1233
MEASUREMENT OF THYROID-STIMULATING
assay. Discs 1
HORMONE IN DRIED BLOOD SPOT KAZUKO ENOMOTO
MINORU IRIE
First Department of Medicine, Toho
University School of
Medicine, Ohmori-Nishi, Tokyo, Japan HIROSHI NARUSE Division of Biological Research on Mental Retardation, National Institute of Mental Health, Ichikawa City,
Chiba-ken, Japan
(thyroid-stimulating hormone) was measured by radioimmunoasdried blood in spotted onto filter-paper and obtained say of the newborn for metabolic disorders. screening during
Summary
Blood
filter-papers were sent by post and kept at -20°C until cm in diameter were punched out, each disc containing approximately 20 µl of whole blood. The blood was eluted from the disc overnight in 0.6 ml of pH 7-6 phosphate buffer 50 mmol/1, containing 0-05% bovine serum albumin. The discs were removed, dried at the upper wall of the test tubes, and discarded. The eluate was subjected to the doubleantibody radioimmunoassay for T.S.H.’ 100 µl of diluted The
T.S.H.
125I-T.S.H. and diluted anti-T.s.H.
serum were
eluate, and the reaction mixture
was
added
incubated
at
to
each
40C for
three days. Then 100 µl of diluted second antibody (anti-rabbit gamma-globulin goat serum) was added and incubated at 4°C for twenty-four hours. After centrifugation, the precipitate was counted by an automated gamma counter and the values of T.S.H. were obtained from a standard curve. T.S.H. values were expressed as jjtU/ml by calculation using a multiple of 5.
By this method, the detection limit for blood T.S.H. was 5-10 µU/ml, which is the approximate upper limit of normal for blood T.S.H. T.S.H. values obtained on dried blood correlated well with those obtained on serum from
of the assay in a single sample is not necessary. This method picked up a case of primary hypothyroidism in a four-year-old girl with symptoms. Since the technique is simple and sensitive enough for the detection of hypothyroidism, it could be valuable in mass-screening for congenital hypothe
same
subjects. Duplication
thyroidism. Introduction HYPOTHYROIDISM is difficult to diagnose in the newborn and is often missed at this time or even in childhood. Early detection is important because brain damage as well as growth disturbance may occur during the first months of life. Dussault et at. 12have reported that the radioimmunoassay of thyroxine from a dried blood spot has been useful for the diagnosis of congenital hypothyroidism, and Klein et al.3 detected an infant with subclinical congenital hypothyroidism by measuring T.S.H. (thyroid-stimulating hormone). in umbilical cord serum. We have used a method to measure T.s.H. in a dried blood spot.
T.S.H. DF DRIED BLOOD SPOT
(µU /ml )
Fig. 1-Correlation between the T.S.H. values obtained by the dried blood spot method and those obtained by the measurement of serum samples from the same
y
=
20.98
+
subjects.
1.09x
(n = 166,
r =
0.841).
Materials and Methods Blood was spotted onto filter-paper.4 To check the validity of the method we assayed blood from adult hypothyroid patients and normal or hypothyroid patients during a 500 µg T.R.H. (thyrotropin-releasing hormone) administration test. To get the background data for a screening programme we also included blood from outpatients of a primary health care centre for children and from infants one day to two months of age from whom specimens had been taken for the multiple screening for metabolic disorders, and umbilical cord blood.
DR WISE AND OTHERS: REFERENCES
1. Goldsmith, R. E. Proc. Staff Meet. Mays Clin. 1972, 47, 953. 2. Jackson, G. L. Am. J. Roentgenol. Radium Ther. Nucl. Med. 1971, 112, 720. 3. Weidinger, P., Johnson, P. M., Werner, S. C. Lancet, 1974, ii, 74. 4. Wise, P. H., Ahmad, A., Pain, R. W. Unpublished. 5 Marion, M. A., Ronai, P. M., Pain, R. W., Wise, P. H. Aust. N. Z.
J. Med.
1974, 4, 379. 6. Mincey, E. K., Wilcox, M. E., Morrison, R. T. J nucl. Med. 1974, 15, 11. 7. Murphy, B. J. Lab. clin. Med. 1965, 66, 165. 8. Oldfield, R., Pain, R. W. Am. J. clin. Path. 1969, 52, 118. 9. Patel, Y. C., Burger, H. G., Hudson, B. J. clin. Endocr. Metab. 1971, 33, 768.
10. Larsen, P. R. J. clin. Invest. 1972, 51, 1939. 11 Tunbridge, W. M. G., Evered, D. Proc. Seventh Med.
1975, p. 101.
T.S.H. OF ANOTHER SPOT
(µU/ml )
2-Correlation between two values of T.S.H. obtained in discs obtained from same individuals.
Fig. int.
Thyroid Conf. Excerpta
y = -1 18 +
1.04x
(n
=
296, r
=
0.978).
two
blood
1234 Results T.S.H. values from the dried blood spot were determined after varying periods of extraction, and the maximum values were obtained after two hours of extraction. Stability of the blood spotted onto filter-paper was determined by measuring the T.s.H. values from two days to fifty-five days after spotting, virtually no change being noted over this period of time. Fig.1 shows the correlation between the T.s.H. values obtained by the dried blood spot method and those obtained by measuring T.S.H. in serum samples from the same subjects. The detection limit for blood T.S.H. was 5-10 µU/ml by the dried blood spot method. Fig. 2 shows the correlation between two values of T.S.H. in two blood discs obtained from same individuals. Using this method, adult hypothyroid patients showed high concentrations of T.S.H., and T.S.H. concentrations rose after the injection of 500 µg synthetic T.R.H. (table). Of BLOOD T.S.H. CONCENTRATION MEASURED ON DRIED BLOOD SPOTS FROM PATIENTS WITH HYPOTHYROIDISM, NORMAL SUBJECTS, AND IN CORD
well as T.s.H. would be necessary, the microdetermination of T. S. II. has substantial value since primary hypothyroidism seems to be much more common than secondary or tertiary hypothyroidism. Klein et al.have shown that treatment before the age of three months could prevent mental retardation among hypothyroid infants. With the method we have described patients with congenital or juvenile primary hypothyroidism could be detected during mass-screening for metabolic disorders, and treatment could begin during the first days or weeks of life.
iodothyronine
as
We thank Dr H. L. Levy of the State Laboratory Institute, Boston, Massachusetts, U.S.A., for his help in the preparation of this paper.
This study was partly supported by a grant from the Ministry of Health of Japan. The materials to measure T.s.H. were supplied by the National Institutes of Health, Bethesda, Maryland, U.S.A., and the Daiichi Radioisotope Company, Tokyo, Japan. We also thank Dr H. Enomoto, Dr K. Inoue, Dr T. Nukui, Dr M. Kuroda, Dr S. Takeda, Dr M. Mizotam, Dr Y. Ito, and Ms S. Oshima for their help; Dr T. Aoki, Dr T. Kumagai, Dr K. Nakayama of the Pædiatric Department of Toho University for their help with the paediatric patients, and Ms Y. litaka and M. Katakura for secretarial work.
BLOOD
Requests for reprints should be addressed to M.l. REFERENCES
1. 2.
*245
µU/ml in the case of hypothyroidism (see text).
227 newborn infants tested none had a raised T.S.H., and values in 5 cord-blood specimens were also normal. From the dried blood spots obtained from outpatients, 1 case of primary hypothyroidism was detected-in a four-year-old girl with clinical signs of hypothyroidism and a T.S.H. of 245 uU/ml.
Dussault, J. H , Laberge, L. Union Meed Can. 1973, 102, 2062 Dussault, J. H., Coulombe, P., Laberge, L., Guyda, H , Khoury, K J. Pediat. 1975, 86, 670. 3. Klein, A. H., Augustin, A. V., Foley, Jr. T. P. Lancet, 1974, ii, 77 4. Guthrie, R., Susi, A., Pediatrics, 1963, 32, 338. 5. Odell, W. D., Wliber, J. F , Utiger, R. D. Rec. Progr Hormone Res 1967, 23, 47. 6. Hershman, J. M., Pittman, Jr. J. A. Ann. intern Med 1971, 74, 481 7. Fisher, D. A., Odell, W D. J. clin Invest 1969, 48, 1670 8. Klein, A. H., Meltzer, S., Kenny, F. M. J Pediat. 1972, 81, 912
NATURAL COURSE OF SYMPTOMLESS AUTOIMMUNE THYROIDITIS
A. GORDIN Discussion We have shown that T.s.H. can be measured in 20 µl of whole blood dried on filter-paper. T.S.H. in blood collected in this manner is stable and extraction is simple. Most importantly T.S.H. values obtained in this way correlate well with those obtained on serum. Though the values of T.S.H. from the dried blood spot are a little lower than T.S.H. values obtained from serum, this method is quite acceptable for use in the detection of primary hypothyroidism by mass screening of the newborne because this method can detect approximately 10 µU/ml Of T.S.H., a value far below that of most cases of primary hypothyroidism.5 Furthermore, the assay does not have to be duplicated, a factor which greatly enhances its value for mass screening. Blood should be obtained three or more days after birth, because serum-T.s.H. levels rise sharply in the postnatal period.67However, a postnatal T.S.H. rise is not a consistent feature because we found normal blood-T.s.H. values in umbilical cord blood and in specimens obtained on the first and second days of life. In patients with secondary or tertiary hypothyroidism, the bloodt. S. H. should be reduced, but such lower than normal levels cannot yet be measured. Although for the diagnosis of all types of hypothyroidism the measurement of blood thyroxine and tri-
B.-A. LAMBERG
Endocrine Research Unit, University of Helsinki; Minerva Foundation Institute for Medical Research, Helsinki; and Third and Fourth Departments of Medicine, University of Helsinki
Summary
Out of 18 subjects with symptomless autoimmune thyroiditis (S.A.T.) 5 (28%) became hypothyroid within 4 to 39 months of observation, whereas 13 (72%) remained euthyroid up to at least 28 to 50 months. In all those who developed hypothyroidism the basal serum level of thyroidstimulating hormone (T.S.H.) was already initially above normal (normal range 1·6-6·9 µU/ml) and 4 had markedly elevated concentrations (>19 µU/ml). All those subjects developing hypothyroidism also had
initially an exaggerated response to thyrotropinreleasing hormone (T.R.H.) (upper normal limit, &Dgr; T.S.H. 30 µU/ml), and in 4 the response was much exaggerated (&Dgr; T.S.H. >70 µU/ml). In 3 of these subjects the basal T.S.H. and the response to T.R.H. were reassessed before starting the substitution therapy and in all there was a further increase in both values. The basal serum T.S.H. was initially also slightly increased in 2 and the response to T.R.H. slightly above normal in 4 subjects who remained euthyroid. The basal T.S.H. level became normal in both cases with
MEASUREMENT OF THYROID-STIMULATING
assay. Discs 1
HORMONE IN DRIED BLOOD SPOT KAZUKO ENOMOTO
MINORU IRIE
First Department of Medicine, Toho
University School of
Medicine, Ohmori-Nishi, Tokyo, Japan HIROSHI NARUSE Division of Biological Research on Mental Retardation, National Institute of Mental Health, Ichikawa City,
Chiba-ken, Japan
(thyroid-stimulating hormone) was measured by radioimmunoasdried blood in spotted onto filter-paper and obtained say of the newborn for metabolic disorders. screening during
Summary
Blood
filter-papers were sent by post and kept at -20°C until cm in diameter were punched out, each disc containing approximately 20 µl of whole blood. The blood was eluted from the disc overnight in 0.6 ml of pH 7-6 phosphate buffer 50 mmol/1, containing 0-05% bovine serum albumin. The discs were removed, dried at the upper wall of the test tubes, and discarded. The eluate was subjected to the doubleantibody radioimmunoassay for T.S.H.’ 100 µl of diluted The
T.S.H.
125I-T.S.H. and diluted anti-T.s.H.
serum were
eluate, and the reaction mixture
was
added
incubated
at
to
each
40C for
three days. Then 100 µl of diluted second antibody (anti-rabbit gamma-globulin goat serum) was added and incubated at 4°C for twenty-four hours. After centrifugation, the precipitate was counted by an automated gamma counter and the values of T.S.H. were obtained from a standard curve. T.S.H. values were expressed as jjtU/ml by calculation using a multiple of 5.
By this method, the detection limit for blood T.S.H. was 5-10 µU/ml, which is the approximate upper limit of normal for blood T.S.H. T.S.H. values obtained on dried blood correlated well with those obtained on serum from
of the assay in a single sample is not necessary. This method picked up a case of primary hypothyroidism in a four-year-old girl with symptoms. Since the technique is simple and sensitive enough for the detection of hypothyroidism, it could be valuable in mass-screening for congenital hypothe
same
subjects. Duplication
thyroidism. Introduction HYPOTHYROIDISM is difficult to diagnose in the newborn and is often missed at this time or even in childhood. Early detection is important because brain damage as well as growth disturbance may occur during the first months of life. Dussault et at. 12have reported that the radioimmunoassay of thyroxine from a dried blood spot has been useful for the diagnosis of congenital hypothyroidism, and Klein et al.3 detected an infant with subclinical congenital hypothyroidism by measuring T.S.H. (thyroid-stimulating hormone). in umbilical cord serum. We have used a method to measure T.s.H. in a dried blood spot.
T.S.H. DF DRIED BLOOD SPOT
(µU /ml )
Fig. 1-Correlation between the T.S.H. values obtained by the dried blood spot method and those obtained by the measurement of serum samples from the same
y
=
20.98
+
subjects.
1.09x
(n = 166,
r =
0.841).
Materials and Methods Blood was spotted onto filter-paper.4 To check the validity of the method we assayed blood from adult hypothyroid patients and normal or hypothyroid patients during a 500 µg T.R.H. (thyrotropin-releasing hormone) administration test. To get the background data for a screening programme we also included blood from outpatients of a primary health care centre for children and from infants one day to two months of age from whom specimens had been taken for the multiple screening for metabolic disorders, and umbilical cord blood.
DR WISE AND OTHERS: REFERENCES
1. Goldsmith, R. E. Proc. Staff Meet. Mays Clin. 1972, 47, 953. 2. Jackson, G. L. Am. J. Roentgenol. Radium Ther. Nucl. Med. 1971, 112, 720. 3. Weidinger, P., Johnson, P. M., Werner, S. C. Lancet, 1974, ii, 74. 4. Wise, P. H., Ahmad, A., Pain, R. W. Unpublished. 5 Marion, M. A., Ronai, P. M., Pain, R. W., Wise, P. H. Aust. N. Z.
J. Med.
1974, 4, 379. 6. Mincey, E. K., Wilcox, M. E., Morrison, R. T. J nucl. Med. 1974, 15, 11. 7. Murphy, B. J. Lab. clin. Med. 1965, 66, 165. 8. Oldfield, R., Pain, R. W. Am. J. clin. Path. 1969, 52, 118. 9. Patel, Y. C., Burger, H. G., Hudson, B. J. clin. Endocr. Metab. 1971, 33, 768.
10. Larsen, P. R. J. clin. Invest. 1972, 51, 1939. 11 Tunbridge, W. M. G., Evered, D. Proc. Seventh Med.
1975, p. 101.
T.S.H. OF ANOTHER SPOT
(µU/ml )
2-Correlation between two values of T.S.H. obtained in discs obtained from same individuals.
Fig. int.
Thyroid Conf. Excerpta
y = -1 18 +
1.04x
(n
=
296, r
=
0.978).
two
blood
1234 Results T.S.H. values from the dried blood spot were determined after varying periods of extraction, and the maximum values were obtained after two hours of extraction. Stability of the blood spotted onto filter-paper was determined by measuring the T.s.H. values from two days to fifty-five days after spotting, virtually no change being noted over this period of time. Fig.1 shows the correlation between the T.s.H. values obtained by the dried blood spot method and those obtained by measuring T.S.H. in serum samples from the same subjects. The detection limit for blood T.S.H. was 5-10 µU/ml by the dried blood spot method. Fig. 2 shows the correlation between two values of T.S.H. in two blood discs obtained from same individuals. Using this method, adult hypothyroid patients showed high concentrations of T.S.H., and T.S.H. concentrations rose after the injection of 500 µg synthetic T.R.H. (table). Of BLOOD T.S.H. CONCENTRATION MEASURED ON DRIED BLOOD SPOTS FROM PATIENTS WITH HYPOTHYROIDISM, NORMAL SUBJECTS, AND IN CORD
well as T.s.H. would be necessary, the microdetermination of T. S. II. has substantial value since primary hypothyroidism seems to be much more common than secondary or tertiary hypothyroidism. Klein et al.have shown that treatment before the age of three months could prevent mental retardation among hypothyroid infants. With the method we have described patients with congenital or juvenile primary hypothyroidism could be detected during mass-screening for metabolic disorders, and treatment could begin during the first days or weeks of life.
iodothyronine
as
We thank Dr H. L. Levy of the State Laboratory Institute, Boston, Massachusetts, U.S.A., for his help in the preparation of this paper.
This study was partly supported by a grant from the Ministry of Health of Japan. The materials to measure T.s.H. were supplied by the National Institutes of Health, Bethesda, Maryland, U.S.A., and the Daiichi Radioisotope Company, Tokyo, Japan. We also thank Dr H. Enomoto, Dr K. Inoue, Dr T. Nukui, Dr M. Kuroda, Dr S. Takeda, Dr M. Mizotam, Dr Y. Ito, and Ms S. Oshima for their help; Dr T. Aoki, Dr T. Kumagai, Dr K. Nakayama of the Pædiatric Department of Toho University for their help with the paediatric patients, and Ms Y. litaka and M. Katakura for secretarial work.
BLOOD
Requests for reprints should be addressed to M.l. REFERENCES
1. 2.
*245
µU/ml in the case of hypothyroidism (see text).
227 newborn infants tested none had a raised T.S.H., and values in 5 cord-blood specimens were also normal. From the dried blood spots obtained from outpatients, 1 case of primary hypothyroidism was detected-in a four-year-old girl with clinical signs of hypothyroidism and a T.S.H. of 245 uU/ml.
Dussault, J. H , Laberge, L. Union Meed Can. 1973, 102, 2062 Dussault, J. H., Coulombe, P., Laberge, L., Guyda, H , Khoury, K J. Pediat. 1975, 86, 670. 3. Klein, A. H., Augustin, A. V., Foley, Jr. T. P. Lancet, 1974, ii, 77 4. Guthrie, R., Susi, A., Pediatrics, 1963, 32, 338. 5. Odell, W. D., Wliber, J. F , Utiger, R. D. Rec. Progr Hormone Res 1967, 23, 47. 6. Hershman, J. M., Pittman, Jr. J. A. Ann. intern Med 1971, 74, 481 7. Fisher, D. A., Odell, W D. J. clin Invest 1969, 48, 1670 8. Klein, A. H., Meltzer, S., Kenny, F. M. J Pediat. 1972, 81, 912
NATURAL COURSE OF SYMPTOMLESS AUTOIMMUNE THYROIDITIS
A. GORDIN Discussion We have shown that T.s.H. can be measured in 20 µl of whole blood dried on filter-paper. T.S.H. in blood collected in this manner is stable and extraction is simple. Most importantly T.S.H. values obtained in this way correlate well with those obtained on serum. Though the values of T.S.H. from the dried blood spot are a little lower than T.S.H. values obtained from serum, this method is quite acceptable for use in the detection of primary hypothyroidism by mass screening of the newborne because this method can detect approximately 10 µU/ml Of T.S.H., a value far below that of most cases of primary hypothyroidism.5 Furthermore, the assay does not have to be duplicated, a factor which greatly enhances its value for mass screening. Blood should be obtained three or more days after birth, because serum-T.s.H. levels rise sharply in the postnatal period.67However, a postnatal T.S.H. rise is not a consistent feature because we found normal blood-T.s.H. values in umbilical cord blood and in specimens obtained on the first and second days of life. In patients with secondary or tertiary hypothyroidism, the bloodt. S. H. should be reduced, but such lower than normal levels cannot yet be measured. Although for the diagnosis of all types of hypothyroidism the measurement of blood thyroxine and tri-
B.-A. LAMBERG
Endocrine Research Unit, University of Helsinki; Minerva Foundation Institute for Medical Research, Helsinki; and Third and Fourth Departments of Medicine, University of Helsinki
Summary
Out of 18 subjects with symptomless autoimmune thyroiditis (S.A.T.) 5 (28%) became hypothyroid within 4 to 39 months of observation, whereas 13 (72%) remained euthyroid up to at least 28 to 50 months. In all those who developed hypothyroidism the basal serum level of thyroidstimulating hormone (T.S.H.) was already initially above normal (normal range 1·6-6·9 µU/ml) and 4 had markedly elevated concentrations (>19 µU/ml). All those subjects developing hypothyroidism also had
initially an exaggerated response to thyrotropinreleasing hormone (T.R.H.) (upper normal limit, &Dgr; T.S.H. 30 µU/ml), and in 4 the response was much exaggerated (&Dgr; T.S.H. >70 µU/ml). In 3 of these subjects the basal T.S.H. and the response to T.R.H. were reassessed before starting the substitution therapy and in all there was a further increase in both values. The basal serum T.S.H. was initially also slightly increased in 2 and the response to T.R.H. slightly above normal in 4 subjects who remained euthyroid. The basal T.S.H. level became normal in both cases with
