Phillips DIW, McLachlan SM, Rees Smith B, Bradbury J, Beeve K. Autoantibodies to thyroglobulin and thyroid peroxidase in blood spots measured with a sensitive, direct radioimmunoassay. Clin Chem 1990;36:823– 4. 10. Mitchell ML, Hermos RJ, Larson CA. Thyroid peroxidase antibodies in dried blood specimens of newborns. Thyroid 2002;12: 609 –11. 11. Package insert. Kronus Kalibre ELISA kit for quantificative determination of serum antibodies to thyroglobulin and thyroid peroxidase. Boise, Idaho; Kronus: 2000. 12. Sherman SI. The laboratory approach to thyroid disorders. In: Cooper DS, ed. Medical management of thyroid disease. New York, NY: Marcel Dekker; 2001:19 –21. 13. McDermott MT, Ridgway EC. Diagnosis and treatment of hypothyroidism. In: Cooper DS, ed. Medical management of thyroid disease. New York, NY; Marcel Dekker; 2001:149 –52. 14. Mandel SJ. Thyroid disease and pregnancy. In: Cooper DS. Medical management of thyroid disease. New York, NY: Marcel Dekker; 2001:411–12. 15. Spencer C, Hollowell J, Nicoloff J, Braverman L. NHANES III: Impact of TSH:TPOAb relationships on redefining the serum TSH normal reference range. In: Abstracts of the 74th Annual Meeting of the American Thyroid Association; October 10 –13, 2002; Los Angeles, Calif. Program 2. p. 111. 16. Danese MD, Powe NR, Sawin CT, Ladenson PW. Screening for mild thyroid failure at the periodic health examination: a decision and cost-effective analysis. JAMA 1996;276:285–92.